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Good riddance to bad proteins : identification of novel protein quality control pathways targeting cytosolic… Fang, Neng 2014

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GOOD RIDDANCE TO BAD PROTEINS: IDENTIFICATION OF NOVEL PROTEIN QUALITY CONTROL PATHWAYS TARGETING CYTOSOLIC MISFOLDED PROTEINS FOR DEGRADATION.  by  Neng Fang  B.Sc, Sun Yat-Sen University, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2014 © Neng Fang, 2014   ii Abstract Protein misfolding is cytotoxic and the accumulation of misfolded proteins threatens cell fitness and viability. Failure to eliminate these polypeptides has been associated with numerous diseases including neurodegenerative disorders. The ubiquitin proteasome system is a major pathway that degrades in the cell these unwanted proteins targeted by protein quality control. Several distinct protein quality control degradation pathways that employ different ubiquitin ligases have been discovered in recent years. Here, we present two novel protein quality control degradation pathways that require the ubiquitin ligases Hul5 and Rsp5 to target cytosolic misfolded proteins for degradation.  We used quantitative mass spectrometry to determine that in Saccharomyces cerevisiae, heat-shock triggered a large increase in the level of ubiquitylation of mainly cytosolic proteins. We discovered that the Hul5 ubiquitin ligase participated in this ubiquitylation response. Hul5 was required to maintain cell fitness after heat-shock and to degrade short-lived misfolded proteins. In addition, the localization of Hul5 in the cytoplasm was important for its quality control function. We also showed that Hul5 targeted low-solubility cytosolic proteins in both heat-shock and unstressed conditions. These data indicate that Hul5 is involved in the degradation of cytosolic misfolded proteins. Beside the Hul5 pathway, we found that Rsp5 ubiquitin ligase also participated in the increase of ubiquitylation levels upon heat-shock. Our results indicated that Rsp5 employed a bipartite recognition mechanism to ubiquitylate heat-induced cytosolic targets via the interaction with the Hsp40 co-chaperone Ydj1 and the PY-motifs iii primarily found in structured regions of these proteins. Notably, we also found that the Rsp5-dependent pathway was dependent on both Ubp2 and Ubp3 deubiquitinases, which acted to mainly reduce the levels of K63-linked ubiquitin chains conjugated to cytosolic misfolded proteins upon heat-shock. The absence of either deubiquitinase led to reduced cell fitness under stress conditions underscoring the importance of the Rsp5-dependent pathway.  All together, we identified the two major yeast ubiquitin ligases that mediated the increase in ubiquitylation of cytosolic misfolded proteins upon heat stress. Our work shed new light on protein quality control and how the cell can mediate the degradation of misfolded proteins.iv Preface Portions of Chapter 1 are based on a published first author review article: Fang NN and Mayor T. (2012) Hul5 ubiquitin ligase: good riddance to bad proteins. Prion. 2012, 6(3):240-4, and on a first author book chapter titled: “Systems-wide analysis of protein ubiquitylation” for a book entitled: “The Molecular Chaperones Interaction Networks in Protein Folding and Degradation” edited by Dr. Walid Houry and published by Springer. The book chapter is currently under editorial processing and the book is intended to be published in early 2014.    Chapter 2 is based on a first author publication: Fang NN, Ng AH, Measday V, Mayor T. (2011) Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins. Nat Cell Biol. 2011,13(11):1344-52. All experiments were designed by myself and Dr.Thibault Mayor. I conducted all the experiments, but the experiments in Figure 2.5b and 2.5c were carried out by Alex Ng, who also helped to generate several other strains and plasmids used in the study (BPM314, 354; YTM 596, 597). Dr. Vivien Measday also helped to design and carried out part of the localization analysis using microscopy in Figure 2.4A.  Chapter 3 is based on a draft of a first author manuscript, a modified version of which has been submitted for revision. Most of the experiments were designed by myself and Dr. Thibault Mayor. I conducted the majority of experiments, while Figure 3.4d and S3.4e were done by Mang Zhu; Figures 3.4a and S3.4f were done by Sophie Comyn; and the bioinformatics analysis in Figures 3.6a, S3.6a, S3.6b and S3.6d were v done by Gerard Chan in collaboration with Dr. Joerg Gsponer based on mass spectrometry data that I generated. In addition, Mang Zhu and Sophie Comyn also contributed to the preparation of several yeast strains and plasmids used in the study (Mang Zhu: BPM564, 566-569, 583, and 584; YTM1132, 1134, 1249, 1295, 1296, and 1308; Sophie Comyn: BPM453, 519, 573, and 575).   vi Table of contents  Abstract ............................................................................................................................ii Preface ............................................................................................................................iv Table of contents .............................................................................................................vi List of tables .................................................................................................................... x List of figures ...................................................................................................................xi List of abbreviations ...................................................................................................... xiii Acknowledgments ......................................................................................................... xix Dedication ..................................................................................................................... xxi Chapter 1: Introduction .................................................................................................... 1 1.1 Protein quality control .................................................................................................................. 1 1.1.1 Definition of protein quality control ..................................................................................... 1 1.1.2 Relevance of the PQC to diseases ......................................................................................... 2 1.2 The ubiquitin proteasome system ................................................................................................ 3 1.2.1 Ubiquitylation and the UPS ................................................................................................... 4 1.2.2 E1, E2 and E3 enzymes .......................................................................................................... 7 1.2.3 Deubiquitinases and deubiquitylation ................................................................................ 10 1.3 Systems-wide approaches to studying ubiquitylation ................................................................ 11 1.3.1 The systems-wide study of the ubiquitome by mass spectrometry ................................... 11 1.3.2 Enrichment of ubiquitylated proteins using tagged ubiquitin ............................................ 13 1.3.3 Enrichment of ubiquitylated peptides using α-diGly antibodies ........................................ 15 1.4 PQC degradation pathways ......................................................................................................... 17 1.4.1 PQC degradation pathways in different cell compartments .............................................. 18 1.4.2 PQC degradation pathways in cytosol ................................................................................ 19 vii 1.4.3 PQC degradation pathways in neurodegenerative diseases and their potential as therapeutic targets ............................................................................................................................. 21 1.4.4 PQC degradation pathways in heat-shock response .......................................................... 23 1.5 Main hypothesis .......................................................................................................................... 24 Chapter 2: Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins ........................................................................ 25 2.1 Introduction ................................................................................................................................ 25 2.2 Methods ...................................................................................................................................... 27 2.2.1 Plasmids .............................................................................................................................. 27 2.2.2 Cells and heat-shock assays ................................................................................................ 27 2.2.3 IMAC—purification of ubiquitylated proteins .................................................................... 28 2.2.4 Mass spectrometry analysis ................................................................................................ 29 2.2.5 Analysis of mass spectrometry data ................................................................................... 30 2.2.6 Pull-down and subcellular fractionation ............................................................................. 31 2.2.7 Microscopy .......................................................................................................................... 32 2.2.8 35S labelling and measurement of protein degradation ..................................................... 32 2.2.9 Statistical analysis ............................................................................................................... 33 2.3 Results ......................................................................................................................................... 34 2.3.1 Heat-induced misfolding triggers a large increase of ubiquitylation levels ........................ 34 2.3.2 HS mainly induces ubiquitylation of cytosolic proteins ...................................................... 36 2.3.3 Hul5 plays a major role in the heat-shock ubiquitylation response ................................... 39 2.3.4 The cytosolic localization of Hul5 is required for its role in the HS ubiquitylation response………………………………………………………………………………………………………………………………………..42 2.3.5 Hul5 is required for targeting misfolded proteins in the absence of SSA-chaperone activity and it is required for misfolded proteins degradation ....................................................................... 45 2.3.6 Hul5 is required for the ubiquitylation of low-solubility cytosolic proteins in physiological conditions ............................................................................................................................................ 49 2.3.7 Ubiquitylated Hul5 substrates accumulate with less-soluble polypeptides ....................... 52 2.4 Discussion .................................................................................................................................... 56 2.5 Supplemental data ...................................................................................................................... 59 Chapter 3: The HECT ubiquitin ligase Rsp5 leads a major PQC degradation pathway that targets cytosolic proteins misfolded due to HS ....................................................... 90 viii 3.1 Introduction ................................................................................................................................ 90 3.2 Methods ...................................................................................................................................... 93 3.2.1 Yeast strains, plasmids and reagents .................................................................................. 93 3.2.2 HS assays ............................................................................................................................. 96 3.2.3 Down-regulation of Tet-Rsp5 expression by doxycycline ................................................... 98 3.2.4 In vitro HS ubiquitylation assay in cell extracts ................................................................... 98 3.2.5 In vivo cross-linking and co-immunoprecipitation experiments ......................................... 99 3.2.6 Yeast growth assays following HS ..................................................................................... 100 3.2.7 35S-labeling and protein turnover assays .......................................................................... 100 3.2.8 Fluorescence based degradation assays ........................................................................... 101 3.2.9 Microscopy ........................................................................................................................ 102 3.2.10 DiGly peptide enrichment for triple SILAC mass spectrometry analysis .......................... 102 3.2.11 IMAC-purification of ubiquitylated proteins ..................................................................... 104 3.2.12 In-gel digestion of IMAC samples for ubiquitin linkage quantification by AQUA-SRM .... 105 3.2.13 Mass spectrometry analysis .............................................................................................. 106 3.2.14 Analysis of mass spectrometry data ................................................................................. 108 3.2.15 Computational analyses of Rsp5 candidate substrates .................................................... 109 3.3 Results ....................................................................................................................................... 110 3.3.1 The RSP5 E3 ligase is required for the HS induced ubiquitylation response .................... 110 3.3.2 Rsp5 directly ubiquitylates heat-induced misfolded proteins .......................................... 113 3.3.3 Rsp5 ubiquitylates heat-induced cytosolic misfolded proteins ........................................ 116 3.3.4 Rsp5 is required for the degradation of cytosolic misfolded proteins ............................. 121 3.3.5 The deubiquitinases Ubp2 and Ubp3 are required for the degradation of misfolded protein and their absence reduces cell fitness upon HS ................................................................... 125 3.3.6 Heat-induced Rsp5 substrates contain PY motifs preferably embedded in structured regions and Hsp40 co-chaperone Ydj1 assists Rsp5 to target heat-induced misfolded proteins .... 130 3.4 Discussion .................................................................................................................................. 136 3.5 Supplemental Data.................................................................................................................... 140 Chapter 4: Conclusions ............................................................................................... 176 4.1 Chapter summary...................................................................................................................... 176 4.2 Common discussion .................................................................................................................. 178 4.2.1 The HS stress ..................................................................................................................... 178 ix 4.2.2 The ligase activity of Hul5 and Rsp5 upon HS stress ......................................................... 179 4.2.3 The ubiquitin linkages catalyzed by Hul5 and Rsp5 .......................................................... 180 4.2.4 The degradation “timer” in the cytosol ............................................................................ 182 4.2.5 The relationship between the Hul5 and Rsp5 pathways .................................................. 184 4.3 Future directions ....................................................................................................................... 185 Bibliography ................................................................................................................ 190    x List of tables Supplementary Table 2.1 Plasmid used in chapter 2 .................................................... 70 Supplementary Table 2.2 Yeast strains used in chapter 2 ............................................ 70 Supplementary Table 2.3 List of heat-shock affected proteins enriched by IMAC ........ 72 Supplementary Table 2.4 List of E3 ubiquitin ligases assessed .................................... 75 Supplementary Table 2.5 List of potential Hul5 substrates under no stress condition enriched by IMAC .......................................................................................................... 77 Supplementary Table 2.6 Method validation of quantitative mass spectrometry and IMAC used in chapter 2 ................................................................................................. 79 Supplementary Table 2.7 Quantitative mass spectrometry data for figure. S2.5b, d ..... 84 Supplementary Table Table 3.1 Yeast strains used in chapter 3 ................................ 152 Supplementary Table 3.2 Plasmids used in chapter 3 ................................................ 154 Supplementary Table 3.3 DiGly peptides quantified in figure. 3.3b ............................. 156 Supplementary Table 3.4 AQUA ubiquitin peptides used in SRM analysis ................. 161 Supplementary Table 3.5 DiGly peptides comparison between ubp2Δ and WT upon HS .................................................................................................................................... 161 Supplementary Table 3.6 DiGly peptides comparison between ubp3Δ and WT upon HS .................................................................................................................................... 169   xi List of figures Figure 1.1 Ubiquitin and ubiquitylation ............................................................................ 6 Figure 1.2 Ubiquitin linkages and functions ..................................................................... 7 Figure 1.3 Different ubiquitin ligases families and their catalytic mechanisms .............. 10 Figure 2.1 Heat-induced misfolding triggers a large increase of ubiquitylation levels ... 35 Figure 2.2 Heat-shock mainly affects cytosolic proteins ................................................ 38 Figure 2.3 HUL5 is required for the full ubiquitylation response and cell fitness after heat-shock ..................................................................................................................... 41 Figure 2.4 Hul5 re-distribution to the cytosol is important for its role in the HS response ...................................................................................................................................... 43 Figure 2.5 HUL5 is essential for the ubiquitylation of proteins misfolded in the absence of SSA-chaperone activity and for the degradation of pulse-labelled misfolded polypeptides .................................................................................................................. 47 Figure 2.6 HUL5 is required for ubiquitylation of low-solubility cytosolic proteins .......... 51 Figure 2.7 Hul5 targets proteins that are specifically ubiquitylated in the low-solubility cellular fraction .............................................................................................................. 54 Figure S2.1 HS induces protein misfolding and polyubiquitylation ................................ 59 Figure S2.2 Identification of HS affected ubiquitylation by quantitative mass spectrometry analysis .................................................................................................... 61 Figure S2.3 HUL5 is required for the full ubiquitylation response and cell fitness after HS ...................................................................................................................................... 63 Figure S2.4 HS causes a re-localization of Hul5 ........................................................... 65 xii Figure S2.5 HUL5 is required for ubiquitylation of misfolded proteins under both HS and unstressed conditions .................................................................................................... 66 Figure S2.6 Hul5 is required for the ubiquitylation of several low solubility proteins in unstressed cells ............................................................................................................ 68 Figure 3.1 Functional Rsp5 is required for the HS ubiquitylation response ................. 112 Figure 3.2 Rsp5 directly ubiquitylates heat-induced misfolded proteins ...................... 115 Figure 3.3 Rsp5 targets cytosolic proteins upon HS ................................................... 119 Figure 3.4 Rsp5 is required for the degradation of cytosolic misfolded proteins ......... 123 Figure 3.5 Ubp2 and Ubp3 are required for the degradation of cytosolic misfolded proteins ....................................................................................................................... 128 Figure 3.6 Rsp5 directly and with Ydj1-adaptor ubiquitylates misfolded proteins upon HS .................................................................................................................................... 134 Figure 3.7 Model for Rsp5 recognition of misfolded proteins ...................................... 137 Figure S3.1 Functional Rsp5 is required for the HS ubiquitylation response ............... 140 Figure S3.2 Rsp5 directly ubiquitylates heat-induced misfolded proteins.................... 142 Figure S3.3 Rsp5 targets cytosolic proteins upon HS ................................................. 143 Figure S3.4 Rsp5 is required for the degradation of cytosolic misfolded proteins ....... 145 Figure S3.5 Ubp2 and Ubp3 are required for the degradation cytosolic misfolded proteins ....................................................................................................................... 147 Figure S3.6 Rsp5 directly and with Ydj1-adaptor ubiquitylates misfolded proteins upon HS ............................................................................................................................... 149 Figure S3.7 Model for Rsp5 proteasome targeting with deubiquitinases Ubp2 and Ubp3 .................................................................................................................................... 151 xiii List of abbreviations % Percentage [PSI+] Self-progagating misfolded form of Sup35p in yeast ≥ Equal to or more than °C Degree Celsius Δ Delta-signifies deletion α Alpha β Beta µg Microgram µl Microliter µM Micromolar μCi Microcurie μm Micrometer 13C Carbon-13, a stable isotope of carbon 13Myc 13 times repeat of a polypeptide protein tag derived from the c-myc gene 14N Nitrogen-14 15N Nitrogen-15, a stable isotope of nitrogen 3' Downstream of a coding sequence 35S Radioactive isotope of sulfur A600 Absorbance at 600nm AQUA Absolute QUAntification Arg-Gly-Gly Arginine-Glycine-Glycine ARS Autonomously Replicating Sequence ART Arrestin-Related Trafficking ATP Adenosine triphosphate ATXN3 Ataxin 3 AUC Area Under the Curve C Cytosol/Cytoplasm C18 Octadecyl carbon chain C2 domain  Calcium binding C2 domain in Nedd4 famaly ubiquitin ligases CaCl2 Calcium Chloride CAN1 CANavanine resistance 1 CDC19 Cell Division Cycle 19 CDC28 Cell Division Cycle 28 CFTR Cystic Fibrosis Transmembrane Regulator CHIP Carboxy terminus of Hsp70-Interacting Protein  CHX Cycloheximide CoCl2 Cobalt(II) Chloride C-terminus/C-terminal Carboxylic acid-terminus of a protein ddH2O Double-distilled water diGly Glycine-Glycine xiv DNA Deoxyribonucleic Acid DOA10  SSM4 (Suppressor of mma Stability Mutant 4) DTA A data file format DTT DiThioThreitol e.g. Exampli gratia (For example) E1 Ubiquitin-activating enzyme E2 Ubiquitin-conjugating enzyme E3 Ubiquitin ligase E4 Ubiquitin-chain elongating enzyme E6AP E6-Associated Protein ECL Enhanced Chemiluminescence EDTA Ethylenediaminetetraacetic acid EGTA Ethylene glycol tetraacetic acid ER Endoplasmic Reticulum ERAD ER-Associated protein Degradation ErbB2 Human epidermal growth factor receptor 2, known as HER2 ESI ElectroSpray Ionization source F Phenylalanine FBP6 Fructose-2,6-bisphosphatase 6 Fig. Figure g Gravitational constant (g-force) GAL4/UAS Yeast transcription activator gene GAL4 and the Upstream Activation Sequence GALp Galactose regulated gene promoter GFP Green Fluorescent Protein GG Glycine-Glycine GPD Glycerol-3-Phosphate Dehydrogenase GST Glutathione S-Transferase GUK1 Guanylate Kinase 1 Hr(s) Hour(s) H2O2 Hydrogen peroxide H3 Histone H3 H8 Octo-histidine HA  a polypeptide tag derived from Human influenza hemagglutinin HCD Higher-energy Collisional Dissociation HECT E6AP Carboxyl Terminus HEK293 Human Embryonic Kidney 293  HeLa Henrietta Lacks HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid  HOIP HOIL-1-interacting Protein HPLC High-Pressure Liquid Chromatography HPV Human Papilloma Virus HRD1 HMG-coA Reductase Degradation 1 HS Heat-Shock Hsp Heat-shock protein Hsp Heat-shock protein xv HU HEPES and Urea HUL4 HECT Ubiquitin Ligase 4 HUL5 HECT Ubiquitin Ligase 5 i.e.  id est (that is) IFN Interferon IgG Immunoglobulin G IMAC  Immobilized Metal Affinity Chromatography IP Immunoprecipitation IPTG IsoPropyl β-D-1-ThioGalactopyranoside ISG15 Interferon-Stimulated Gene of 15kDa K Lysine Kar2/Bip2 KARyogamy 2 kDa Kilo Dalton KOAc Potassium Acetate KOS Kaufman Oculocerebrofacial Syndrome K-ε-GG  Lysine covelently linked to Glycine-Glycine  LANA Leucine-Alanine-Asparagine-Alanine log2 Binary logarithm LPNY Leucine-Proline-Asparagine-Tyrosine LPxY Leucine-Proline-any a.a.-Tyrosine LSM7 Like SM 7 LTQ Linear Trap Quadropole M Membrane M Molar M/P Mitochondria or Peroxisome MG132 Proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal MgAc Magnesium Acetate min Minute ml Milliliter ml-1 Per milliliter mM Millimolar MOPS 3-(N-MOrpholino)PropaneSulfonic acid mRNA messenger RNA ms millisecond MS/MS Tandem mass spectrometry MUP1 Methionine Uptake MVB MultiVesicular Body Mw Molecular weight N Nuclei/Nucleus n Number Na2HPO4 Sodium phosphate dibasic NaAc Sodium Acetate NAB N-Aryl Benzimidazole NaCl Sodium Chloride NEDD4 Neuronal precursor cell-expressed Developmentally Downregulated 4 NEDD8 Neural-precursor-cell-Expressed and Developmentally Down-xvi regulated gene 8 NF-κB  Nuclear Factor Kappa-light-chain-enhancer of activated B cells NH4HCO3 Ammonium bicarbonate NLS SV40 Nuclear Localization Signal NP-40 Nonyl phenoxypolyethoxylethanol N-terminus/N-terminal Amine-terminus of a protein ORFs Open Reading Frames P Pellet PaeI-R Parkin-associated endothelin receptor-Like Receptor PAGE PolyAcrylamide Gel Electrophoresis  PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PDC1 Pyruvate DeCarboxylase 1 PDR5 Pleiotropic Drug Resistance 5 PGK1 3-Phosphoglycerate Kinase pH Potential of Hydrogen PhD Doctor of Philosophy pi Percentage PIN3 Psi+ Inducibility 3 PIPKY Proline-Isoleucine-Proline-Lysine-Tyrosine PMSF PhenylMethaneSulfonyl Fluoride poly(A) Multiple Adenosine monophosphate PP/GG mutate two Proline to two Glycine PPARα Peroxisome Proliferator Activated Receptor alpha  ppm Parts-Per Million PPPP Proline-Proline-Proline-Proline PPR Proline-Proline-Arginine PPxF Proline-Proline-any a.a.-Phenylalanine PPxY Proline-Proline-any a.a.-Tyrosine PQC Protein Quality Control PrP Prion Protein PTM Protein Translational Modification PxY Proline-any a.a-Tyrosine PY Proline-rich PY/AA mutate Proline and Tyrosine to two Alanine  QQQ Triple quadrupole  RBRs RING-between-RINGs RFP Red Fluorescent Protein RING Really Interesting New Gene RKR1/LTN1 RING domain mutant Killed by rtf1 deletion 1 RMA1 RING Membrane-Anchor protien 1 RNA Ribonucleic Acid RPS7B Ribosomal Protein 7B of the Small subunit  RPT6 Regulatory Particle Triphosphatase 6 RSP5 Reverses Spt-Phenotype 5 xvii s Second S Soluble s.d Standard deviation s.e.m. Standard Error of the Mean SAN1 Sir Antagonist 1 SC Synthetic Complete media SCX  Strong Cation-Exchange SD-MET Yeast synthetic minimal media without methionine SDS Sodium Dodecyl Sulfate SGD Saccharomyces Genome Database SEC23 SECretory protein 23 SILAC Stable Isotope Labeling by Amino acid in Cell culture SIS1 SIt4 Suppressor 4 SLH1 SKI2-Like Helicase 1 SPxF Serine-Proline-any a.a.-Phenylalanine SRM Selected Reaction Monitoring SSA Stress Seventy subfamily A SSE1 A cytosolic member of Hsp70 family STE6 STErile 6 STI1 Stress Inducible 1 STY Serine, Threonine, Tyrosine SUMOs Small Ubiquitin-Like Modifiers Sup. Supplementary SUP45 SUPpressor 45 T Total cell extract t0 Time 0 TAP Tandem Affinity Purification TBS Tris-Buffered Saline TCA Trichloroacetic Acid TCEP 3,3′,3′′-Phosphanetriyltripropanoic acid Th Thomson unit ti TCA-soluble signal at indicated time point TOM1 Trigger of Mitosis 1 TRAP Ion Trap mass spectrometer ts Temperature Sensitive TSA2 Thiol-Specific Antioxidant 2 TWEEN 20 Polysorbate 20 TWEEN 80 Oleic acid precursor: Polyethylene glycol sorbitan monooleate UBE3B UBiquitin ligase E3B UBE3C UBiquitin ligase E3C Ubi Ubiquitin Ubls Ubiquitin-Like Proteins UBP14 Ubiquitin-specific Protease 14 UBP2 Ubiquitin-specific Protease 2 UBP3 Ubiquitin-specific Protease 3 UBP6 Ubiquitin-specific Protease 6 xviii  UBR1 E3 Ubiquitin ligase (N-recognin) 1 UBR2 An cytosolic ubiquitin ligase UCHL1 Ubiquitin Carboxy-terminal Hydrolase L1 UFD2 Ubiquitin Fusion Degradation 2 UFD4 Ubiquitin Fusion Degradation protein 4 UPS Ubiquitin Proteasome System URA Uracil URA3  URAcil requiring 3 UTR Untranslated Region VHL Von Hippel-Lindau VPxF Valine-Proline-any a.a.-Phenylalanine VPxY Valine-Proline-any a.a.-Tyrosine WT Wild-type WW Tryptophan Tryptophan x Times YDJ1 Yeast DnaJ 1 YKO Yeast Knockout YNB Yeast Nitrogen Base (Yeast synthetic minimal media)  YOS9 Yeast OS-9 homolog YPD Rich media for yeast gorwth containing Yeast extract, Peptone and Dextrose       xix Acknowledgments I would like to thank people listed here who have helped me during my PhD studies. Their support was invaluable to completion of this work.  Most importantly, I would like to thank Dr. Thibault Mayor, my research supervisor, for his mentorship, guidance and insight. I am especially grateful to Dr. Mayor for his persistent support and patience for my research ventures, which admittedly often took me off the beaten path. I am convinced that this experience will be essential to my future career.   I would also like to thank my supervisory committee, Dr. Leonard Foster and Dr. Chris Loewen as well as our collaborator Dr. Vivien Measday for their insight, criticism and advice on this work.   I am grateful to Dr. Jason. Winget, Dr. Nikolay Stoynov and members of the Foster lab for their technical assistance with mass spectrometry analysis that was invaluable to completion of this work.    I want to say thank you to members of the Mayor lab, past and present, including Sophie Comyn, Gerard Chan, Mang Zhu, Dr. Patrick Hau Wing Chan, Dr. Razvan Albu Patrizio Panelli, Alex Ng, Dr. Jason Winget, Nicolas Coutin, Nelson Chow, Dr. Inga Wilde, Farzin Khosrow-Khavar, Chris Hao Pu and Coustance Couture for their support xx and critique. I would especially like to thank Alex Ng, Sophie Comyn, Gerard Chan and Mang Zhu for their essential contributions to this work.   I would like to extend my gratitude to Dr. E. Jan, Dr. L. Howe, Dr. L. Conibear, Dr. M. Roberge, Dr. C. Boone, Dr. S. Jentsch, E. Craig, Dr. D.Finley, Dr. R. Deshaies, Dr. P. Hieter, Dr. H.R. Pelham, Dr. J. Huibregtse, and Dr. T. Zodalek for their generous support in providing reagents or access to equipment that made this work possible. I would also like to thank Dr. P. Kaiser and Dr. J. Gsponer for comments and discussion on this work.   I also want to say thank you to Dr. Adam Chruscicki and Dr. Matthew Dahabieh whose input and advice were invaluable to my development as a scientist.   I want to extend my gratitude to the funding agencies that supported this work, the Canada Institutes of Health Research (CIHR) and the University of British Columbia.   Finally, and most importantly, I want to thank my parents, Jianzhi Ma and Nigong Fang as well as my Uncle Martin Feng and his family for their everlasting support, love and encouragement through all these years of work.     xxi Dedication I dedicate this thesis to my family.            “The important thing is not to stop questioning. Curiosity has its own reason for existing. One cannot help but be in awe when he contemplates the mysteries of eternity, of life, of the marvelous structure of reality. It is enough if one tries merely to comprehend a little of this mystery every day. Never lose a holy curiosity.”           Albert Einstein   1 Chapter 1: Introduction  1.1 Protein quality control As the working horses of most biological processes in the cell, proteins are essential elements for any living organism. Most proteins must adopt specific three-dimensional structures to be functional, also called the native fold or native state. After synthesis, most polypeptides adopt different folding conformations that may include distinct structural intermediates before reaching their native state. Even after proper folding, proteins can still switch between different conformational states due to post-translational modification or binding to interaction partners[1, 2]. As the energy barriers between native and non-native conformations are generally very small, proteins are at constant risk of misfolding under various cellular stress conditions such as mutations, translational errors and environmental stresses[1, 3]. Cellular protein quality control pathways play a major role in sensing and sorting misfolded proteins to prevent their accumulation in the cell[4-6].   1.1.1 Definition of protein quality control Facing constant cellular challenges, eukaryotic cells must keep the level of misfolded proteins in check to maintain protein homeostasis and cell viability. Protein homeostasis (proteostasis) is an integrated network within the cell comprises of the folding system, degradation systems, signaling pathways, and compartmentalized modules that control the biosynthesis, folding, trafficking and degradation of proteins[7, 2 8]. The task of maintaining low level of misfolded proteins is performed by an elaborate system in the cell called Protein Quality Control (PQC) that consists of a network of effector proteins that monitor and maintain the integrity of the proteome. PQC requires the collaboration of molecular chaperones/co-chaperones and proteolytic systems to mediate folding or eliminate aberrant species[6, 9]. The network of chaperones and co-chaperones is responsible for detecting; folding or help with folding of the misfolded proteins. In some cases, the terminally misfolded species (i.e., that cannot fold or refold) will be subjected to degradation, a process that may also be assisted by chaperone and co-chaperone proteins. There are different routes that PQC adopts to eliminate misfolded proteins, the ubiquitin proteasome system (UPS), chaperone-mediated autophagy, and selective macroautophagy[9, 10]. Among these degradation systems, the UPS is the primary method that is responsible for the proteolysis of majority of the misfolded proteins in the cell[11-13].  1.1.2 Relevance of the PQC to diseases The progressive accumulation of misfolded proteins can lead to the formation of protein aggregates, which is a common feature in numerous neurodegenerative diseases, as well as other disorders[10, 14, 15]. Cytosolic and nuclear inclusion bodies are found in polyglutamine diseases; Lewy bodies is a typical hallmark for Parkinson disease and related synucleinopathies; and neurofibrillary tangles and plaques can be found in Alzheimer disease and tauopathies respectively. These examples underscore the importance of PQC. To prevent accumulation of misfolded proteins, functional protein degradation systems such as the UPS are crucial, as eliminating aberrant 3 proteins is perhaps a better way (than e.g. assisting misfolded proteins to refold) to prevent their accumulation and aggregation. In line with this argument, the impairment of the UPS has been associated with the progression of many protein-misfolding disorders including neurodegenerative diseases[10, 13, 14, 16, 17]. Therefore, it is crucial that we improve our knowledge of PQC and how the misfolded proteins are targeted for proteolysis.   1.2 The ubiquitin proteasome system The proteome is far more complex than the genome from which it is derived due to alternative splicing and extensive protein post-translational modifications. Importantly, post-translational modifications fulfill the key roles by controlling protein-protein interactions, protein localization, enzymatic activity and protein turnover. To date, ubiquitylation is among the most abundantly identified post-translational modifications following phosphorylation, of which there are more than 51,000 reported protein modification sites[18]. Ubiquitin is a 76 amino acid, highly structured protein with a molecular weight of about 8.5 kDa (Fig. 1.1a, MMDB ID:57540)[19]. It was named after its ubiquitous expression in eukaryotes and its common role as a protein modifier in numerous biological processes. There are also other similar protein modifiers, called ubiquitin-like proteins (Ubls), such as SUMOs (small ubiquitin-like modifiers), ISG15 (interferon-stimulated gene of 15kDa) and NEDD8 (neural-precursor-cell-expressed and developmentally down-regulated gene 8) in cells. These Ubls have both a sequence and a structural homology to ubiquitin, and modify substrates via similar mechanisms.  4 However, ubiquitin is the best-characterized member in the family, especially for its role in proteolysis.   1.2.1 Ubiquitylation and the UPS Ubiquitylation is a process in which ubiquitin is covalently attached to another protein through the formation of an isopeptide bond between the carboxyl group of the last glycine residue of ubiquitin and, typically, the epsilon amino group of a target lysine residue (Fig.1.1b). Substrate proteins can be modified by a single ubiquitin on a single lysine (mono-ubiquitylation) or multiple lysine residues (multi-monoubiquitylation). Alternatively, the lysine residues within ubiquitin itself can be used to covalently attach subsequent ubiquitin molecules, forming multimeric chains conjugated to a single lysine residue on the targeted protein (poly-ubiquitylation)(Fig. 1.2)[20, 21]. The seven lysine residues (K6, 11, 27, 29, 33, 48 and 63) and the N-terminus of ubiquitin (Fig. 1.1a) can be used to generate ubiquitin chains that are either homogenous (one linkage type throughout), mixed (with different linkage types) or branched (arising when two ubiquitins are conjugated to two separate lysines on the same ubiquitin moiety, thus creating a branching point) (Fig. 1.2). This unique, built-in ability to generate linkages of diverse architecture enables ubiquitylation to be a versatile post-translational modification that affects most cellular pathways in some way. A major function of ubiquitylation is to target substrates for proteasomal degradation. In addition (or in tandem), a plethora of different processes are regulated by ubiquitylation, including: endocytosis, selective macro-autophagy, cell cycle control, inflammation and NF-κB 5 activation, DNA repair, transcription, antigen processing, viral infection, and ribosome and peroxisome biogenesis (Fig. 1.2)[21-23].  In the UPS, to be recognized and degraded by the proteasome, most of the substrates are needed to be linked to ubiquitin chains that usually contain at least four ubiquitins and are assembled through K48, K29 or K11 on ubiquitins (Fig. 1.2 highlighted in orange)[24, 25]. There are some exceptions such as some oxidatively damaged proteins can be degraded by the proteasome in an ATP- and ubiquitin-independent manner[26-29]. The ubiquitylation-dependent proteasome targets can be divided into two major classes based on the outcome of the degradation. One is the regulatory targets, which are degraded by the UPS for regulatory and signaling purposes. A perhaps most well-known example is the degradation of cyclins, inhibitors of cyclin-dependent kinases and anaphase inhibitors by UPS for cell-cycle progression[30]. Another major class of UPS substrates are misfolded proteins that are targeted by PQC[11].              6 Figure 1.1 Ubiquitin and ubiquitylation    (a). Backbone structure of human ubiquitin (according to its crystal structure; MMDB ID: 57540) with all lysines highlighted in yellow and N/C-terminus indicated. (b). Schematic representation of the ubiquitin enzyme cascade involved in the ubiquitylation process.   7 Figure 1.2 Ubiquitin linkages and functions   Schematic representation of diverse ubiquitin chains linked through different lysine residues and N-terminus on ubiquitins and their known cellular functions. The linkages known to be involved in UPS are highlighted in orange.   1.2.2 E1, E2 and E3 enzymes Three classes of enzymes are required for the ubiquitylation cascade that leads to substrate modification, namely an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an ubiquitin ligase (E3). Prior to substrate conjugation, ubiquitin first needs to be activated by the E1 enzyme via an ATP-dependent reaction, in which a thioester linkage is formed between the C-terminus of ubiquitin and a 8 cysteine residue of the E1[31, 32]. The E1 then mediates the transfer of ubiquitin to a cysteine residue of an E2 enzyme via a trans-thioesterification reaction[33, 34]. Finally, an E3 ligase will recruit the target substrate for the last ligation step (Fig. 1.1b). The substrate specificity and the type of linkage conjugated are determined either by E3 alone or in conjunction with the associated E2. In mammalian genome, at least 600 genes are estimated to encode for E3 ligases and about 40 for E2s[35]. Therefore, the ubiquitin system relies on a highly developed and complex network of proteins.  E3 ligases have historically been classified into two families based on their catalytic mechanism and structure. The majority of E3 ligases belong to the Really Interesting New Gene (RING) family. A RING or RING-related ligase facilitates the direct transfer of the ubiquitin molecule from the E2 to the associated substrate (Fig. 1.3a). The other family of the Homologous to the E6AP Carboxyl Terminus (HECT) domain E3s transiently accepts the ubiquitin from the E2 prior to transferring it to the associated substrate[35] (Fig. 1.3b). Based on recent knowledge, RING-between-RINGs (RBRs) ubiquitin ligases also transiently accept ubiquitin prior to substrate conjugation[36, 37]. The ligases in this family such as Parkin[38] and HOIP[39] use a hybrid RING/HECT mechanism to transfer ubiquitin to substrates (Fig. 1.3c). In addition, some ubiquitin ligases also function as poly-ubiquitin chain elongation factors called E4s that conjugate ubiquitin previously attached to the substrate by other E3 ligases[40]. A challenge in the field is to distinguish which UPS pathways are involved in the degradation of PQC targets and to understand the molecular mechanisms that dictate substrate recognition and selection.  9 The two novel pathways discovered in this work, which are involved in targeting cytosolic misfolded proteins in yeast, depend on two HECT ligases Hul5 and Rsp5. There are around 30 HECT E3 ligases in humans[41] and five (RSP5, HUL4, HUL5, TOM1 and UFD4) in Saccharomyces cerevisiae. The first HECT ubiquitin ligase discovered was the human papilloma virus (HPV) E6-associated protein (E6AP)[42]. HECT is a domain that consists of around 350 to 450 amino acids found at the C-terminus of the ligase. It contains an E2 binding site located in a larger N-lobe of the domain (located at the N-terminus of the domain) and an active-site cysteine in a smaller C-lobe[43]. The sizes of HECT E3s are ranging from 90 kDa to over 500 kDa. The region on the N-terminus of HECT ligases usually determines substrate specificity, the ligase cellular localization and may be involved in regulation of the ligase activity[41, 43]. Despite of the small family size, HECT ligases are involved in numerous essential cellular processes. For example, Rsp5, the only Nedd4-subfamily member in yeast, is an essential gene and is involved in multiple cellular processes such as protein trafficking, endocytosis, mitochondrial inheritance, transcription regulation[41]. Nedd4 family is the best-characterized subfamily of HECT ligases, while the functions and regulation of many other HECT ligases are less well known.   10 Figure 1.3 Different ubiquitin ligases families and their catalytic mechanisms   (a) A RING family ligase catalyzes ubiquitin transfer directly from an E2 to a substrate. (b) A HECT family ligase first accepts the ubiquitin before transferring it onto a substrate. (c) A RBR family ligase uses a hybrid mechanism.   1.2.3 Deubiquitinases and deubiquitylation Ubiquitylation is a dynamic process that can be reversed by a class of enzymes called deubiquitinases (Fig.1.1b). So far, there have been 98 deubiquitinases identified in humans[44] and 17 in yeast. Deubiquitinases are involved in maintaining ubiquitin homeostasis by processing newly-synthesized ubiquitin, recycling used ubiquitin, controlling or modulating the fate of ubiquitylated proteins[45], antagonizing the ligase 11 activity[46] and modulating proteasome processivity[47]. It was shown that some deubiquitinases had linkage specificity[48, 49]. Therefore, deubiquitinases may contribute to the substrate-recognition specificity of different UPS degradation pathways. For example, the yeast Ubp2 specifically removes K63-linkages built by the HECT ligase Rsp5 to promote mono-ubiquitylation of the substrates[50].  While our general understanding of the enzymatic mechanisms of ubiquitylation has advanced immensely, thanks to detailed biochemical analyses, a more comprehensive view of the relationships between the different components of the ubiquitin system is still lacking. Therefore, to study PQC degradation pathways and their misfolded targets, there is an increasing need to integrate systems-wide approaches such as mass-spectrometry-based proteomics, which can potentially provide a broader understanding of this intricate system.  1.3 Systems-wide approaches to studying ubiquitylation To date, the systems-wide analyses of ubiquitylated proteins were mainly based on three major types of proteomic approaches: mass spectrometry analysis, in vitro protein/peptide arrays and the newly arising bioinformatics methods. We, for instance, relied on different mass spectrometry-based approaches that I will introduce here in order to identify ubiquitylated proteins targeted by PQC.  1.3.1 The systems-wide study of the ubiquitome by mass spectrometry Most mass spectrometry-based protein analyses use a top-down approach in which proteins are digested into peptides, then separated by liquid chromatography 12 prior to their analysis by the mass spectrometer. Using tandem mass spectrometry (MS/MS), peptides are further fragmented into second order mass spectra, which act as “fingerprints” that are subsequently searched against a database to obtain a potential sequence match. The peptides can, in turn, be used to identify the proteins from which they are derived. Mass spectrometry can thus be used to identify the protein composition of the analyzed biological sample, and, in some cases, also to quantify differences between two or more samples[51].  Analysis of PTMs is well suited for mass spectrometry when modified peptides can be characterized by a difference in mass (compared to their unmodified counterparts) that can be detected by the instrument. In the case of ubiquitylation, a characteristic amino acid “tail” is left on the lysine residue of the substrate peptide, which was first used to detect in vivo ubiquitylation sites by mass spectrometry in 1993[52]. Different proteases have been used to generate peptides with remnant ubiquitin tails for mass spectrometry analysis[52]. However, due to its robust catalytic activity and cleavage site specificity, trypsin is now the most commonly used enzyme (among many other applications) for proteomic studies of ubiquitylation. Trypsin is a serine protease that cuts peptide chains on the carboxyl side of lysine and arginine. Because the carboxy-terminal end of ubiquitin has the amino acid sequence Arg-Gly-Gly, tryptic digestion of ubiquitylated peptides will generate a “tail” consisting of the last two glycine residues. This di-glycine “tail” (hereafter referred to as the remnant or diGly tail) adds a signature mass shift of +114 Da on the modified lysine residues. At the same time, trypsin is unable to cleave ubiquitylated lysines, and thus modified peptides also contain a missed cleavage site. One potential problem is that the same mass shift of 114 Da can also 13 occur due to the alkylation of lysine residues during sample preparation when using iodoacetamide (commonly used to protect reduced cysteine residues prior to mass spectrometry)[53]. Therefore, sample alkylation is commonly performed at no more than room temperature, and iodoacetamide is substituted by chloroacetamide, which generally does not cross-react as readily. By the early 2000s, most of the effector-enzymes (E1s, E2s, E3s, Deubiquitinases) in the ubiquitin system had been uncovered[54]. However, only a small fraction of their substrates were characterized. To fill in the gap—and taking advantage of major progresses in mass spectrometry instrumentation—systems-wide proteomic approaches were developed. Given the transient, and mostly low-abundant nature of ubiquitylation in the cell, conjugated proteins and their ubiquitylation sites are difficult to detect by mass spectrometry analysis without any pre-enrichment. Therefore, the depth and coverage of the ubiquitome is tightly dependent on the enrichment method.   1.3.2 Enrichment of ubiquitylated proteins using tagged ubiquitin Taking advantage of yeast genetics, Steven Gygi and colleagues were among the first to intracellularly express tagged ubiquitin, enabling them to enrich for ubiquitin conjugates for large-scale analysis of the ubiquitome[55]. A yeast strain, which an ectopic 6xHis-myc-ubiquitin was expressed instead of wild-type ubiquitin, was used to purify ubiquitin conjugates under denaturing conditions using nickel-affinity chromatography. 1,075 proteins were identified by mass spectrometry in the study, including 110 ubiquitylation sites (i.e., diGly-containing peptides) in 72 proteins. Analysis of ubiquitin conjugates is not constrained to single cell model organisms. The GAL4/UAS tissue-14 targeted expression system was also used to study the ubiquitin proteome in Drosophila melanogaster[56]. In this study, ubiquitin that contains a specific BirA recognition sequence was over-expressed solely in the nervous system, together with the Escherichia coli birA gene to biotinylate tagged ubiquitin. Consequently, 48 novel neuronal-specific ubiquitylation substrates were identified in this pioneering proteomic study conducted in a multi-cellular organism.  To reduce the levels of non-specifically-bound proteins (binding to the tag or to the affinity column) during the enrichment of tagged ubiquitin, which is notoriously problematic with the histidine-tag system, different approaches have subsequently been used. For instance, Peter Kaiser’s group developed a tandem affinity tag approach by employing a two-step purification, using 6xhistine and biotin tags[57]. In the second step, the extraordinarily high affinity between biotin and streptavidin allows the enrichment of targets under very stringent conditions, such as 2% SDS and 8M urea. Over 150 ubiquitylated proteins were identified under these conditions. This approach was then applied to mammalian tissue cultures to identify over 600 ubiquitylated conjugates in HeLa cells[58]. We developed a simpler approach using an octo-histidine (H8) tagged ubiquitin that enables the introduction of SDS during the washes to reduce the background (See Chapter 2). Strong cation exchange was used by Stanley Fields and colleagues in order to further fractionate ubiquitylated peptides after nickel chromatography (also using a H8 tagged ubiquitin), as the amino group of the ubiquitin remnant tail adds an additional positive charge at low pH in comparison to other peptides[59]. In their study, 870 ubiquitylation sites were identified among 438 proteins. The number of analyzed fractions may also influence the output. After tagging ubiquitin 15 with a tandem tag (streptavidin and HA) and separating the purified proteins through gel electrophoresis, Danielsen and colleagues identified over 700 ubiquitylation sites after analyzing 20 gel fractions[60]. Overall, due to its relative simplicity, the purification of proteins conjugated to histidine-tagged ubiquitin (as well as to other tags) remains widely used. For instance, we used the H8-ubiquitin approach in Chapter 2 in order to identify which proteins are ubiquitylated upon heat-shock (HS) and Hul5 ubiquitin ligase substrates.   1.3.3 Enrichment of ubiquitylated peptides using α-diGly antibodies A major breakthrough was the introduction of antibodies that directly bind to ubiquitylated peptides. After the first 110 ubiquitylation sites identified by Gygi and colleagues[55], the uncovering of new ubiquitylation sites had largely stalled until antibodies that specifically enrich for ubiquitylated peptides were developed.  In the antibody-based approach, ubiquitylated peptides of low abundance are greatly enriched prior to identification by mass spectrometry using antibodies that recognize the ubiquitin remnant tail left on trypsin-cleaved peptides. The laboratory of Samie Jaffrey was the first to publish an antibody-based approach to enrich for diGly peptides in 2010[61]. In this study, 374 ubiquitylation sites on 236 proteins were identified from HEK293 cells. To generate the antibodies, the authors synthesized a lysine rich protein antigen (histone) containing multiple K-ε-GG that was then injected into mice. In the following year, both Steven Gygi’s and Chuna Ram Choudhary’s groups successfully and independently conducted notable large scale studies: around 19,000 ubiquitylation sites in ~5,000 human proteins, and ~11,000 sites in ~4,200 proteins were 16 mapped, respectively[62, 63]. One reason the diGly-antibody approach is so potent is that trypsin digestion essentially abolishes most other protein-protein interactions and, combined with the high-affinity of the antibody, modified peptides are effectively enriched, despite their low abundance in the cell. Other major advantages are that no extra experimental controls are required to distinguish ubiquitylated sites from the rest of the identified peptides in the sample (the detection of the +114 Da mark is sufficient), it does not rely on ectopic expression of a tagged ubiquitin, it is applicable to all eukaryotic organisms or tissues. While the antibody-based approach is very potent and already widely used, it also has a few shortcomings. Since the NEDD8 and ISG15 Ubls share the same carboxyl-terminal sequence (Arg-Gly-Gly) with ubiquitin, proteins conjugated to these Ubls will also generate indistinguishable remnant tails after trypsin digestion. Therefore, some ubiquitylated peptides may be mis-assigned. Fortunately, levels of ISG15 are usually undetectable in cells, unless stimulated by interferon (IFN)-α/β[64]. Furthermore, Gygi’s group determined that over 95% of the sites which they identified were conjugated to ubiquitin and not NEDD8 (which is mainly conjugated to cullins). The antibodies against the ubiquitin remnant may also introduce a bias for some sites, since Choudhary’s group found that these antibodies have a slight sequence preference[65]. Our lab also noted that, using this approach, proteins ubiquitylated at multiple sites may be less prevalent compared to proteins conjugated at a single lysine, as peptide ion intensities would be lower (and thereby possibly not detected by the mass spectrometer) in the former case[66]. Another consideration is that the information regarding the chain linkage on a particular conjugation site (for polyubiquitylation) is lost when using this approach. 17 In addition, atypical sites (N-terminal, for instance) are also not selected. Nevertheless, the antibody-based approach has been key to the recent advancements and has now been adopted by many groups around the world. We have for instance adopted this approach in order to identify potential substrates of Rsp5 E3 ligase and Ubp2, 3 deubiquitinases in Chapter 3. The advance of systems-wide approaches described in this section have led to a significantly better understanding of the ubiquitin system, often by providing unique information that could not have been obtained by other means. For instance, we can now identify which proteins in the cells are targeted by specific PQC degradation pathways.  1.4 PQC degradation pathways To ensure the efficient targeting of terminally misfolded proteins for degradation, cells have developed multiple strategies in which distinct components of the UPS are involved in targeting misfolded proteins based on their localization. A number of misfolding diseases affect primarily different cellular compartments according to the loci of the protein misfolding events. For example, Huntington’s disease is due to the accumulation of misfolding and aggregation of the N-terminal fragments of huntingtin protein preferentially in the nucleus[15]. Cystic fibrosis caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, the overexpression of which or inhibition of proteasome activity was shown to cause accumulation of CFTR variant leading to the formation of aggresomes[15]. One of the most common features of Parkinson disease is the accumulation of α-synuclein in the cytosol of dopamine-18 producing neurons[15]. Therefore, the understanding of the molecular mechanisms of different degradation pathways that target these distinct misfolded proteins might help advancing our knowledge on the causes behind these diseases.    1.4.1 PQC degradation pathways in different cell compartments So far, several PQC degradation pathways that target misfolded proteins according to their localization have been discovered. Perhaps the best-characterized system is the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway that specifically targets misfolded or unassembled ER proteins[67]. ERAD is conserved in eukaryotes and key studies made in yeast have determined that there are two major ERAD pathways for mediating misfolded substrate-recognition. The Hrd1 E3-ligase associated complex targets membrane and soluble proteins exposing unfolded peptides in the ER lumen, which are recognized by two chaperone proteins in the ER: Yos9 and Kar2/Bip2[68, 69]. The E3 ligase Doa10 targets membrane proteins with misfolded cytosolic domains. It has been shown that Hsp70 associated to Ydj1 is required for the ubiquitylation of Ste6, a misfolded 12-transmembrane ERAD substrate, by Doa10[70]. However, not all Doa10 substrates require Hsp70[70] and it remains unclear whether other cytosolic chaperone proteins are involved in the ERAD pathways. Mammalian Hrd1 can ubiquitylate and induce degradation of neurodegenerative disease-linked proteins like huntingtin, Parkin-associated endothelin receptorlike receptor (PaeI-R), prion protein (PrP) as well as tau protein and is recruited to these protein aggregates and amyloids[10]. ERAD pathway seems to be far more complex in higher eukaryotes. Over 16 mammalian ubiquitin ligases have been proposed to have a 19 role in ERAD[71]. In addition, additional pathways may mediate degradation depending on the localization of the misfolded protein. For example, it was showed that the membrane-anchored ligase RMA1 and cytosolic ligase CHIP (carboxy terminus of Hsp70-interacting protein) act sequentially to monitor the folding status of CFTR Delta F508 (a single amino acid deletion of the chIoride-ion channel that causes cystic fibrosis) to target the misfolded ones for degradation[72, 73]. A recent study also discovered a novel plasma membrane quality control pathway that targets misfolded integral membrane proteins post-ER quality control. In this pathway, the ubiquitin ligase Rsp5 recognizes and ubiquitylates aberrant plasma membrane proteins with the help of a network of Arrestin-Related Trafficking (ART) adaptor proteins to target them for lysosomal degradation[74]. In yeast, the nuclear misfolded proteins are mainly ubiquitylated by the San1 ubiquitin ligase[75].  San1 is able to recognize directly the abnormal toxic proteins via its intrinsically disordered N- and C-terminal domains in a chaperone-independent manner[76]. The plasticity feature of San1 may allow it to bind many proteins with variable misfolded conformations. San1 together with a cytosolic E3 ligase Ubr1 also ubiquitylates cytosolic proteins that are translocated into the nucleus for proteasomal degradation[77]. However, San1 does not have homologs in mammalian systems, and its functional equivalents are yet to be discovered.  1.4.2 PQC degradation pathways in cytosol It is becoming apparent that in the cytoplasm a variety of different PQC degradation pathways have been deployed. In metazoans CHIP is a major PQC 20 ubiquitin ligase that can ubiquitylate misfolded proteins for proteasomal degradation in both, chaperone-dependent and -independent manner[78-80]. CHIP, which is not conserved in the lower eukaryotes like yeast, is not the sole E3 that targets cytosolic misfolded proteins to the proteasome. For instance, the N-end rule Ubr1 ubiquitin ligase not only targets polypeptides with destabilizing N-terminal amino acids (e.g., arginine) for degradation[81], but also mediates chaperone-dependent ubiquitylation of cytosolic proteins that are internally misfolded[77, 82]. In mammalian cells, the Ubr1 and CHIP pathways are redundant (at least for a subset of misfolded proteins)[83]. In yeast, Ubr1 can function together with the Ubr2 or the nuclear-localized San1 ubiquitin ligases to target cytosolic misfolded proteins[77, 82, 84-86]. In addition, another PQC pathway in the cytoplasm requires the ubiquitin ligase Rkr1/Ltn1 that directly associates with ribosomes to specifically target aberrant newly synthesized polypeptides that contain a translated poly(A) tail due to the lack of or missed stop codon[87-89]. Besides Rkr1/Ltn1, the other ribosome-associated ubiquitin ligases that were shown to participate in targeting these aberrant poly(A) tail-containing polypeptides, include Hel2, Hul5 and Hrd1. However, single deletion of these ligases only led to a partial impairment of the ubiquitylation of the poly(A) tail-containing species[90]. These data suggest that multiple known pathways are involved and presumably additional novel pathways are yet to be discovered. Similarly, the other misfolded proteins in the cytosol are also likely to be targeted by multiple PQC pathways. For instance, a previous study in our lab showed that some cytosolic misfolded temperature-sensitive mutants are targeted for proteasomal degradation by unknown ligase(s)[84]. In addition, deletion of either yeast Ubr1 and Ltn1 21 did not fully suppress the ubiquitylated of nascent polypeptides associated to ribosomes that are presumably not folded properly[90].   1.4.3 PQC degradation pathways in neurodegenerative diseases and their potential as therapeutic targets As mentioned earlier, protein aggregates are a common hallmark of neurodegenerative diseases related to protein misfolding. In many of these disorders, the accumulation of misfolded proteins not only leads to a loss-of-function of the proteins in question, but can also cause a gain-of-function with a pathogenic outcome[11, 15]. These aberrant species can either 1) adopt non-native conformations that interfere with the function of the corresponding native protein pool, 2) form oligomers and aggregates that impair cellular functions, or 3) sequester critical PQC components such as chaperones and the degradation machinery[15].  As the primary degradative route for PQC targets, UPS components are commonly found in the disease-related protein aggregates/inclusions. For example, cytosolic E3 ubiquitin ligase CHIP has been shown to associate with aggregation-prone proteins such as tau and expended polyglutamine proteins[91, 92]. Another example is E3 ubiquitin ligase Parkin, inactivation of which is a major cause of early onset Parkinson’s disease[93], and which was found enriched in cytosolic protein aggregates[94] suggesting that it might be involved in the clearance of misfolded proteins. Although, the more recent studies suggest that Parkin might function in mitophagy that removes damaged mitochondria via autophagy[95, 96], the role of Parkin in quality control is not yet fully understood. Besides Parkin, the Ubiquitin Carboxy-terminal Hydrolase L1 (UCHL1) is 22 one of the most abundant proteins in the brain (1-2% of the total soluble proteins). A down-regulated and oxidatively damaged form of UCHL1 was found in some Alzheimer’s and Parkinson’s patients[97]. It was originally identified as a deubiquitinase[98] and was later also shown to potentially function as an ubiquitin ligase[99] and a mono-ubiquitin stabilizer[100]. A recent study from our lab, showed that UCHL1 was enriched in the ubiquitin-containing aggregates induced by chemical inhibition of the proteasome in human neuroblastoma tissue culture cells[101]. Although different functional models of UCHL1 have been proposed, its role in diseases has not yet been clearly elucidated. Clearly then, understanding of the molecular mechanisms of the PQC degradation pathways helps us gain insight into the pathogenesis and progression of these devastating diseases, and provides avenues for the development of novel therapeutics.  Some pilot studies have shown that promoting UPS activity using chemical compounds could improve clearance of misfolded/aggregating proteins. For example, peroxisome proliferator activated receptor α (PPARα) agonists has been shown to promote the degradation of myofibrillar protein by UPS in rodents[102]. Doxorubicin was also shown to activate UPS by enhancing functions of both the ubiquitylation apparatus and proteasome[103]. Recently, a compound that targets Rsp5 as been shown to reduce cytoxicity caused by alpha-synuclein[104]. Beside chemical compounds, other studies have also showed that the clearance of Hsp70/Hsp90 substrates like CFTR, the glucocorticoid receptor and the ErbB2 receptor is enhanced when overexpressing CHIP E3 ligase in cultured cells[105, 106] suggesting the upregulation of a PQC E3 could reduce levels of misfolded proteins in the cell. Modulation of the activity of other UPS 23 components, such as deubiquitinases, could also have therapeutic potential. One of the functions of deubiquitinases is to remove the ubiquitin(s) attached to the PQC-target proteins. For example, in yeast, a proteasome-associated deubiquitinase Ubp6 is known to modulate proteasome processivity by removing ubiquitin chains on the proteasome substrates[107, 108]. Recent studies showed that the chemical inhibition of Ubp14, the mammalian ortholog of Ubp6, accelerates degradation of the aberrant proteins[109, 110]. This highlights how advances in the knowledge of the PQC degradation pathways could extend the search for drugable-targets to treat diseases caused by the protein misfolding.  1.4.4 PQC degradation pathways in heat-shock response Instead of specific model substrates, we reasoned that environmental stresses may be well suited to identify novel PQC pathways, as certain environmental stresses such as heat shock (HS) can induce global protein misfolding. Differential scanning calorimetry and other techniques had been used over a decade ago to show that protein denaturation events do occur in vivo upon thermal-stress[111]. Heat-induced protein misfolding should lead to a strong PQC response. Indeed, HS has long been known to cause an increase in polyubiquitylation in the cell[112]. It is also known that thermal-stress induces proteasomal degradation of mainly newly-synthesized proteins[113]. However, despite being discovered over 25 years ago the HS-induced increase in ubiquitylation has yet to be further characterized. It is not known which proteins are ubiquitylated upon HS or which pathway(s) is(are) responsible for targeting these HS-24 induced misfolded proteins. Therefore, HS could potentially be an excellent stress model to study PQC in the context of an elevated level of protein misfolding.  1.5 Main hypothesis  We hypothesized that additional and yet unknown PQC pathway(s) may target misfolded cytosolic proteins. As mentioned earlier, diverse PQC degradation pathways were found in yeast; however, several cytosolic, misfolded temperature-sensitive mutants characterized in our lab are targeted for proteasomal degradation by unknown pathway(s)[84]. Most of the knowledge that we gained about these cytosolic PQC pathways is based on studies of model substrates, which may not fully encapsulate the whole spectrum of physiological substrates. We reasoned that systems-wide approach may help to uncover novel pathway(s) that can target a wide spectrum of misfolded substrates.  My PhD project aims at uncovering novel degradation pathways involved in cytosolic PQC using proteomic coupled with biochemical and genetic approaches  25 Chapter 2: Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins  2.1 Introduction PQC degradation pathways eliminate misfolded and damaged proteins to prevent their cytotoxic accumulation and aggregation in the cell[11, 114]. Failure to degrade these non-native proteins is associated with numerous diseases, especially age-related neurodegenerative pathologies such as Parkinson’s and Huntington’s. Modulating proteostasis may help treat these diverse proteopathies; therefore, there is an increasing need for deeper insight into PQC mechanisms[7]. In eukaryotes, the UPS plays a major role in the degradation of misfolded proteins[115, 116]. Selective recognition of aberrant polypeptides is cardinal to avoid degradation of proteins that can refold into their native state. It is becoming apparent that several quality control pathways target misfolded proteins in the cytoplasm. The CHIP ubiquitin ligase plays a major role in eliminating cytosolic misfolded proteins in metazoans[78]. Other ubiquitin ligases, such as Ubr1, were shown to target cytosolic misfolded proteins in yeast[77, 82, 85]. Moreover, the Ltn1 (also called Rkr1) ligase was found to associate with ribosomes to specifically target nascent non-stop polypeptides for degradation[87]. These distinct and specialized quality 26 control pathways probably complement each other to eliminate the wide spectrum of aberrant cytosolic polypeptides. In this chapter, we describe that HS triggered a large increase in the level of ubiquitylation linked to misfolding of cytosolic proteins in yeast cells. We discovered that Hul5 (HECT ubiquitin ligase 5) played a major role in this stress response, whereas none of the other known ubiquitin ligases functioning in protein quality control were involved. Cytosolic Hul5 was important for cell fitness and increased ubiquitylation levels after HS. We identified several potential physiological substrates to show that Hul5 was also important for the ubiquitylation and degradation of low-solubility cytosolic proteins in unstressed cells. Together, these findings indicate that Hul5 is a component of a novel cytosolic PQC pathway.     27 2.2 Methods 2.2.1 Plasmids All plasmids are further described in supplementary Table 2.1. HUL5 (BPM309) and hul5-C878A (BPM310) were subcloned into pRS316 with endogenous promoter and termination regions from pJH84 and pJH85 (D. Finley), respectively. For 13Myc-Hul5 (BPM325), HUL5 was inserted into a pRS316 plasmid that contains the high-expression GPD1 promoter, the N-terminal Myc13 tag and the terminator sequence from PGK1. For GFP-HUL5 (BPM341) and GFP-NLS-HUL5 (BPM345), the HUL5 promoter region, the GFP+[117] with or without a 3′ NLS (SPKKKRKVEAS), and the HUL5 coding sequence with the 3′ UTR containing an ARS sequence were inserted into pRS306.  2.2.2 Cells and heat-shock assays All yeast strains are listed in supplementary Table 2.2. Hul5-GFP and Nic96-RFP were generated by a genomic insertion of GFP+[117] and RFP[118] at the 3′ end of the coding sequences by homologous recombination. For HS assays, overnight-saturated cultures were diluted and grown to exponential phase in YPD at 25 °C to an A600 of 1-1.5 before heat-shock at 45 °C for the indicated times (typically 15 min). For western and dot blots, cells were washed twice and snap frozen in liquid nitrogen. Lysis was carried out with glass beads in pre-warmed 1× SDS-PAGE Laemmli sample buffer without reducing agent and dye. All samples were normalized by a Bradford assay before multiplex analysis with mouse anti-ubiquitin (1:2,500; MAB1510) and rabbit anti-  28 Pgk1 or anti-PSTAIR (Cdc28; 1:1,000) antibodies. Fluorescent secondary antibodies (1:10,000; LI-COR) were used for quantification analysis by an Odyssey Infrared Imaging System. For dot blot assay, 3 μl of the normalized samples (5-10 μg proteins) was spotted and dried overnight on a nitrocellulose membrane. Membranes were rehydrated with 1×TBS and processed as other western blots. For the proteasome inhibition experiment, 20 μM MG132 was added to pdr5Δ cells as indicated. Cells for the SSA experiment were similarly processed and incubated at the indicated temperatures. To assess solubility, cells were lysed (100 mM HEPES, 1% Triton X-100, 300 mM NaCl and protease inhibitors) with glass beads, pre-cleared at 2,000g and fractionated at 16,000g for 10 min. For the cell fitness assay, exponentially growing cells in SD-URA media were incubated for 20 min at 25 °C or 45 °C and transferred to a 96-well plate placed on the Infinite 200 PRO plate reader system (Tecan; Fig. 2.3c) or in an incubator (Fig. 2.4f) with constant shaking at 25 °C.   2.2.3 IMAC—purification of ubiquitylated proteins In all experiments, about 1 μl of MagneHis (Promega) was used per 100 μg of protein extract. For mass spectrometry analysis, cell metabolic labelling (each in 500 ml) was done in YNB media at 25 °C as previously literature described[119]. Equal amounts of cells were mixed and lysed in HU buffer (8 M urea, 100 mM HEPES at pH 8, 0.05% SDS, 10 mM chloroacetamide, 1 mM phenylmethylsulphonyl fluoride, 10 mM imidazole and protease inhibitors cocktail) by glass beads. Following 90 min incubation at ambient temperature, nickel beads were washed three times in HU buffer with 1% SDS, followed by three washes in HU buffer with 0.5% Triton X-100 and three washes in HU buffer.   29 For pellet pre-enrichment, cells were first lysed in 100 mM HEPES at pH 8, 1% Triton X-100, 300 mM NaCl, 1 mM phenylmethylsulphonyl fluoride, 1 mM phenanthroline, 10 mM chloroacetamide and protease inhibitors by glass beads and centrifuged at 16,000g. Cell pellets were then resolubilized in HU buffer with 0.05% SDS. For TAP validation, 150 ml of cells carrying H8-Ubi plasmid (RDB1851/BPM30)[119] or empty vector were grown in SD-URA before lysis in HU buffer with 0.05% SDS, and elution from the beads was carried out after adding one volume of 2 M imidazole, one volume of HU buffer and one volume of 3× SDS-PAGE sample buffer. IMAC from both the insoluble and soluble fractions was carried out with a 1:1 mixture of the HU and lysis buffers with 0.05% SDS. Samples were also analysed with rabbit anti-TAP antibody (1:1,000; CAB1001).  2.2.4 Mass spectrometry analysis Samples were prepared as before[119]. The IMAC samples of Hul5 substrate identification experiments (Fig. 2.6a and S2.5d) were purified and fractionated on SCX stage tips to generate five fractions as described previously[120, 121]. All of the whole-cell lysate samples (Fig. 2.2, S2.2, S2.5 and S2.6) and the other IMAC samples (Fig. 2.2, S2.2 and S2.5b) were purified with C18 stage tips without fractionation. Purified peptides were analyzed using a linear-trapping quadrupole Orbitrap mass spectrometer (LTQ-Orbitrap; ThermoFisher Scientific) or a LTQ-Obitrap Velos (Fig. 2.6a) on-line coupled to an Agilent 1100 Series nanoflow HPLC using a nanospray ionization source (ThermoFisher Scientific). SCX fractions were run with a 90 min gradient, and other unfractionated samples were run for a 240 min gradient or 120 min with the Velos Instrument. The LTQ-Orbitrap was set to acquire a full-range scan at 60,000 resolution   30 from 300 to 1,600 Th and to fragment the top five peptide ions in each cycle in the Orbitrap, and the top 15 ions (90 min gradient) or the top 10 ions (120 min gradient) in the Velos instrument. Parent ions were then excluded from tandem mass spectrometry for the next 30 s, as well as singly charged ions. The Orbitrap was continuously recalibrated using the lock-mass function[122].  2.2.5 Analysis of mass spectrometry data  Centroided fragment peak lists were processed to Mascot generic format using DTA Supercharge (http://msquant.sourceforge.net) with LTQ-Orbitrap data or Proteome Discover with the Velos instrument data. Fragment spectra were searched using the Mascot algorithm against the Saccharomyces Genome Database (SGD_051107) using the following parameters: peptide mass accuracy 10 ppm; fragment mass accuracy 0.6 Da; trypsin; two 13C; fixed modification (carbamidomethyl); variable modifications (deamidation and oxidation; oxidation only for Velos data); ESI-TRAP fragment characteristics. The typical false-positive rate for peptide identification (ion score ≥15, peptide length ≥6 amino acids) was estimated between 0.5 and 1.3% by using a Decoy database with Mascot. Mascot results were modified by a 15N-labelling script (http://msquant.sourceforge.net/#N15support) and 14N/15N ratios of identified peptides were quantified by MSQuant (v1.5b7; minimum of two unique peptides per protein to be selected). All peptide quantifications were manually validated and the averaged log2 (14N/15N) values of the quantified proteins were set to a maximum of 5. As none of the perturbations affected the ratio distributions of the proteins in the whole-cell lysates, we used these ratio distributions as a reference to estimate the confidence of 14N/15N   31 enrichment in the IMAC for a given threshold[101]. We reported the portion of ratios in the whole-cell lysate of each sample that were above the established threshold (for example, log2 (14N/15N) ≥0.5) as an estimate of the false-positive rates for the enrichment (all between 3 and 5% in mass spectrometry analysis tables).   2.2.6 Pull-down and subcellular fractionation For co-immunoprecipitation, cells with BPM325 (13MYC-HUL5) or pRS316, after a 20 min incubation at 45 °C, were lysed in IP buffer (100 mM HEPES at pH 8, 20 mM MgAc, 300 mM NaAC, 10% glycerol, 1% NP-40, 10 mM EGTA, 0.1 mM EDTA, 1× protease inhibitor cocktail and 1 mM phenylmethylsulphonyl fluoride), and TAP-tagged proteins were pulled-down with IgG-coupled Dynal magnetic beads (Invitrogen) incubated for 3 h at 4 °C followed by three washes with IP buffer. The nuclei and cytosolic fractions were prepared as previously described[123] from Hul5-GFP cells collected at exponential phase with additional procedures as follows. The spheroblasts were lysed (20 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 1 mM phenylmethylsulphonyl fluoride and 1× protease inhibitor cocktail) with 8 strokes in a chilled tight-fitting pestle dounce homogenizer, after 15 min incubation on ice. Unbroken cells and debris were removed by centrifugation for 5 min at 300g at 4 °C. The cytosolic fraction (supernatant) was collected after a spin at 13,000g for 20 min at 4 °C and the nuclear-enriched fraction (pellet) was washed once with lysis buffer before collection. Normalized volumes of cytosolic fraction and nuclei fraction were then subjected to western-blotting analysis and also probed with mouse anti-MYC (9E10;   32 1:5,000), anti-GFP (1:3,000; Roche), anti-mouse ECL-coupled IgGs (1:3,000; BioRad) and rabbit anti-H3 (1:3,000) antibodies. 2.2.7 Microscopy  Hul5-GFP,Nic96-mRFP cells were grown to log phase at 25 °C and either imaged immediately or subjected to heat-shock at 42 °C for 30 min. GFP-NLS-Hul5-expressing cells were similarly treated but incubated with Hoechst 33342 (2.5 μg ml-1) for 30 min before imaging. Cells were imaged with a Zeiss Axio Observer inverted microscope equipped with a Zeiss Colibri LED illuminator and a Zeiss Axiocam ultrahigh-resolution monochrome digital camera Rev 3.0. image stacks were acquired with a ×40 or ×63 objective, at a step of 0.3 μm or in single stack, and analyzed with Zeiss Axiovision software.  2.2.8 35S labelling and measurement of protein degradation The procedure was adapted with minor modifications from a previous study[113]. Yeast cells were grown in SD medium supplemented with six essential amino acids, uracil and 20 g ml-1 tyrosine[124] to an A600 of 0.8. Cells were washed twice and incubated in SD-Met media with tyrosine for 50 min. EXPRE35S35S protein labelling mix (50 μCi ml-1, PerkinElmer) was added for 5 min to label newly synthesized proteins before two washes with ice-cold chase media containing methionine (6 mg ml-1), cysteine (0.5 mg ml-1) and cycloheximide (0.5 mg ml-1). Cells were then incubated at 25 °C or 38 °C. Cell aliquots were mixed to give a final concentration of 10% TCA and incubated overnight at 4 °C. Radioactivity in both TCA-soluble and -insoluble fractions was measured in a MicroBeta2 radiometric detector (PerkinElmer). The percentage (pi)   33 of short-lived protein degradation was calculated by subtracting the background TCA-soluble signal in time0 (t0) from the TCA-soluble signal (ti), and then dividing it by the t0 signal of the TCA-insoluble fraction.  2.2.9 Statistical analysis Data were statistically analyzed as indicated (mean, s.d. and s.e.m.) and the statistical significance for the ubiquitylation response was assessed by a two-tailed unpaired Student’s t-test.    34 2.3 Results 2.3.1 Heat-induced misfolding triggers a large increase of ubiquitylation levels To gain holistic insight into the targeting of misfolded proteins by the ubiquitin proteasome system, we sought to identify a cellular stress that induces a strong ubiquitylation response. We found a near twofold increase in the level of polyubiquitylation in Saccharomyces cerevisiae cells after a 15 min 45 °C HS treatment, as determined by both western and dot blots (Fig. 2.1a). The HS ubiquitylation response was much stronger when compared with other tested stresses (Fig. S2.1a). We ruled out that this phenomenon was caused by a decrease in the level of proteasome activity at high temperature, as a larger increase in the level of polyubiquitylation was observed in cells treated with a proteasome inhibitor (Fig. S2.1b,c). Notably, most of these HS-induced polyubiquitylated species were poorly soluble (Fig. 2.1b). These data indicate that heat-induced protein misfolding causes a rapid and strong increase in the level of polyubiquitylation mediated by a PQC pathway (Fig. 2.1c). We further characterized this stress response by determining which E2 is involved, because several E2 enzymes participate in PQC in different cell compartments[75, 125-127]. Deletion of both UBC4 and UBC5 fully abolished the HS ubiquitylation response (Fig. 2.1d), whereas single deletions of these two E2 enzymes had no effect (data not shown). Deletion of UBC6 and UBC7, which are E2 enzymes involved in ERAD[125, 126], had no effect either (Fig. 2.1d). These data indicate that HS may induce an increase in the level of ubiquitylation of cytosolic proteins, as Ubc4 and Ubc5 target short-lived misfolded proteins in the cytoplasm[127].    35 Figure 2.1 Heat-induced misfolding triggers a large increase of ubiquitylation levels   (a) BY4741 cells were subjected to heat-shock (HS; 15 min at 45 °C) or not (No HS). Left, experimental triplicates were analyzed by western (top) and dot blots (bottom) with anti-ubiquitin and anti-Pgk1 antibodies. Right, the region above Mw70K in the western blot and the whole spotted signal in the dot blot were quantified. Ubiquitylation signals were normalized to Pgk1 levels and standard deviations are shown. (b) Ubiquitylation levels in total cell extract (T), soluble (S) and pellet (P) fractions after 16,000g centrifugation from both unstressed and HS-treated (15 min 45 °C) BY4741 cells shown by anti-ubiquitin western blot. (c) Schematic diagram of the proposed relationship between heat-shock and the increased ubiquitylation response. (d) Relative increases of Pgk1-normalized ubiquitylation levels after a 15 min 45 °C HS in the indicated E2 double-deletion strains were quantified by dot blot and averaged values from three replicates are shown with standard deviations.   36 2.3.2 HS mainly induces ubiquitylation of cytosolic proteins To determine whether HS affects cytosolic proteins, we adapted a quantitative mass spectrometry method[119] to identify proteins that were further ubiquitylated (Fig. 2.2a). We used cells expressing an octahistidine tag fused to the amino terminus of ubiquitin (H8-Ubi) and carried out immobilized metal ion affinity chromatography (IMAC) to purify tagged ubiquitin conjugates from cells that were metabolically labelled with either 14N or 15N for quantitative analysis. We first confirmed that neither the labelling nor IMAC was introducing a bias, as most proteins were equally enriched in the two differentially labelled cell populations (Fig. S2.2a, sup. Table 2.6). To identify proteins ubiquitylated after HS, we compared stressed cells (14N; 20 min at 45 °C) with unstressed control cells (15N). Using this approach, we identified 155 proteins that were ubiquitylated or further ubiquitylated following HS treatment in at least two of three independent experiments (Fig. 2.2b, S2.2b and sup. Table 2.3). In contrast, most of the proteins in the whole-cell lysate were unaffected by HS. By comparing H8-Ubi (14N) and untagged (15N) cells, we verified that most of the quantified proteins were specifically enriched owing to the ubiquitin tag (80%; Fig. S2.2c and sup. Table 2.6) and were more ubiquitylated after HS (Fig. S2.2d and sup. Table 2.6). Representative quantified peptides from two ubiquitylated proteins further enriched in HS-treated cells as shown in Fig. S2.2e. Rps7B is a ribosomal protein that is targeted to the proteasome in unstressed cells[119]. Pin3 (also called Lsb2) is a prion-like protein that contains short polyglutamine stretches[128, 129], and is further ubiquitylated following heat-stress[130]. We confirmed that endogenous TAP-tagged Pin3 was noticeably less soluble after HS, indicating that the increase in the level of ubiquitylation may be due to augmentation of protein misfolding (Fig. 2.2c). Nearly 80% of the HS-affected proteins are cytosolic,   37 according to data from a yeast genome-wide GFP analysis[118]. The enrichment of cytosolic proteins when compared with the localization profile of the proteome (Fig. 2.2d) indicates that heat-stress causes the rapid ubiquitylation of mostly cytosolic proteins, and does not predominantly affect proteins in other compartments. This result indicates that an efficient PQC machinery exists that targets misfolded cytosolic proteins after HS.      38 Figure 2.2 Heat-shock mainly affects cytosolic proteins   (a) A schematic diagram of the workflow of the quantitative mass spectrometry analysis. HS: heat-shock; No HS: no heat-shock. (b) Percentage of proteins above the corresponding log2 values of the 14N HS/15N no HS ratios for three independent experiments (I: light; II: medium; and III: dark). Analysis of proteins in the whole-cell lysate (grey: I, 486; II, 730; and III, 399) and of IMAC-enriched ubiquitylated proteins (green: I, 302; II, 481; and III, 219) are shown. Proteins with a log2 ratio ≥0.5 are considered HS affected. (c) Pin3-TAP solubility was assessed before and after a 15 min 45 °C HS. An equal portion of each fraction (T: total; S: soluble; P: pellet) was loaded on an SDS-PAGE gel for western-blot analysis with the anti-TAP antibody. (d) Subcellular localization of 155 proteins affected by HS (green; identified as enriched in at least two of the three experiments in b), compared with the whole proteome (grey)-cytosol (C), nucleus (N), membrane (M) and mitochondria or peroxisome (M/P). Note that several proteins localize to more than one compartment.   39 2.3.3 Hul5 plays a major role in the heat-shock ubiquitylation response To better understand the targeting mechanism of these misfolded proteins, we next identified which ubiquitin ligase(s) may be involved in this HS response. Using the dot blot HS assay, we assessed 82 yeast strains that each carried a deletion of a verified or putative ubiquitin ligase gene (Sup. Table 2.4). Surprisingly, none of the known or suspected ubiquitin ligases involved in PQC[75-77, 82, 85, 87, 131-133]showed any significant perturbation of the HS ubiquitylation response (Fig. S2.3a). Deletion of HUL5, a gene encoding a HECT (homologous to E6AP C terminus) ubiquitin ligase[134], led to a significant decrease in the level of the HS ubiquitylation response (Fig. S2.3a). Hul5 associates with the proteasome, and has been implicated in processing ERAD substrates and promoting proteasomal processivity[107, 108, 135, 136]. We confirmed that the heat-induced ubiquitylation level was decreased in two independent hul5Δ deletion strains using the dot blot assay (Fig. 2.3a). In agreement with their role in the HS ubiquitylation response, Ubc4-TAP and Ubc5-TAP interact with 13Myc-Hul5 (Fig. 2.3b and S2.3b). Deletion of HUL5 led to a decrease in the level of the HS ubiquitylation response between 5 and 30 min (Fig. S2.3c). These data indicate that HUL5 is required at the early stage of the HS response. Deletion of HUL5 resulted in a 20–50% decrease in the level of the ubiquitylation response, indicating that Hul5 is a major ubiquitin ligase involved in targeting misfolded proteins after HS. Although HS treatment did not cause a significant loss of viability in hul5Δ cells (Fig. S2.3d), we observed a growth delay following recovery from the stress. To quantify this effect, we measured the growth rate of wild-type (WT) and hul5Δ cells following HS treatment during the exponential growth phase. In WT cells, a 20 min HS at 45 °C   40 induced a 50-min delay of the first cell doubling after incubating the cells back to 25 °C (Fig. 2.3c and S2.3e). In contrast, hul5Δ cells exhibited a much longer growth delay of 190 min after HS, whereas unstressed hul5Δ cells grew normally. Similar results were obtained with an independent hul5Δ strain (data not shown). The delay in hul5Δ cells was rescued to the normal growth rate when a wild-type copy of HUL5 expressed from its own promoter was introduced (Fig. 2.3c and S2.3e). Conversely, the addition of HUL5 mutated on a conserved catalytic cysteine residue of its HECT ligase domain (C878A) did not rescue the HS-induced growth delay of hul5Δ cells (Fig. 2.3c and S2.3e). Correspondingly, cells carrying catalytically inactive Hul5 exhibited a decreased HS ubiquitylation response (Fig. 2.3d). Together these results indicate that Hul5 ligase activity is required for ubiquitylation of misfolded proteins and maintenance of cell fitness after HS treatment.   41 Figure 2.3 HUL5 is required for the full ubiquitylation response and cell fitness after heat-shock   (a) Ubiquitylation levels in unstressed (No HS) and heat-shocked (HS) cells are compared between WT and hul5Δ strains using the dot blot assay. HUL5 was deleted by two different cassettes (KanMX6 and NatMX4). Standard deviation is shown for three replicates and P values were calculated using an unpaired Student’s t-test. (b) TAP-immunoprecipitation (TAP-IP) experiments with cells expressing or not Ubc4-TAP (left) or Ubc5-TAP (right) at endogenous levels with or without a plasmid expressing 13Myc-Hul5 were analyzed by western blot using 9E10 or anti-TAP antibodies. Inputs (1%) are shown below. (c) The A600 of the first cell doubling following a 20 min incubation at 45 °C  HS (black) or at 25 °C (grey) of HUL5 (round) and hul5Δ::NAT (square) cells. hul5Δ::NAT cells carrying a centromeric plasmid (dashed lines) with HUL5 (round) or hul5C878A (square) are also compared. Growth delay is defined by the difference of the first doubling time between the corresponding unstressed and stressed cells. Each data point is averaged from three replicates. (d)  hul5Δ cells carrying either WT HUL5 or the catalytically inactive hul5C878A were subjected to HS (15 min at 42 °C). The increase in ubiquitylation levels (with standard deviations) was measured by dot blots with anti-ubiquitin and anti-Pgk1 antibodies in three replicates. An unpaired Student’s t-test was used to assess the significance of the difference between the cell populations.   42 2.3.4 The cytosolic localization of Hul5 is required for its role in the HS ubiquitylation response We observed by fluorescence microscopy that, whereas the localization of GFP-tagged Hul5 prevailed in the nucleus of unstressed cells, the pool of cytosolic Hul5 increased after HS (Fig. 2.4a,b and S2.4a). The change of Hul5 distribution after HS was verified by subcellular fractionation of the nuclei and cytosol (Fig. 2.4c). To determine the importance of Hul5 cytosolic localization, we added the SV40 nuclear localization signal (NLS) to a GFP-Hul5 fusion protein. The NLS led to a further enrichment of Hul5 in the nucleus of unstressed cells, and blocked Hul5 relocalization to the cytoplasm after HS (Fig. 2.4d). The presence of the NLS also led to a decrease in the level of the heat-induced ubiquitylation response and a slower growth recovery after HS (Fig. 2.4e,f and S2.4b). These data indicate that ubiquitylation of misfolded proteins after HS requires localization of Hul5 to the cytoplasm.               43 Figure 2.4 Hul5 re-distribution to the cytosol is important for its role in the HS response    44 (a) Localization of Hul5-GFP was assessed in a strain with Nic96-mRFP as a nuclear periphery marker, in both unstressed and HS (30 min at 42 °C) cells. Images were taken with a ×40 objective and in focus z-stacks were flattened with the wavelet extended function. The nuclear positions are outlined. Scale bar, 5 μm. (b) Histograms of the mean Hul5-GFP signal intensities (per pixel; with standard error) measured in a 0.7 μm2 area of the nuclei (N) and cytoplasm (C) of unstressed (No HS; 25 °C) and heat-shocked (HS; 30 min at 42 °C) cells (n=100), after subtraction of the average background signal from untagged cells. (c) Levels of Hul5-GFP in both nuclear and cytosolic fractions were analyzed by western blot using anti-GFP, anti-Pgk1 and anti-histone H3 antibodies after subfractionation of both unstressed and heat-shocked (30 min at 42 °C) cells. (d) The localization of GFP-Hul5 (upper panels) and GFP-NLS-Hul5 (lower panels) expressed from a plasmid was assessed in hul5Δ cells grown at 25 °C and subjected to HS (30 min at 42 °C). DNA was stained with Hoechst (shown below GFP images); single-stack images were taken with a ×63 objective; arrowheads point to the nucleus. Scale bar, 5 μm. (e) The increase of ubiquitylation levels after HS (15 min at 45 °C) is compared using the dot blot assay between HUL5 and hul5Δ cells with an empty vector, and hul5Δ cells expressing GFP-HUL5 or GFP-NLS-Hul5 from a plasmid. Standard deviation is shown for three replicates and P values were calculated using an unpaired Student’s t-test (in black or in grey when comparing with HUL5- or GFP-HUL5-expressing cells, respectively). (f)  A600, following a 20 min HS at 45 °C (black) or 20 min at 25 °C (grey), of hul5Δ cells carrying a plasmid with GFP-HUL5 (round) or GFP-NLS-HUL5 (square) are compared. Each data point is averaged (with standard deviations) from three replicates.   45 2.3.5 Hul5 is required for targeting misfolded proteins in the absence of SSA-chaperone activity and it is required for misfolded proteins degradation To further characterize the function of HUL5 in protein homeostasis, we assessed its role in the ubiquitylation of proteins misfolded in the absence of SSA (stress seventy subfamily A)-chaperone activity. The Hsp70 subfamily, which is composed of four members (Ssa1–4), is a major folding system in the cytosol[137, 138]. We reasoned that inactivation of SSA-chaperone activity should be followed by an increased level of ubiquitylation of misfolded polypeptides (Fig. 2.5a). Indeed, we observed a twofold increase of ubiquitylation levels in thermo-sensitive cells carrying the ssa1-45 mutant allele, in which the three SSA2, 3 and 4 genes are deleted, after a shift to the non-permissive temperature (37 °C) for 40 min (Fig. 2.5b). In contrast, there was only a minor increase in the level of ubiquitylation in the control cells (ssa2Δ,3Δ,4Δ) expressing WT SSA1. We next assessed whether HUL5 is important for the ubiquitylation response caused by SSA-chaperone inactivation. Remarkably, we saw a marked decrease in the level of ubiquitylation in two independent hul5Δ strains carrying the ssa1-45 allele (Fig. 2.5c). These data indicate that HUL5 has an important role in targeting misfolded cytosolic proteins in an SSA-chaperone-independent pathway (because the ubiquitylation level increase occurs in the absence of SSA activity). We next determined whether HUL5 is important for the degradation of misfolded ubiquitylated polypeptides. It was previously shown that newly synthesized proteins have a higher turnover rate at high temperature owing to increased misfolding[113]. We therefore carried out pulse-chase labelling to determine whether HUL5 is important for the short half-life of these misfolded proteins. As reported, shifting cells from 25 °C to   46 38 °C led to a higher proteolysis rate of 35S pulse-labelled proteins in WT cells (Fig. 2.5d). Conversely, this increase in the level of proteolysis was strongly decreased in hul5Δ cells, indicating that HUL5 plays a major role in the degradation of short-lived misfolded proteins. To further examine whether Hul5 solely targets newly synthesized proteins, we repeated the HS-induced ubiquitylation assay in the absence of protein synthesis. Pre-treatment with the translational elongation inhibitor cycloheximide led to a lower heat-induced ubiquitylation response that was further decreased in the absence of HUL5 (Fig. 2.5e). These results indicate that Hul5 probably targets both newly synthesized and long-lived misfolded proteins. Remarkably, degradation of short-lived proteins was also affected at ambient temperature in hul5Δ cells (Fig. 2.5d). A substantial portion of short-lived proteins is probably degraded owing to misfolding in physiological conditions and Hul5 may play an important role in their proteolysis and in protein homeostasis in general.             47 Figure 2.5 HUL5 is essential for the ubiquitylation of proteins misfolded in the absence of SSA-chaperone activity and for the degradation of pulse-labelled misfolded polypeptides   (a) Schematic diagram of the proposed model for the SSA-hsp70-chaperone inactivation. (b) The histogram shows Cdc28-normalized ubiquitylation levels in SSA1 and ssa1-45 cells (ssa2-4Δ) measured before and after shifting cells from 25 °C to 37 °C for 40 min. The ubiquitylation levels were quantified by dot blot assay from four replicates and are shown with standard errors. (c) The increased ubiquitylation levels in ssa1-45 HUL5 or ssa1-45 hul5Δ cells (two independent strains) were compared using   48 five replicates for each assessed strain as described for b. The P-values were determined by Student’s t-test. (d) WT (BY4741) and hul5Δ cells were subjected to 35S-pulse labelling (5 min) followed by a 3 h chase. The graph shows the averaged percentage (with standard errors) of pulse-labelled proteins (corresponding to newly synthesized protein) that were degraded at each time point from cells incubated at 25 °C or 38 °C from three replicates. (e) Histograms of averaged ubiquitylation signals measured by dot blots in unstressed (no HS; light grey) and heat-shocked (HS; dark grey) cells in three replicates are shown with standard deviations. Cells were grown at 25 °C and pre-treated or not for 15 min with 100 μg ml−1 cycloheximide (CHX) before HS (15 min at 45 °C). Student’s t-test was used to assess the significance of the ubiquitylation level differences between the indicated strains.   49 2.3.6 Hul5 is required for the ubiquitylation of low-solubility cytosolic proteins in physiological conditions We next identified misfolded proteins that are ubiquitylated by Hul5 in physiological conditions. We reasoned that Hul5 substrates should be more ubiquitylated in WT cells when compared with hul5Δ cells. We also supposed that misfolded or aggregated proteins should be enriched in the cell pellet owing to their lower solubility. We therefore compared metabolically labelled WT (14N) and hul5Δ (15N) cells expressing H8-Ubiquitin grown at 25 °C and not subjected to HS stress, then lysed in a native buffer before fractionation by centrifugation. Proteins in the cell pellet were next resolubilized in a denaturing buffer for IMAC followed by mass spectrometry analysis (Fig. S2.5a). We identified several HUL5-dependent ubiquitylated proteins (Fig. 2.6a). In contrast, we found that deletion of HUL5 caused no significant perturbation of the ubiquitylation profile when isolating conjugated proteins from the whole-cell lysate (Fig. S2.5b and Sup. Table 2.7). These results indicate that, in unstressed conditions, Hul5 specifically affects ubiquitylation of low-solubility proteins, but not the overall ubiquitylation levels in the cell. In three independent experiments, we identified a total of 95 candidate proteins that were less ubiquitylated in hul5Δ cells, including thirteen proteins that were identified in at least two analyses (Fig. 2.6b and Sup. Table 2.5). A small pool of Hul5 candidate physiological substrates, including Pin3, was also identified in the original HS analysis (Fig. S2.5c). We confirmed that HUL5 plays an important role in targeting misfolded proteins after HS (20 min, 45 °C), as approximately 20% of the ubiquitylated proteins quantified by mass spectrometry were less abundant in hul5Δ cells (Fig. S2.5d and Sup. Table 2.7). Furthermore, only a small portion of Hul5   50 candidate substrates was identified in both stressed and unstressed conditions (Fig. S2.5e). These data indicate that predominantly different proteins are ubiquitylated owing to misfolding by Hul5 in unstressed and heat-stressed conditions. Notably, most proteins requiring HUL5 for ubiquitylation in unstressed or stressed cells were cytosolic (Fig. 2.6c), further emphasizing the importance of Hul5 in targeting cytosolic misfolded proteins.             51 Figure 2.6 HUL5 is required for ubiquitylation of low-solubility cytosolic proteins (a) 14N and 15N metabolic labelling was carried out for WT and hul5Δ cells, respectively. Percentage of proteins above the corresponding log2 values of the 14N/15N ratios in three independent experiments (I: light; II: medium; and III: dark). Analysis of proteins in the whole-cell lysate (grey: I, 345; II, 346; and III, 240) and of IMAC-enriched ubiquitylated proteins (green: I, 661; II, 430; and III, 267) are shown. Proteins with an IMAC log2 ratio ≥1 are considered Hul5 candidate substrates. (b) Venn diagram representing all 95 proteins identified as more ubiquitylated in experiments I to III in a. Bold protein names are confirmed Hul5 substrates in Fig. 2.7 and Supplementary Fig. S2.6. (c) Histogram showing the subcellular localization of the 99 (light green) and 95 (dark green) Hul5 candidate substrate proteins identified in HS-stressed (HS) and unstressed cells (No HS), respectively: cytosol (C), nucleus (N), membrane (M) and mitochondria or peroxisome (M/P).      52 2.3.7 Ubiquitylated Hul5 substrates accumulate with less-soluble polypeptides To verify the data, we selected a few identified proteins for additional analysis. We first focused on Lsm7, a small protein involved in cytosolic RNA decay[139], which was identified as a Hul5 candidate substrate in unstressed cells. For validation, we subjected cells that expressed H8-Ubi and endogenous C-terminally TAP-tagged Lsm7 to IMAC, followed by an anti-TAP western blot. High-molecular-weight species corresponding to polyubiquitylated Lsm7-TAP (Ubin-Lsm7) were detected at greater levels in WT cells when compared with hul5Δ cells, confirming the data obtained by mass spectrometry (Fig. 2.7a). Remarkably, ubiquitylated Lsm7-TAP species (after IMAC) were found only in the low-solubility fraction, whereas most unmodified Lsm7-TAP and most ubiquitylated proteins were soluble in the cell (Fig. 2.7b). This result indicates that ubiquitylation of Lsm7 is associated with less-soluble polypeptides. We also found that polyubiquitylation of TAP-tagged Pin3 required HUL5 (Fig. 2.7c). We confirmed that these ubiquitylated species were present only in the pellet fraction (Fig. 2.7d). As Pin3 was less soluble and further ubiquitylated in a HUL5-dependent manner after HS (Fig. 2.2c and S2.5d), we repeated the IMAC after shifting the cells to 45 °C for 20 min. Indeed, Pin3-TAP was further ubiquitylated after HS and this increase was mostly abrogated in hul5Δ cells (Fig. 2.7c). We also verified that HUL5 is required for the ubiquitylation of two other Hul5 substrate candidates, Tsa2 (thioredoxin peroxidase) and Fbp26 (fructose-2,6-bisphosphatase; Supplementary Fig. S2.6a,b). These four TAP-tagged proteins have long half-lives in our growth conditions (data not shown), indicating that only a small portion of these proteins may misfold to be then degraded. In contrast, levels of Slh1-TAP, another Hul5 candidate substrate both in physiological and   53 HS conditions, were found to decrease after a 2 h cycloheximide treatment (Fig. 2.7e) in a proteasome-dependent manner (data not shown). Slh1 is a putative RNA helicase that inhibits translation of non-poly(A) messenger RNA in the cytoplasm[140]. We confirmed that ubiquitylation levels of Slh1-TAP were decreased by HUL5 deletion (Fig. S2.6c) and only low-solubility Slh1-TAP was ubiquitylated (Fig. S2.6d). As Slh1-TAP ubiquitylation levels increase after HS (Fig. S2.5d) and its protein half-life decreases at higher temperature (Fig. S2.6e), we reasoned that Slh1 turnover may be associated with misfolding. In agreement, we observed that deletion of HUL5 stabilized Slh1-TAP (Fig. 2.7e). Slh1 was also stable when Hul5 localization was constrained to the nucleus following the addition of the NLS (Fig. 2.7f), further indicating that Hul5 targets substrates in the cytoplasm. Together, these results confirm that Hul5 is important for the targeting of low-solubility cytosolic proteins.            54 Figure 2.7 Hul5 targets proteins that are specifically ubiquitylated in the low-solubility cellular fraction   55 (a–d) Validation of the Hul5 substrate candidates Lsm7 and Pin3 using TAP-tagged strains expressing H8-Ubi. IMAC was carried out in denaturing conditions to pull down ubiquitylated proteins, and anti-TAP or anti-ubiquitin antibody was used for western-blot analysis. The asterisk denotes unspecific signal. Corresponding signal intensities for poly- and mono-ubiquitin were measured by subtracting the background signal in control cells (a,c). Solubility of ubiquitylated Lsm7-TAP (b) and Pin3-TAP (d) was assessed by comparing both soluble (S) and pellet (P) fractions subjected to IMAC and analyzed by western blots with anti-TAP (b,d) and anti-ubiquitin (b). A 20 min 45 °C  HS was also applied to Pin3-TAP-expressing cells (c,d). No HS, no heat-shock. (e,f) Turnover of Slh1 is dependent on cytosolic Hul5. Protein levels of Slh1-TAP were monitored by western-blot analysis after the addition of 100 μg ml-1 cycloheximide to both HUL5 and hul5Δ cells grown at 25 °C (e), and to hul5Δ cells expressing either GFP-Hul5 or GFP-NLS-Hul5 and shifted to 38 °C (f). Relative averaged signal intensities (with standard deviations) were quantified and normalized to Pgk1 levels in three independent experiments.   56 2.4 Discussion Previous studies on yeast cytosolic PQC were mainly based on ‘model’ or ‘bait’ substrates[77, 85, 87]. Our approach, instead, primarily focused on global misfolding responses in the cell. We identified HUL5 as having an important role in the ubiquitylation of cytosolic misfolded proteins in unstressed conditions and in response to stresses that induce misfolding (HS and SSA-inactivation). Moreover, the Hul5 catalytic HECT domain and its cytosolic localization are important for cell fitness following HS stress. The absence of Hul5 also strongly decreases the degradation rate of short-lived misfolded proteins. We found that Hul5 substrates are mainly cytosolic and these ubiquitylated species are specifically associated with the low-solubility cellular fraction. Overall, our data show that Hul5 participates in a quality control degradation pathway targeting misfolded cytosolic proteins. Deletion of other ubiquitin ligases involved in cytosolic quality control pathways, such as Ubr1 and Ltn1, shows no significant defect in the HS ubiquitylation response, indicating that these ligases are unlikely to be involved in this pathway. The Hul5 quality control machinery is also possibly distinct from the previously identified pathway responsible for the degradation of the misfolded VHL (Von Hippel–Lindau) model substrate. Turnover of VHL requires SSA-chaperone activity as well as the Sse1 and Sti1 cofactors[141]. Similarly, deletion of SSE1 or STI1 did not affect the increase of ubiquitylation levels after HS (data not shown). Together, these results further indicate that there are several distinct cytosolic PQC pathways in the cell. Hul5 binds to the proteasome and has been proposed to antagonize the DUB Ubp6[107, 108]. Interestingly, chemical inhibition of Usp14, the mammalian Ubp6   57 orthologue, was recently found to enhance proteasome function and to accelerate the degradation of proteins involved in proteotoxic stress such as ATXN3[110]. Hence, the possible role of Hul5 in the ubiquitylation of misfolded proteins and its Ubp6-antagonistic activity are consistent. It will be interesting to determine whether Hul5 remains associated with the proteasome during the ubiquitylation of low-solubility proteins. In unstressed cells, most Hul5 localizes to the nucleus. Strikingly, only about 5% of Hul5 candidate substrates are solely nuclear (most nuclear proteins affected by HUL5 deletion are also cytosolic)[118]. One possible explanation for Hul5 not targeting more nuclear proteins is that it requires another cofactor in the cytoplasm. The low levels of cytosolic Hul5 may be sufficient for targeting a basal level of misfolded proteins in the cytoplasm of unstressed cells. In agreement, turnover of Slh1 is abrogated when GFP-NLS-Hul5 is sequestered to the nucleus, and blocking Hul5 cytosolic localization mimics the hul5Δ phenotype after HS. Our results indicate that Hul5 ubiquitylates low-solubility cytosolic substrates within the cytoplasm. Intriguingly, most Hul5 candidate substrates were mono-ubiquitylated and the mono-ubiquitylation levels were not affected by HUL5 deletion to the same extent as the poly-ubiquitylation signal (Fig. 7a-d and S6a,b). These results indicate that Hul5 may, in agreement with previous work[108], act as an E4 ubiquitin ligase to further ubiquitylate misfolded proteins targeted by another E3 ligase. Noticeably, deletion of Ufd2, another E4 ligase, did not affect the heat-induced ubiquitylation response (Fig. S3a), indicating that Hul5 is specific for this quality control pathway. One possibility is that misfolded proteins are targeted by this quality control machinery in a two-step ubiquitylation process that precludes the ‘en masse’ targeting of transiently misfolded polypeptides that can refold (Fig. S7f). In addition, the redistribution of Hul5 to the cytoplasm during   58 HS may permit the rapid targeting of a larger number of misfolded proteins. The discovery of cofactors that work in conjunction with Hul5 will provide further insight into the recognition and targeting mechanisms of these misfolded proteins in cells.  Human Ube3b and Ube3c ubiquitin ligases are the two homologs that share the highest sequence similarity with the HECT region of Hul5. Loss of function of UBE3B was suggested to be associated with diseases such as a developmental disorder called Kaufman oculocerebrofacial syndrome (KOS)[142], autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome[143], and autism[144]. This suggests that Ube3b might play an important role in neurodevelopment. Despite these finding, the function of Ube3b remains unknown. A very recent study found that in the absence of ligase Ube3C, a destabilizing domain-containing reporter protein was degraded more slowly and incompletely by the proteasome, indicating that Ube3C might promote proteasome processivity[145]. A similar phenomenon has also been observed for Hul5 in yeast[135]. As a future direction, it would be interesting to further investigate whether Ube3B and/or Ube3C are also involved in HS-induced ubiquitylation increase and whether Ube3C specifically enhances the degradation of misfolded proteins, highlighting the conserved function of Hul5.      59 2.5 Supplemental data Figure S2.1 HS induces protein misfolding and polyubiquitylation    (a) BY4741 cells were subjected to heat-shock (45 °C, HS), 1 mM H2O2, 5 mM paraquat, 10 mM CoCl2 and 2 M NaCl for 30 min or no stress. Experimental triplicates were analyzed by dot blot assay with anti-ubiquitin and anti-Pgk1 antibodies (left). The whole spotted signals in the dot blot were quantified (right). Ubiquitylation signals were normalized to Pgk1 levels and averaged (with standard deviations). (b) Schematic diagram of two possible mechanisms to account for the increase of poly-ubiquitylation following heat shock stress: increased ubiquitylation is due to misfolding (1), or accumulation of ubiquitylated proteins is due to proteasome inhibition (2). (c) MG132 experimental procedure to examine whether HS only causes dysfunction of proteasome   60 (left). BY4741 cells were exposed to HS (15 min at 45 °C) or no HS with or without the presence of 20 μM MG132. Experimental triplicates were analyzed by dot blots with anti-ubiquitin and anti-Pgk1 antibodies (middle). Ubiquitylation signals were normalized to Pgk1 levels and averaged (with standard deviations). One group of samples was also subjected to analysis by Western blot (right). The region above 60 kDa in the Western blot was quantified, and the relative levels, normalized to Pgk1, are indicated above (grey). There is no major difference in the two quantification approaches indicating that the dot-blot is adequate to quantify the HS ubiquitylation response. If proteasome inhibition was the sole cause of the increased ubiquitylation, there should not be any difference between samples 2 and 3. In this experiment, HS induced a higher increase of ubiquitylation in MG132-treated cells (compare sample 3 with samples 2 and 6). In this case, the increase of ubiquitylation is due to a combination of misfolding due to the stress and absence of proteasome activity (due to MG132). Regardless of the exact contribution of each phenomenon, the increased ubiquitylation after HS cannot be solely accounted for by the inhibition of the proteasome. Hence, an increase in misfolding (and not proteasome inhibition) plays a major role in inducing poly-ubiquitylation after HS.  61 Figure S2.2 Identification of HS affected ubiquitylation by quantitative mass spectrometry analysis    62 (a and c) Two experiments were conducted to validate the purification method for identifying ubiquitylated proteins using H8-Ubi and metabolic labeling. The percentage of proteins above the corresponding log2 of the 14N/15N ratios in each experiment is shown. Analysis of proteins in the total cell lysate (grey; 481 proteins in a, 479 proteins in c) and of IMAC enriched ubiquitylated proteins (green; 253 proteins in a, 259 proteins in c) in each experiment are shown. Note, that the few log2(14N/15N) values greater than five were converted to the fixed value of five, as large ratio differences are often inaccurate due to the background signal noise, as well as to accommodate near “infinite” ratio values. In one experiment (a), unstressed H8-Ubi expressing cells (YTM434) were differentially labeled. IMAC purified ubiquitylated proteins were expected to be equally enriched in both cell populations to confirm that no bias was introduced due to the labeling or the IMAC. Indeed, most ratios were close to one (0 in log2), with the exception of a small tail (corresponding to false positives that are also present in the total cell lysate analysis). In the other experiment (c), we compared cells expressing H8-Ubi (YTM434; 14N labeled) to cells expressing untagged ubiquitin (YTM419; 15N labeled). We expected that truly ubiquitylated proteins would be further enriched in 14N labeled cells (log2(ratio) ≥ 0.5). The few proteins that were not specifically enriched (log2(ratio) ~ 0) mainly consisted of proteins containing short histidine stretches. In this particular data set, less than 4% of proteins quantified in the total cell lysate were considered enriched using the cut off threshold (log2(ratio) ≥ 0.5). Note that we specifically selected a lower ratio threshold value to account for the fixed ubiquitin concentration in the cell, which precludes high enrichment of all conjugated proteins during the global heat-shock stress. (b) Venn diagram representing all 387 proteins identified as more ubiquitylated (log2(ratio) ≥ 0.5) in three independent heat-shock experiments (experiments I to III in Fig. 2.2b). There were 155 proteins that were found enriched in at least two of three experiments, which were then further analyzed for their localization (Fig. 2.2d). (d) Two MS datasets are plotted in a scatter diagram. Y axis is the ratio of proteins (log2(14N/15N)) identified in HS vs. noHS experiment with H8-Ubi cells (same data as experiment I in Fig. 2.2b). X axis is the ratio of proteins (log2 (14N/15N)) in H8-Ubi vs. untagged cells that were both subjected to HS (20 min at 45 °C; 342 proteins). A majority of proteins that were found ubiquitylated (X axis) were further ubiquitylated after HS (Y axis). Proteins that were not further ubiquitylated after HS, de-ubiquitylated after HS and unspecific are circled in green, blue and red, respectively. (e) Full MS scan spectra (MS1) of representative peptides from two HS affected proteins Rps7B (top) and Pin3 (bottom) in experiment I (Fig. 2.2b) are shown. The quantification of peptides was done by comparing peaks in MS1 scans using MSQuant. 14N/15N ratios of these two peptides at the selected retention times are also indicated.   63   Figure S2.3 HUL5 is required for the full ubiquitylation response and cell fitness after HS (a) WT, hul5Δ and strains with single deletion of E3s that are known or suspected to be involved in protein quality control were subjected to HS (15 min at 45 °C) or no HS. Biological triplicates were analyzed by dot blots with anti-ubiquitin and anti-Pgk1   64 antibodies. Quantified increase of ubiquitylation levels of each strain (HS - noHS) is shown with standard deviations. Student’s t-test was used to assess the significance of differences between each deletion and WT strain. While deletion of SAN1 or RKR1/LTN1 led to a decrease of the response, it was not significant. We also tested strains carrying a double deletion of HUL5 and either SAN1 or RKR1/LTN1, and found no significant differences (of HS ubiquitylation response) compared to cells carrying the single HUL5 deletion (data not shown), confirming that both SAN1 and RKR1/LTN1 are unlikely to be involved in this stress response pathway. (b) MYC IP experiments with cells expressing or not Ubc4TAP at endogenous levels with or without a plasmid expressing 13MycHul5 were analyzed by Western blot using 9E10 anti-MYC and anti-TAP antibodies. Inputs (1%) are shown below. Pull-down of 13MycHul5 was performed using 9E10 antibody bound to protein-G agarose (Roche) similar to TAP pull downs. We were not able to co-immunoprecipitate Ubc5TAP with 13MycHul5 presumably due to the low expression levels of Ubc5TAP. All the pull-down experiments were also positive when using cells not treated by HS (Fig. S2.7; see noHS lanes). We also verified that 13MycHul5 does not interact with the TAP tag, as it does not co-immunoprecipitate with Sik1TAP, which is expressed at slightly higher levels than Ubc4 in our conditions (data not shown). (c) Time course HS experiment (45 °C) was used to compare hul5Δ to the BY4741 WT strain. The region above 45 kDa in the Western blot was quantified (left). Ubiquitylation signals were normalized to Pgk1 levels and the increase in ubiquitylation at each time point from both strains is shown (right). (d) Cell viability before and after HS was analyzed for WT, hul5Δ, hul5Δ + hul5-C878A (BPM310) and hul5Δ + HUL5 (BPM309) strains. Cells were spotted in a 3 fold dilution series on YPD plates after or without HS (30 min at 45 °C) and were incubated at 25 °C for 3 days. (e) Average growth delays caused by HS with standard deviations (n=3) are indicated using data derived from Fig. 2.3c.  65 Figure S2.4 HS causes a re-localization of Hul5    (a) Representative images of Hul5GFP cells that were used for quantification in Fig. 2.4b are shown for unstressed (25 °C) and heat-shocked (30 min at 42 °C) cells. Merge (left), Hul5GFP (middle) and Nic96RFP (right) panels are shown. Untagged WT cells (marked with asterisks) are shown. The scale bar is 5 μm. For quantification, single-stack images were taken with a 40x oil EC Plan- Neofluar objective, and 100 cells in each condition were quantified in two separate image fields. For each cell in focus (based on Nic96RFP signal), the averaged GFP signal intensity was measured in a 0.7 μm2 area in the nucleus (defined by Nic96RFP signal) and in the cytosol. Average background signal was also measured from untagged cells (n=25/image), which were mixed and imaged with the tagged cells, then subtracted from the GFP signal intensities measured in tagged cells. (b) The average delay in growth of the indicated strains caused by HS is indicated (with standard deviations, n=3) using data derived from Fig. 2.4f. For comparison, the growth delay for HUL5 and hul5Δ cells carrying an empty pRS316 plasmid is also shown.        66 Figure S2.5 HUL5 is required for ubiquitylation of misfolded proteins under both HS and unstressed conditions   67 (a) A schematic diagram of the workflow to identify, by quantitative mass spectrometry, Hul5 misfolded substrates. (b) 14N and 15N metabolic labeling was performed in WT and hul5Δ cells, respectively. IMAC was directly performed with the whole cell lysate (instead of the low solubility cell fraction) derived from unstressed cells. The graph represents the percentage of proteins above the corresponding log2 values of the 14N/15N ratios. Analysis of proteins in the total cell lysate (grey; 457 proteins) and of IMAC enriched ubiquitylated proteins (green; 179 proteins) are shown. In contrast to Fig. 2.6a, here deletion of HUL5 does not abrogate the ubiquitylation of a significant fraction of the ubiquitin proteome. In unstressed cells, most ubiquitylated polypeptides are unlikely to correspond to low solubility proteins, as most conjugates remain soluble after centrifugation (Fig. 2.1b). Therefore deletion of HUL5 does not affect the overall ubiquitylation in the cell, but only perturbs the conjugation of a subpopulation that corresponds to low solubility proteins. (c) Venn diagram representing 155 proteins that are more ubiquitylated after HS (Fig. 2.2b; light green) and 95 proteins identified as Hul5 candidate substrates in unstressed cells in Fig. 2.6a (dark green). (d) 14N and 15N metabolic labeling was performed for WT and hul5Δ cells, respectively. Cells were treated with HS (20 min at 45 °C) before equal mixing and lysis. IMAC was performed from low solubility cell fraction. The graph represents the percentage of proteins above the corresponding log2 values of the 14N/15N ratios. Analysis of proteins in the total cell lysate (grey; 374 proteins) and of IMAC enriched ubiquitylated proteins (green; 490 proteins) are shown. 99 proteins with ratios that are higher than the cut-off (log2(14N/15N) ≥ 0.5) are considered candidate Hul5 substrates after HS induced misfolding (dotted box). (e) Venn diagram representing 99 Hul5 substrate candidates that are misfolded after HS in Fig. S2.5c (light green) and 95 Hul5 substrate candidates that are enriched in the low solubility fraction in unstressed cells in Fig. 2.6a (dark green). Five proteins were enriched in both approaches such as the prion-like protein Pin3, which may be susceptible to misfolding and therefore readily detected in both unstressed and HS stressed cells.  68 Figure S2.6 Hul5 is required for the ubiquitylation of several low solubility proteins in unstressed cells      69 (a, b, c) Validation of the Hul5 candidate substrates Tsa2, Fbp26, and Slh1 using TAP-tagged strains expressing H8-Ubi. IMAC was performed to pull down ubiquitylated proteins and anti-TAP antibody was used for Western blots; corresponding signal intensities for poly- and mono-ubiquitin were measured by subtracting the background signal in control cells (a and b). HS (45 °C, 20 min) was also performed on Slh1TAP expressing cells prior to cell lysis (c). High contrast of the Tsa2TAP image is also presented to show the high molecular weight bands in cells with HUL5 (a, right). The asterisks denote unspecific signals. (d) Solubility of ubiquitylated Slh1TAP was assessed by comparing both soluble and pellet fractions (after 16,000 g centrifugation) subjected to IMAC and analyzed by anti-TAP. (e) Turnover of Slh1TAP was assessed in exponentially growing cells maintained at 25 °C or shifted to 38 °C after the addition of 100 μg/ml cycloheximide. Samples were collected at the indicated times. Cells were lysed in 1x SDS-PAGE Laemmli sample buffer and Slh1TAP relative averaged signal intensities (with standard errors) were quantified after Western blotting and normalized to Pgk1 levels in three independent experiments. (f) Schematic representation of a possible model for the Hul5 quality control pathway.   70 Supplementary Table 2.1 Plasmid used in chapter 2 Strain ID Name Source & Note BPM30 pRS316-H8-ubiquitin Deashies Lab, H8-ubiquitin with GPD promotor and PGK terminator is insert into pRS316  yeast URA3, CEN, ARS vector in the EcoRI and SmaI sites BPM42 pRS316 yeast URA3, CEN, ARS vector w/ polylinker used for empty control BPM309 pRS316-HUL5  This study. Wild-type copy of HUL5 with endogenous promoter and terminator was subcloned from pJH84 (kindly provided by Dr. Finley) using SacI and ApaI sites and inserted into pRS316 (yeast URA3, CEN, ARS vector) cut at the SacI and SmaI sites BPM310 pRS316-HUL5-C878A This study.  Catalytically inactive mutant of HUL5 (C878A)  with endogenous promoter and terminator was subcloned from pJH85 (kindly provided by Dr. Finley) using SacI and ApaI sites and inserted into pRS316 (yeast URA3, CEN, ARS vector) cut at the SacI and SmaI sites BPM325 pRS316-GPDp-13Myc-HUL5-PGKt This study. Wild type HUL5 PCR amplyfied from BPM309 was first cloned with NotI and SalI in BPM173,  a pRS313 plasmid that contains the high expression GPD promoter, the N-terminal Myc13 tag and the terminator sequence from gene PGK1, then sub-cloned into pRS316 (yeast URA3, CEN, ARS vector) using SacI and XbaI. BPM341 pRS306-HUL5p-GFP-HUL5 This study. The PCR amplified fragments of the HUL5 promoter region (-400 to -1) with SacII and XbaI, the GFP+ with XbaI and SmaI, and the wild-type HUL5 coding sequence with 3’UTR (+1 to + 304) with SmaI and KpnI were inserted into pRS306 (yeast URA3  vector) in the SacII and KpnI restriction sites. As the 3'UTR region of Hul5 also contains and ARS sequence (ARS712), this plasmid was maintained in cells gown in selective media. The 3' GFP+ and 5' HUL5 linker region translates to the following sequence:  ...DELYKTRggLNFTGQX... BPM345 pRS306-HUL5p-GFP-NLS-HUL5 This study. The PCR amplified fragments of the HUL5 promoter region (-400 to -1) with SacII and XbaI, the GFP+ followed by a NLS sequence with XbaI and SmaI, and the HUL5 coding sequence with 3’UTR (+1 to + 304) with SmaI and KpnI were inserted into pRS306 (yeast URA3 vector) in the SacII and KpnI restriction sites. As the 3'UTR region of Hul5 also contains and ARS sequence (ARS712), this plasmid was maintained in cells gown in selective media. The 3' GFP+ with NLS and 5' HUL5 linker region translates to the following sequence:  ...DELYKTRggSPKKKRKVEASggLNFTGQX...    Supplementary Table 2.2 Yeast strains used in chapter 2 Strain ID Alias Genotype Background & Mating Type Source & Note YTM4 W303 can1-100, leu2-3,-112, his3-11,-15, trp1-1, ura3-1, ade2-1, pdr5∆::HIS3-MX6 W303, a  YTM408 BY4741 his3∆1, leu2∆0, met15∆0, ura3∆0 S288C, a  YTM547 YWO1 his3-∆200, leu2-3,2-112, lys2-801, trp1-1(am), ura3-52 DF5, DBY1829, alpha Dr. Jentsch Lab YTM548 ubc4∆ubc5∆ his3-∆200, leu2-3,2-112, lys2-801, trp1-1(am), ura3-52, ubc4∆:: HIS3, ubc5∆::LEU2 DF5, DBY1829, a Dr. Jentsch Lab YTM549 ubc1∆ubc4∆ his3-∆200, leu2-3,2-112, lys2-801, trp1-1(am), ura3-52, ubc1∆:: HIS3, ubc4∆::TRP1 DF5, DBY1829, alpha Dr. Jentsch Lab YTM550 ubc1∆ubc5∆ his3-∆200,  lys2-801, trp1-1(am), ura3-52, ubc1∆:: URA3, ubc5∆::LEU2 DF5, DBY1829, alpha Dr. Jentsch Lab YTM551 ubc6∆ubc7∆ leu2-3,2-112, lys2-801, trp1-1(am), ura3-52, ubc6∆:: HIS3, ubc7∆::HIS3 DF5, DBY1829, alpha Dr. Jentsch Lab YTM419 Untagged (MS)  can1-100, his3-11,-15, ADE2, LEU2, URA3, trp1::TRP1, pdr5∆::HIS3-MX6 W303, a  this study YTM434 H8Ubi (MS) can1-100, his3-11,-15, ADE2, LEU2, TRP1, pdr5∆::HIS3-MX6, ura3-1::H8-Ubiquitin::URA3 W303, a  this study   71 Strain ID Alias Genotype Background & Mating Type Source & Note YTM439 H8Ubi, hul5∆ (MS) can1-100, his3-11,-15, ADE2, LEU2, TRP1, ∆pdr5::HIS3-MX6, ura3-1::H8-Ubiquitin::URA3, hul5∆::KanMX6 W303, a  this study YTM645 hul5∆ his3∆1, leu2∆0, met15∆0, ura3∆0, hul5∆::KanMX6 S288C, a Open Biosystems  YTM515 Y7092 can1∆::STE2pr-Sp_HIS5, lyp1∆, his3∆1 leu2∆0 ura3∆0 met15∆0 S288C, alpha Dr. Boone Lab YTM459 hul5∆ can1∆::STE2pr-Sp_HIS5, lyp1∆, his3∆1 leu2∆0 ura3∆0 met15∆0, hul5∆::NatMX4 S288C, alpha Dr. Boone Lab YTM685 HUL5GFP leu2∆0, met15∆0, ura3∆0, arg4∆::KanMX6, HUL5-GFP::HIS3-MX6 BY4741, S288C, a this study YM1922 HUL5GFP, NIC96RFP leu2∆0, ura3∆0, HUL5-GFP::HIS3-MX6 NIC96-RFP::kanMX6 BY4741, S288C, a this study YM1935 background leu2∆0, ura3∆0, his3∆1, lys2∆0 BY4741, S288C, alpha this study YTM695 UBC4TAP his3∆1, leu2∆0, met15∆0, ura3∆0, UBC4-TAP::HIS3-MX6 BY4741, S288C, a Open Biosystems  YTM696 UBC5TAP his3∆1, leu2∆0, met15∆0, ura3∆0, UBC5-TAP::HIS3-MX6 BY4741, S288C, a Open Biosystems  YTM444 SSA1 his3-11,3-15,  leu2-3,2-112,  ura3-52,  trp1-∆1,  lys2, SSA1, ssa2∆::LEU2, ssa3∆::TRP1, ssa4∆::LYS2 DS10, alpha Dr. Craig Lab YTM445 ssa1-45 his3-11,3-15,  leu2-3,2-112,  ura3-52,  trp1-∆1,  lys2, ssa1-45BKD, ssa2∆::LEU2, ssa3∆::TRP1, ssa4∆::LYS2 DS10, alpha Dr. Craig Lab YTM596 ssa1-45, hul5∆ his3-11,3-15,  leu2-3,2-112,  ura3-52,  trp1-∆1,  lys2, ssa1-45BKD, ssa2∆::LEU2, ssa3∆::TRP1, ssa4∆::LYS2, hul5∆::KanMX6 DS10, alpha this study YTM597 ssa1-45, hul5∆ his3-11,3-15,  leu2-3,2-112,  ura3-52,  trp1-∆1,  lys2, ssa1-45BKD, ssa2∆::LEU2, ssa3∆::TRP1, ssa4∆::LYS2, hul5∆::KanMX6 DS10, alpha this study YTM575 PIN3TAP his3∆1, leu2∆0, met15∆0, ura3∆0, PIN3-TAP::HIS3-MX6 BY4741, S288C, a Open Biosystems  YTM681 PIN3TAP, hul5∆ his3∆1, leu2∆0, met15∆0, ura3∆0, PIN3-TAP::HIS3-MX6, hul5∆::KanMX6 BY4741, S288C, a this study YTM552 LSM7TAP his3∆1, leu2∆0, met15∆0, ura3∆0, LSM7-TAP::HIS3-MX6 BY4741, S288C, a Open Biosystems  YTM682 LSM7TAP, hul5∆ his3∆1, leu2∆0, met15∆0, ura3∆0, LSM7-TAP::HIS3-MX6, hul5∆::KanMX6 BY4741, S288C, a this study YTM562 SLH1TAP his3∆1, leu2∆0, met15∆0, ura3∆0, SLH1-TAP::HIS3-MX6 BY4741, S288C, a Open Biosystems  YTM814 SLH1TAP, hul5∆ his3∆1, leu2∆0, met15∆0, ura3∆0, SLH1-TAP::HIS3-MX6, hul5∆::KanMX6 BY4741, S288C, a this study YTM553 TSA2TAP his3∆1, leu2∆0, met15∆0, ura3∆0, TSA2-TAP::HIS3-MX6 BY4741, S288C, a Open Biosystems  YTM683 TSA2TAP, hul5∆ his3∆1, leu2∆0, met15∆0, ura3∆0, TSA2-TAP::HIS3-MX6, hul5∆::KanMX6 BY4741, S288C, a this study YTM560 FBP26TAP his3∆1, leu2∆0, met15∆0, ura3∆0, FBP26-TAP::HIS3-MX6 BY4741, S288C, a Open Biosystems  YTM684 FBP26TAP, hul5∆ his3∆1, leu2∆0, met15∆0, ura3∆0, FBP26-TAP::HIS3-MX6, hul5∆::KanMX6 BY4741, S288C, a this study       72 Supplementary Table 2.3 List of heat-shock affected proteins enriched by IMAC ORF Name I_pep num II_pep num III_pep num I_LOG II_LOG III_LOG YAL038W CDC19  49 112 132 0.767 0.385 0.537 YBL085W BOI1   24 78 35 1.101 0.619 1.006 YBL101C ECM21  0 6 4 N.D. 1.226 1.112 YBR025C OLA1   4 19 8 1.533 1.967 1.695 YBR059C AKL1   15 8 4 1.533 1.036 0.901 YBR072W HSP26  9 7 2 4.139 6.639 4.051 YBR086C IST2   65 145 198 1.299 1.088 0.868 YBR127C VMA2   3 18 6 1.261 1.644 1.343 YBR177C EHT1   4 4 0 1.155 0.781 N.D YBR225W YBR225W 37 72 60 1.757 1.066 1.192 YBR255W YBR255W 20 51 44 1.35 0.502 -0.02 YCL014W BUD3   0 3 3 N.D. 3.361 1.601 YCL030C HIS4   2 9 3 0.835 0.766 0.979 YCL051W LRE1   11 11 0 1.579 0.911 N.D YCR012W PGK1   32 74 89 1.123 0.424 0.59 YCR030C SYP1   3 12 3 2.853 2.972 3.843 YDL014W NOP1   6 5 0 0.684 0.512 N.D YDL025C YDL025C 76 116 189 1.436 0.908 0.833 YDL124W YDL124W 2 5 0 2.278 2.884 N.D YDL126C CDC48  9 30 22 1.874 1.895 2.202 YDL185W TFP1   17 37 20 0.75 0.447 0.534 YDL223C HBT1   114 192 134 2.884 1.157 1.077 YDL224C WHI4   0 7 4 N.D. 0.713 1.276 YDR050C TPI1   5 3 0 1.285 1.122 N.D YDR123C INO2   2 6 10 -0.28 0.599 0.727 YDR127W ARO1   2 19 0 1.353 0.692 N.D YDR150W NUM1   15 54 50 1.55 2.066 1.521 YDR155C CPR1   9 28 32 1.296 1.183 0.862 YDR171W HSP42  50 65 99 1.641 0.697 0.659 YDR205W MSC2   0 9 3 N.D. 1.261 1.089 YDR266C YDR266C 7 10 0 2.874 3.518 N.D YDR293C SSD1   18 32 23 1.261 0.967 1.118 YDR320C SWA2   2 3 0 2.906 1.569 N.D YDR326C YSP2   5 2 6 1.567 1.928 1.251 YDR379W RGA2   7 4 0 1.306 1.233 N.D YDR388W RVS167 5 6 0 2.579 1.967 N.D YDR422C SIP1   5 10 7 1.504 1.343 1.284 YDR475C JIP4   30 35 19 1.55 0.892 0.89 YDR477W SNF1   33 122 115 1.018 0.556 0.489 YDR497C ITR1   2 6 0 1.296 1.644 N.D YDR524C AGE1   24 22 21 1.095 0.646 0.601 YDR533C HSP31  4 6 0 1.217 0.776 N.D YEL060C PRB1   28 47 53 0.794 0.732 0.683 YER025W GCD11  2 2 0 0.827 0.508 N.D YER043C SAH1   8 36 26 0.597 0.469 0.524 YER056C FCY2   4 5 0 1.299 1.465 N.D YER068W MOT2   11 40 56 0.992 0.898 0.717 YER088C DOT6   29 102 98 1.123 0.608 0.615 YER125W RSP5   5 9 4 1.484 1.639 1.438 YER132C PMD1   4 8 3 1.207 0.502 1.725   73 ORF Name I_pep num II_pep num III_pep num I_LOG II_LOG III_LOG YER143W DDI1   2 7 3 1.748 1.608 1.715 YER151C UBP3   36 54 172 1.204 1.289 1.302 YER155C BEM2   136 330 324 1.292 0.742 0.603 YER165W PAB1   10 46 34 1.733 2.852 2.887 YFL039C ACT1   15 17 8 0.895 0.385 0.587 YFL045C SEC53  2 8 0 1.398 2.884 N.D YFR019W FAB1   0 4 6 N.D. 0.646 0.554 YGL009C LEU1   4 14 8 1.306 2.972 2.944 YGL026C TRP5   6 6 0 1.056 0.59 N.D YGL055W OLE1   4 4 0 1.456 0.936 N.D YGL105W ARC1   10 13 0 2.071 3.289 N.D YGL157W YGL157W 3 12 7 0.947 1.497 2.175 YGL178W MPT5   28 58 45 1.364 0.895 1.07 YGL206C CHC1   2 4 0 1.224 0.871 N.D YGL234W ADE5,7 2 4 0 1.23 0.573 N.D YGL245W GUS1   2 20 7 1.659 1.51 1.574 YGR097W ASK10  11 25 11 1.292 1.122 1.115 YGR161C RTS3   8 7 5 2.041 1.879 1.328 YGR178C PBP1   76 237 231 2.271 1.461 1.565 YGR234W YHB1   10 43 56 0.925 0.437 0.659 YGR237C YGR237C 8 44 5 1.282 1.001 0.851 YGR240C PFK1   4 22 12 1.104 0.678 0.671 YGR254W ENO1   21 27 23 0.663 0.832 0.918 YGR285C ZUO1   0 9 6 N.D. 2.761 4.003 YHL015W RPS20  0 28 16 N.D. 1.776 1.991 YHR020W YHR020W 0 9 4 N.D. 0.655 0.78 YHR042W NCP1   2 11 2 3.342 2.072 3.251 YHR064C SSZ1   0 8 2 N.D. 3.603 3.168 YHR073W OSH3   19 17 4 1 0.704 0.603 YHR119W SET1   4 6 0 1.217 0.837 N.D YHR174W ENO2   28 38 35 0.47 0.666 0.827 YHR183W GND1   12 19 3 0.944 0.473 0.86 YIL045W PIG2   7 5 0 1.251 1.552 N.D YIL053W RHR2   2 10 5 1.289 2.193 1.434 YIL078W THS1   0 2 5 N.D. 1.324 1.843 YJL005W CYR1   34 103 46 1.429 1.103 1.105 YJL042W MHP1   45 156 139 1.516 1.094 0.841 YJL080C SCP160 0 28 13 N.D. 2.186 2.652 YJL083W TAX4   0 17 6 N.D. 1.088 1.739 YJL165C HAL5   8 14 6 1.619 1.426 1.53 YJR027W YJR027W 10 6 0 1.142 1.112 N.D YJR059W PTK2   27 41 21 1.623 1.141 0.952 YJR137C ECM17  6 33 16 2.023 2.723 2.347 YKL056C TMA19  2 9 0 3.164 3.007 N.D YKL060C FBA1   18 27 19 0.545 0.756 0.622 YKL081W TEF4   6 11 0 2.44 2.267 N.D YKL152C GPM1   0 22 6 N.D. 0.824 0.755 YKL198C PTK1   6 3 0 1.145 1.626 N.D YKR031C SPO14  2 4 0 1.177 1.01 N.D YKR039W GAP1   12 21 13 1.65 1.842 1.629 YLL013C PUF3   45 80 100 1.204 0.597 0.69 YLL024C SSA2   15 34 51 1.247 1.617 1.402   74 ORF Name I_pep num II_pep num III_pep num I_LOG II_LOG III_LOG YLL026W HSP104 0 7 2 N.D. 2.03 1.018 YLR089C ALT1   2 7 0 1.08 0.852 N.D YLR153C ACS2   6 9 8 0.822 0.391 1.027 YLR180W SAM1   6 15 8 1.597 1.433 1.643 YLR219W MSC3   2 3 0 1.949 2.06 N.D YLR249W YEF3   20 75 34 1.484 1.569 1.466 YLR328W NMA1   10 67 19 1.254 0.418 0.78 YLR354C TAL1   2 8 5 0.514 0.541 0.509 YLR371W ROM2   72 176 126 1.148 0.713 0.811 YLR403W SFP1   4 7 0 1.9 1.084 N.D YLR421C RPN13  2 2 0 1.762 1.206 N.D YML008C ERG6   6 5 3 0.903 1.354 1.276 YML016C PPZ1   57 34 81 1.114 1.075 0.71 YML028W TSA1   4 8 7 0.992 -0.01 0.629 YML072C TCB3   6 5 2 1.289 1.23 1.973 YMR012W CLU1   0 15 3 N.D. 0.569 1.099 YMR031C YMR031C 0 7 5 N.D. 2.368 1.961 YMR070W MOT3   11 47 44 1.429 0.72 0.524 YMR079W SEC14  0 5 2 N.D. 1.933 2.148 YMR109W MYO5   10 9 0 1.496 1.109 N.D YMR205C PFK2   2 10 0 1.029 0.858 N.D YMR273C ZDS1   5 13 16 1.224 0.614 0.018 YMR275C BUL1   2 10 3 3.126 2.485 2.967 YNL055C POR1   4 9 5 1.092 0.917 1.125 YNL096C RPS7B  25 82 96 2.278 1.573 1.944 YNL103W MET4   2 10 0 1.575 0.811 N.D YNL183C NPR1   10 18 11 1.053 0.776 0.396 YNL192W CHS1   2 4 0 1.452 1.141 N.D YNL209W SSB2   7 39 40 1.61 2.685 2.251 YNL298W CLA4   15 6 0 0.958 1.299 N.D YNL321W VNX1   13 9 0 1.433 1.001 N.D YNR016C ACC1   2 28 5 0.466 1.858 1.705 YNR047W YNR047W 4 3 5 1.174 1.1 0.75 YNR050C LYS9   3 13 2 0.871 1.23 1.347 YOL059W GPD2   55 74 159 0.845 0.422 0.506 YOL086C ADH1   14 29 41 0.927 0.632 0.817 YOR018W ROD1   0 3 2 N.D. 2.199 2.615 YOR023C AHC1   12 15 19 1.074 0.811 0.86 YOR070C GYP1   33 70 50 1.456 1.199 1.151 YOR096W RPS7A  32 104 152 2.237 1.449 1.876 YOR133W EFT1   14 41 19 0.892 0.692 1.128 YOR134W BAG7   6 6 2 2.35 1.418 1.458 YOR198C BFR1   2 11 4 2.139 3.852 1.112 YOR204W DED1   9 23 6 2.12 2.742 2.34 YOR230W WTM1   2 7 0 0.595 0.522 N.D YOR267C HRK1   63 159 169 1.133 0.68 0.524 YOR317W FAA1   2 12 0 1.797 0.606 N.D YOR359W VTS1   26 42 94 1.306 0.972 0.827 YPL106C SSE1   4 20 13 1.9 2.811 3.643 YPL231W FAS2   13 28 7 1.31 0.328 0.72 YPR035W GLN1   7 16 2 1.177 0.735 0.72 YPR065W ROX1   13 8 7 2.44 1.816 1.822   75 ORF Name I_pep num II_pep num III_pep num I_LOG II_LOG III_LOG YPR154W PIN3   6 6 4 1.614 0.67 0.66 * I, II, III represent three independent MS analyses; ORF: yeast open reading frame; Name: gene name; Pep Num: peptide number quantified; Log: Log2(HS/noHS);  *Proteins with ratio above threshold (log2> or = 0.5) in 2 out of 3 biological replicates are considered HS-affected proteins.    Supplementary Table 2.4 List of E3 ubiquitin ligases assessed  ORF NAME Motif Background YAL002W VPS8 RNF-ring finger A YBR062C YBR062C RNF-ring finger A YBR114W RAD16 RNF-ring finger A YBR158W AMN1 F BOX B YBR203W COS111 F BOX A YBR280C SAF1 F BOX A YCR066W RAD18 RNF-ring finger A YDL008W APC11 RNF-ring finger C YDL013W SLX5 RNF-ring finger B YDL074C BRE1 RNF-ring finger A YDL190C UFD2 U-box A YDR049W YDR049W Zinc FINGER,C2H2 A YDR103W STE5 RNF-ring finger B YDR131C YDR131C F-BOX A YDR132C YDR132C BTB B YDR143C SAN1 RNF-ring finger A YDR219C MFB1 F BOX A YDR255C RMD5 RNF-ring finger B YDR265W PEX10 RNF-ring finger A YDR266C YDR266C RNF-ring finger B YDR306C YDR306C F BOX A YDR313C PIB1 RNF-ring finger A YDR457W TOM1 HECT A YDR460W TFB3 RNF-ring finger A YER116C SLX8 RNF-ring finger A YER125W RSP5 HECT C YFL009W CDC4 F BOX C YGL003C CDH1 WD40 repeat,APC/C complex component A YGL116W CDC20 WD41 repeat,APC/C complex component C YGL131C SNT2 RNF-ring finger B YGL141W HUL5 HECT A YGR003W CUL3 CULLIN REPEAT B YGR184C UBR1 RNF-ring finger A YHL010C YHL010C RNF-ring finger A YHR115C DMA1 RNF-ring finger A YIL001W YIL001W BTB A YIL030C SSM4 (DOA10) RNF-ring finger A YIL046W MET30 F BOX C YJL047C RTT101 CULLIN B   76 ORF NAME Motif Background YJL149W DAS1 F BOX A YJL157C FAR1 RNF-ring finger B YJL204C RCY1 F BOX A YJL210W PEX2 RNF-ring finger B YJR036C HUL4 HECT A YJR052W RAD7 F BOX B YJR090C GRR1 F BOX A YKL010C UFD4 HECT A YKL034W TUL1 RNF-ring finger A YKR017C YKR017C RNF-ring finger A YLL036C PRP19 U-box C YLR024C UBR2 RNF-ring finger A YLR032W RAD5 RNF-ring finger A YLR097C HRT3 F BOX A YLR108C YLR108C BTB B YLR224W YLR224W F BOX A YLR247C IRC20 RNF-ring finger A YLR323C CWC24 RNF-ring finger C YLR352W YLR352W F BOX A YLR368W MDM30 F BOX A YLR427W MAG2 RNF-ring finger A YML068W ITT1 RNF-ring finger B YML088W UFO1 F BOX A YMR026C PEX12 RNF-ring finger B YMR094W CTF13 F BOX C YMR119W ASI1 RNF-ring finger A YMR247C YMR247C RING Zinc FINGER A YMR258C YMR258C F BOX A YNL008C ASI3 RNF-ring finger A YNL023C FAP1 RNF-ring finger B YNL116W DMA2 RNF-ring finger A YNL230C ELA1 F BOX A YNL311C SKP2 F BOX A YOL013C HRD1 RNF-ring finger A YOL054W PSH1 RNF-ring finger A YOL133W HRT1 RNF-ring finger C YOL138C RTC1 RNF-ring finger B YOR080W DIA2 F BOX A YOR191W ULS1 RNF-ring finger B YPL046C ELC1 ELONGIN C,BTB,SKP1 COMPPNENT B YPR093C ASR1 RNF-ring finger A YMR247C RKR1 RNF-ring finger A YMR080C NAM7 CH-rich domain (RING-related domain) B * Background: A= BY4741 his3∆1, leu2∆0, met15∆0, ura3∆0, S288C a; B=BY4742 his3∆1, leu2∆0, lys2∆0, ura3∆1, S288C alpha; C=BY4743 his3∆1/his3∆1, leu2∆0/leu2∆0, met15∆0/MET15, ura3∆0/ura3∆1, lys2∆0/LYS2, S288C diploid        77 Supplementary Table 2.5 List of potential Hul5 substrates under no stress condition enriched by IMAC  ORF Name I_pep num II_pep num III_pep num I_LOG II_LOG III_LOG YBR072W HSP26 3 2 0 2.64 3.4 N/A YDL223C HBT1 27 24 6 1.4 1.68 1.16 YDR453C TSA2      2 0 2 2.49 N/A 1.14 YGR264C MES1 5 0 2 1 N/A 1.47 YGR271W SLH1 3 4 0 1.75 5 N/A YHL047C ARN2 4 2 0 5 5 N/A YJL052W TDH1 5 3 0 1.25 1.88 N/A YLR205C HMX1 5 0 2 2.38 N/A 2.52 YNL147W LSM7 2 2 0 1.06 1.17 N/A YOR046C DBP5 2 2 0 1.18 1.48 N/A YPR154W PIN3 3 4 0 1.07 1.7 N/A YPR160W GPH1 4 3 0 1.35 1.07 N/A YPR184W GDB1 3 2 0 1.02 2.32 N/A YAL060W BDH1 3 0 0 1.39 N/A N/A YBL011W SCT1 3 0 0 1.07 N/A N/A YBL086C YBL086C 0 3 0 N/A 1.1 N/A YBR121C GRS1 6 2 3 0.52 0.65 1.06 YBR166C TYR1 3 0 0 2.05 N/A N/A YBR214W SDS24 4 2 0 0.94 1.28 N/A YCL034W LSB5 2 0 0 1.16 N/A N/A YCR021C HSP30 0 2 0 N/A 2.71 N/A YDL160C DHH1 4 0 0 1.05 N/A N/A YDR001C NTH1 3 2 0 0.12 1.03 N/A YDR117C YDR117C 2 0 0 5 N/A N/A YDR229W IVY1 2 0 0 1.51 N/A N/A YDR258C HSP78 0 2 0 N/A 1.07 N/A YDR270W CCC2 9 0 0 2.2 N/A N/A YDR320C SWA2 2 0 0 1.08 N/A N/A YDR388W RVS167 5 5 0 0.77 1.17 N/A YDR432W NPL3 3 0 0 1.02 N/A N/A YEL047C YEL047C 2 0 0 2.12 N/A N/A YER062C HOR2 2 2 0 5 0.67 N/A YER143W DDI1 2 0 0 1 N/A N/A YFR053C HXK1 3 2 0 0.92 1.42 N/A YGL141W HUL5 3 0 0 3.1 N/A N/A YGL181W GTS1 2 0 0 1.02 N/A N/A YGR145W ENP2 2 0 0 1.06 N/A N/A YGR161C RTS3 4 2 0 1.56 0.52 N/A YGR237C YGR237C 16 4 10 0.54 0.95 1.46 YHL001W RPL14B 6 5 6 0.18 1.08 0.58 YHR005C GPA1 4 0 0 5 N/A N/A YHR063C PAN5 5 2 0 1.02 0.11 N/A YHR107C CDC12 0 4 0 N/A 1.14 N/A YHR111W UBA4 2 2 0 1.35 0.43 N/A YHR170W NMD3 4 0 0 1.11 N/A N/A YIL074C SER33 0 2 4 N/A 0.77 1.08 YIL129C TAO3 0 3 0 N/A 1.32 N/A YIL142W CCT2 2 5 0 1.68 0.72 N/A   78 ORF Name I_pep num II_pep num III_pep num I_LOG II_LOG III_LOG YIR036C YIR036C 2 0 0 1.7 N/A N/A YJL016W YJL016W 2 0 0 4.42 N/A N/A YJL155C FBP26 2 0 0 1.52 N/A N/A YJR096W YJR096W 0 2 0 N/A 1.09 N/A YJR109C CPA2 13 7 2 0.39 0.6 1.08 YKR018C YKR018C 2 0 0 1.47 N/A N/A YLL008W DRS1 2 2 0 4.4 0.09 N/A YLL026W HSP104 15 9 4 0.86 1.42 0.76 YLL048C YBT1 2 0 0 1 N/A N/A YLR172C DPH5 0 2 0 N/A 1.15 N/A YLR214W FRE1 2 0 0 4.74 N/A N/A YLR259C HSP60 2 0 0 1.36 N/A N/A YLR347C KAP95 2 0 0 1.89 N/A N/A YLR351C NIT3 2 0 0 1.22 N/A N/A YLR380W CSR1 0 2 0 N/A 1.01 N/A YLR388W RPS29A 2 3 0 1.65 0.17 N/A YLR454W YLR454W 2 0 0 4.1 N/A N/A YML004C GLO1 2 0 0 1 N/A N/A YML123C PHO84 2 0 0 1.14 N/A N/A YMR080C NAM7 3 0 0 1.16 N/A N/A YMR096W SNZ1 3 0 0 1.18 N/A N/A YMR099C YMR099C 2 2 0 2.83 0.53 N/A YMR105C PGM2 2 0 0 1.33 N/A N/A YMR140W SIP5 2 0 0 1.08 N/A N/A YMR169C ALD3 0 3 0 N/A 1.13 N/A YMR171C YMR171C 2 0 0 1.68 N/A N/A YMR178W YMR178W 4 0 0 1.13 N/A N/A YMR226C YMR226C 4 0 0 1.02 N/A N/A YMR229C RRP5 15 0 2 0.34 N/A 1.54 YMR250W GAD1 2 0 0 1.21 N/A N/A YMR315W YMR315W 2 3 0 4.3 0.55 N/A YNL007C SIS1 2 0 0 1.28 N/A N/A YNL087W YNL087W 3 2 0 1.03 0.51 N/A YNL096C RPS7B 8 10 7 0.1 0.52 1.06 YNL190W YNL190W 2 0 0 1.23 N/A N/A YNL274C YNL274C 5 7 2 0.65 0.15 1.39 YNL298W CLA4 2 0 0 4.67 N/A N/A YOL059W GPD2 9 7 3 -0.19 0.23 1.21 YOR109W INP53 0 3 0 N/A 1.43 N/A YOR116C RPO31 2 0 0 2.48 N/A N/A YOR160W MTR10 2 0 0 1.91 N/A N/A YOR326W MYO2 3 0 0 4.18 N/A N/A YOR375C GDH1 20 5 0 0.26 1.05 N/A YPL086C ELP3 5 0 0 1.17 N/A N/A YPL145C KES1 5 3 2 0.68 0.21 1.22 YPL226W NEW1 12 4 2 0.54 0.93 1.07 YPR008W HAA1 2 0 0 1.4 N/A N/A * I, II, III represent three independent MS analyses; ORF: yeast open reading frame; Name: gene name; Pep Num: peptide number quantified; Log: Log2 (WTpellet/hul5Δpellet).  *Proteins with ratio above threshold (log2> or = 1) are considered potential Hul5 substrates. Highlighted in green, are proteins found enriched in at least 2 experiments.   79 Supplementary Table 2.6 Method validation of quantitative mass spectrometry and IMAC used in chapter 2 S2.2a S2.2c S2.2d ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  YMR081C 3 2.2 YLR342W 6 5 YLR180W 6 5 YER068W 10 0.49 YDL223C 52 1.78 YNR006W 3 4.88 YBR157C 2 5 YDR487C 3 0.49 YJL153C 4 1.62 YBL075C 2 4.54 YGR279C 2 4.79 YEL046C 3 0.48 YGR136W 2 1.3 YGL045W 2 4.35 YLR019W 2 3.97 YML024W 3 0.48 YLR109W 5 1.1 YGL253W 2 4.32 YDR320C 2 3.02 YHR208W 8 0.48 YGR149W 2 1.09 YGR136W 2 3.74 YDR266C 7 2.97 YHR174W 20 0.47 YEL060C 15 0.91 YNL192W 4 3.68 YHR042W 4 2.97 YGR234W 9 0.46 YGR032W 2 0.83 YLR327C 2 3.59 YHR064C 2 2.97 YLR327C 2 0.46 YBR072W 3 0.81 YMR160W 5 3.16 YHL015W 3 2.83 YDR037W 9 0.46 YGL253W 3 0.76 YOR096W 15 3.07 YMR031C 2 2.83 YCR031C 2 0.46 YBL002W 2 0.74 YKL210W 8 2.74 YPL226W 2 2.83 YDL061C 4 0.46 YDR171W 15 0.74 YNL096C 7 2.72 YLR248W 2 2.79 YER086W 7 0.46 YDR432W 6 0.73 YKR021W 2 2.67 YMR275C 3 2.77 YBR135W 2 0.46 YMR186W 5 0.73 YGR032W 13 2.63 YJR137C 5 2.71 YGL058W 2 0.46 YDR533C 2 0.7 YIL108W 3 2.63 YNL096C 8 2.6 YPL055C 2 0.46 YJL052W 7 0.63 YBR082C 2 2.59 YDL126C 7 2.46 YGR124W 8 0.45 YKR042W 4 0.63 YPR154W 4 2.56 YCR030C 5 2.43 YDL185W 11 0.45 YOR375C 7 0.58 YKR039W 6 2.55 YBR072W 6 2.41 YAL038W 27 0.45 YIL045W 3 0.56 YBL007C 3 2.37 YOR096W 16 2.36 YCL030C 5 0.44 YIL051C 3 0.54 YBR177C 2 2.27 YDR205W 2 2.28 YDL083C 3 0.43 YLR298C 3 0.51 YBR072W 4 2.24 YPR154W 3 2.23 YGR204W 4 0.43 YPL224C 2 0.49 YEL046C 2 2.16 YMR081C 6 2.22 YLR029C 4 0.43 YOR063W 13 0.48 YGR175C 3 1.93 YGL157W 3 2.17 YBR011C 13 0.43 YMR160W 2 0.43 YDR345C 4 1.91 YOR204W 7 2.16 YLR058C 12 0.43 YAL005C 12 0.41 YHL015W 2 1.88 YDL223C 61 2.12 YER131W 4 0.42 YOR134W 2 0.38 YOR316C 2 1.86 YBL101C 3 2.12 YGR061C 2 0.42 YOR209C 3 0.37 YMR315W 2 1.83 YMR295C 2 2.09 YNL308C 4 0.42 YNL055C 3 0.36 YFL018C 2 1.79 YDR092W 2 2.04 YGR254W 6 0.42 YKL129C 27 0.34 YKL062W 2 1.64 YOR018W 4 2.02 YHR073W 14 0.42 YPR043W 2 0.31 YLR167W 13 1.62 YER067W 3 2.01 YJR145C 3 0.42 YBR177C 4 0.3 YBR126C 2 1.54 YIL118W 2 1.96 YGR237C 5 0.41 YER177W 3 0.3 YMR079W 2 1.54 YDR388W 4 1.95 YDL014W 5 0.4 YJR065C 2 0.3 YBL002W 2 1.51 YMR022W 2 1.95 YOL059W 20 0.39 YBR170C 3 0.3 YDR432W 7 1.5 YBR079C 3 1.93 YDR436W 2 0.39 YCL012C 2 0.28 YDR032C 2 1.45 YGL105W 3 1.91 YNL067W 2 0.38 YOR125C 2 0.28 YDR123C 3 1.44 YKR039W 5 1.91 YDL125C 2 0.38 YLR089C 2 0.27 YLR109W 3 1.44 YNL209W 6 1.89 YHR010W 4 0.38 YJL172W 10 0.27 YDR533C 2 1.41 YMR305C 2 1.85 YOL140W 12 0.38 YNR050C 2 0.26 YOR322C 2 1.34 YKL081W 3 1.85 YOL120C 6 0.38 YGL008C 21 0.26 YBR225W 16 1.31 YIL045W 7 1.84 YGL037C 3 0.38 YLR303W 11 0.25 YNL208W 2 1.29 YHR108W 2 1.79 YLR244C 2 0.38 YDL131W 5 0.25 YMR105C 2 1.27 YBR025C 6 1.78 YOL087C 10 0.37 YBR196C 3 0.25 YDL126C 6 1.27 YIL140W 2 1.78 YER091C 12 0.37 YJR009C 2 0.24 YEL060C 14 1.25 YBL002W 2 1.78 YOL086C 9 0.37 YAL003W 2 0.24 YLR403W 2 1.25 YER165W 7 1.71 YNL074C 4 0.37 YGR254W 5 0.24 YER151C 17 1.24 YJL083W 2 1.7 YPL224C 4 0.36 YGR124W 3 0.23 YML008C 3 1.24 YML097C 2 1.66 YCL037C 16 0.36   80 S2.2a S2.2c S2.2d ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  YPL079W 3 0.22 YPR065W 5 1.23 YOR322C 2 1.65 YMR104C 20 0.35 YLR328W 14 0.22 YBR059C 3 1.23 YDL161W 2 1.64 YGR085C 2 0.33 YER080W 2 0.22 YLR340W 3 1.22 YBL047C 2 1.61 YPL079W 3 0.32 YKL214C 3 0.21 YGL157W 3 1.21 YML008C 3 1.59 YLR089C 3 0.31 YDR205W 3 0.21 YHR007C 5 1.21 YIL078W 2 1.56 YLR355C 4 0.31 YPL061W 10 0.21 YMR318C 2 1.19 YNL192W 4 1.54 YDR477W 22 0.3 YFL039C 5 0.21 YPL061W 7 1.19 YIL109C 4 1.51 YLR075W 6 0.3 YGR240C 7 0.2 YNL103W 2 1.17 YNL097C 2 1.5 YDR035W 2 0.28 YOR096W 16 0.2 YOR375C 7 1.17 YMR250W 3 1.48 YLR432W 8 0.26 YGL035C 3 0.2 YKL214C 3 1.17 YBR196C 2 1.46 YER080W 2 0.26 YBR191W 4 0.18 YGL245W 5 1.16 YGR032W 2 1.45 YDR475C 28 0.23 YOR142W 2 0.16 YLL026W 4 1.16 YMR219W 2 1.45 YBR238C 15 0.23 YDL136W 2 0.16 YHR183W 11 1.16 YAL005C 13 1.45 YIL051C 8 0.21 YNL096C 3 0.15 YIL053W 2 1.15 YBR140C 10 1.42 YBL099W 2 0.21 YLR219W 4 0.15 YGL008C 18 1.14 YCL051W 3 1.42 YFL021W 2 0.2 YCR012W 28 0.15 YLR337C 3 1.13 YDR050C 4 1.39 YGL076C 2 0.19 YDR155C 5 0.14 YAR010C 5 1.09 YDR379W 6 1.39 YBR249C 11 0.17 YKL152C 5 0.13 YCR012W 21 1.08 YNR047W 5 1.38 YFR049W 2 0.16 YDR477W 16 0.13 YJL165C 4 1.07 YGR161C 5 1.37 YJL190C 3 0.16 YOR042W 5 0.13 YMR062C 2 1.06 YDL095W 2 1.36 YMR273C 2 0.15 YAL038W 23 0.13 YAL005C 11 1.04 YER169W 2 1.36 YJL026W 7 0.1 YLL024C 2 0.13 YDR155C 4 1.04 YDR497C 3 1.34 YDR234W 18 0.08 YGL103W 11 0.13 YOR042W 2 1.03 YJL052W 5 1.34 YNL183C 7 0.07 YGL030W 2 0.12 YGL156W 2 1.02 YNL321W 9 1.34 YLR303W 15 0.06 YLR029C 5 0.12 YBR127C 4 1.01 YIL053W 2 1.32 YDR123C 4 0.04 YLR167W 12 0.11 YBR196C 3 1.01 YER056C 2 1.32 YKL214C 2 0.04 YER131W 3 0.1 YBR025C 5 0.97 YLR219W 4 1.31 YJL172W 19 0.03 YOR322C 2 0.1 YNL178W 7 0.96 YNR016C 4 1.31 YGR149W 2 -0.03 YHR183W 7 0.1 YER165W 5 0.96 YER125W 8 1.3 YBL066C 3 -0.04 YJL190C 3 0.1 YML028W 5 0.95 YLR249W 18 1.3 YKL210W 12 -0.07 YGL147C 2 0.09 YLR180W 6 0.94 YMR037C 4 1.3 YMR012W 4 -0.1 YLR340W 4 0.09 YNL134C 2 0.92 YGL009C 4 1.3 YER090W 2 -0.15 YJR145C 4 0.09 YJL084C 12 0.92 YLL026W 3 1.28 YJR016C 12 -0.2 YER165W 5 0.09 YLR298C 3 0.92 YMR070W 6 1.28 YKL062W 3 -0.25 YDR050C 8 0.09 YBL086C 2 0.91 YLR403W 7 1.27 YHL023C 2 -0.31 YOL120C 4 0.09 YPL231W 10 0.88 YGL035C 4 1.27 YDL171C 8 -0.45 YKL180W 4 0.09 YML128C 2 0.87 YMR105C 3 1.25    YJR017C 2 0.09 YDL229W 6 0.87 YLR109W 2 1.25    YEL034W 6 0.08 YLR048W 2 0.86 YBR225W 24 1.24    YGR034W 2 0.08 YKL182W 18 0.86 YOL103W-B 8 1.24    YHR174W 18 0.08 YCL009C 12 0.85 YGL178W 16 1.23    YBR011C 9 0.08 YGR097W 3 0.84 YDR326C 8 1.23    YER043C 6 0.08 YBR170C 3 0.84 YDR096W 3 1.22    YGR237C 9 0.08 YBL099W 2 0.84 YOR227W 11 1.22    YPR154W 3 0.08 YMR186W 7 0.82 YGL245W 5 1.22    YML119W 2 0.08 YNL055C 3 0.81 YJL068C 2 1.21    YNL308C 6 0.07 YLR153C 3 0.8 YER008C 2 1.2    YMR226C 3 0.07 YER183C 2 0.79 YOL113W 2 1.2    YCL030C 2 0.07 YDL223C 45 0.78 YHR021C 2 1.2    YLR048W 3 0.07 YGL026C 3 0.78 YBR127C 5 1.19      81 S2.2a S2.2c S2.2d ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  YLR180W 3 0.07 YJR123W 3 0.76 YPR161C 4 1.19    YDR233C 2 0.07 YNL104C 3 0.76 YBR182C 2 1.19    YLR075W 7 0.07 YLR354C 3 0.75 YBR106W 2 1.18    YDL185W 3 0.07 YER043C 6 0.74 YHR119W 2 1.18    YOL086C 11 0.07 YDL082W 2 0.74 YMR109W 4 1.18    YML126C 2 0.07 YAL012W 3 0.74 YOL019W 3 1.18    YLR044C 20 0.07 YOR209C 5 0.74 YPR035W 3 1.17    YLR092W 4 0.06 YGR085C 2 0.74 YEL031W 4 1.14    YNL178W 4 0.06 YJR145C 5 0.73 YGR240C 6 1.13    YFL010C 2 0.06 YER088C 11 0.73 YEL060C 13 1.13    YCL009C 13 0.06 YBL087C 2 0.72 YLR167W 12 1.13    YER091C 20 0.06 YOR063W 17 0.72 YMR246W 3 1.13    YER132C 2 0.06 YGL123W 4 0.7 YOR233W 4 1.12    YIL052C 2 0.06 YOL087C 10 0.69 YML016C 21 1.11    YHR010W 6 0.06 YGR124W 4 0.69 YPL106C 3 1.1    YNL209W 2 0.05 YJL080C 2 0.69 YOR374W 2 1.1    YDR037W 5 0.05 YGR240C 7 0.69 YGL008C 14 1.07    YHR007C 2 0.05 YDL083C 4 0.69 YOR023C 11 1.07    YML026C 3 0.05 YLL024C 4 0.67 YGL055W 4 1.06    YMR037C 2 0.05 YJL153C 3 0.67 YGR097W 7 1.06    YMR116C 7 0.05 YLL050C 2 0.67 YBR010W 2 1.05    YGL026C 2 0.05 YMR205C 5 0.67 YBL085W 24 1.05    YDL125C 2 0.05 YKL129C 25 0.67 YPR065W 10 1.05    YOL140W 9 0.05 YNR050C 2 0.67 YDR480W 2 1.02    YBR031W 5 0.05 YBR255W 13 0.67 YLR225C 3 1.02    YDL060W 4 0.05 YMR120C 9 0.67 YLR298C 2 1.02    YPL090C 3 0.05 YLR371W 34 0.66 YDR348C 12 1.01    YOL040C 3 0.05 YJR077C 2 0.66 YLR328W 10 1.01    YBL027W 3 0.05 YGR237C 9 0.66 YOL060C 8 1.01    YMR273C 3 0.04 YNR016C 4 0.66 YOR375C 5 1    YGR234W 2 0.04 YGR178C 20 0.65 YBR177C 4 0.99    YGL206C 2 0.04 YPL081W 3 0.65 YLL029W 2 0.99    YOR133W 13 0.04 YJL190C 3 0.65 YNL309W 2 0.98    YDL061C 5 0.04 YOR133W 11 0.64 YBR066C 2 0.97    YNL103W 3 0.04 YIL078W 2 0.64 YBR059C 11 0.96    YOR023C 3 0.04 YDR050C 6 0.64 YER082C 9 0.96    YLR256W 3 0.04 YOR128C 2 0.63 YEL054C 3 0.95    YMR120C 8 0.04 YBL027W 3 0.63 YER032W 4 0.94    YGR185C 3 0.03 YDR025W 4 0.63 YBL054W 8 0.94    YOL087C 9 0.03 YLR044C 19 0.63 YNR006W 4 0.94    YGR192C 16 0.03 YJR121W 3 0.62 YCL061C 2 0.93    YML008C 2 0.03 YGR118W 2 0.62 YGL045W 4 0.93    YHL015W 4 0.03 YNL067W 2 0.61 YLR153C 4 0.93    YCR031C 2 0.03 YPR074C 7 0.61 YJL084C 18 0.92    YBL030C 4 0.02 YML017W 23 0.6 YNL178W 5 0.92    YBL092W 10 0.02 YDL131W 7 0.6 YOL040C 2 0.92    YOL019W 2 0.02 YHR073W 9 0.59 YOR014W 2 0.92    YDR025W 7 0.02 YOR023C 5 0.59 YBR255W 12 0.92    YLL045C 6 0.02 YER082C 3 0.59 YGR178C 38 0.92    YMR242C 3 0.01 YJR016C 7 0.59 YHR020W 6 0.92      82 S2.2a S2.2c S2.2d ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  YDR023W 2 0.01 YML063W 5 0.59 YNL055C 4 0.92    YLR342W 18 0.01 YLL045C 6 0.58 YJR059W 16 0.92    YLR058C 8 0.01 YJR010W 2 0.58 YJR109C 2 0.92    YML017W 18 0.01 YDR353W 2 0.58 YKR021W 3 0.9    YEL054C 2 0.01 YLR249W 17 0.58 YML059C 6 0.89    YKL182W 7 0.01 YOR040W 2 0.58 YOR040W 3 0.89    YMR104C 14 0.01 YJR059W 14 0.58 YGL206C 2 0.89    YDL175C 6 0.01 YBR191W 5 0.57 YDR293C 12 0.88    YBR127C 2 0 YLR256W 3 0.57 YDR422C 3 0.88    YGR118W 3 0 YOR142W 3 0.57 YOR359W 11 0.88    YNL154C 25 0 YGR254W 5 0.56 YDR524C 10 0.87    YLR355C 2 0 YGL076C 5 0.56 YCR012W 19 0.87    YBR255W 14 -0.01 YOL127W 3 0.56 YDR155C 6 0.86    YIL018W 11 -0.01 YDL136W 2 0.56 YHL007C 2 0.86    YBR249C 14 -0.01 YIL069C 4 0.56 YJL042W 26 0.85    YGL076C 4 -0.01 YFL039C 5 0.55 YFL010C 3 0.83    YER068W 8 -0.03 YBR135W 2 0.55 YJL165C 7 0.83    YJR123W 2 -0.03 YOL058W 2 0.55 YLR342W 11 0.83    YJL042W 21 -0.04 YJL177W 3 0.55 YDR208W 4 0.81    YLL011W 3 -0.05 YKL152C 6 0.55 YFR019W 3 0.81    YHR097C 22 -0.05 YHR174W 19 0.54 YHR183W 9 0.81    YHR141C 4 -0.06 YLR328W 17 0.54 YKL152C 4 0.81    YOL059W 17 -0.07 YOR341W 4 0.54 YKL182W 16 0.8    YJL084C 14 -0.07 YOL040C 4 0.54 YMR318C 2 0.79    YGR085C 2 -0.07 YDR475C 18 0.54 YER088C 16 0.78    YBR059C 5 -0.07 YLR355C 6 0.53 YMR315W 2 0.78    YLR185W 3 -0.07 YHL001W 2 0.53 YCL009C 16 0.78    YIR034C 2 -0.08 YGL103W 12 0.53 YER132C 5 0.78    YOR267C 29 -0.08 YDR158W 4 0.53 YMR120C 10 0.78    YBR225W 9 -0.08 YER155C 47 0.53 YDR345C 3 0.77    YPR080W 16 -0.09 YDR205W 2 0.53 YLR371W 42 0.77    YFR032C-A 3 -0.09 YML126C 3 0.52 YBR086C 35 0.77    YDL025C 22 -0.1 YDR023W 5 0.51 YGL048C 2 0.77    YHR216W 2 -0.1 YML024W 4 0.5 YPL061W 6 0.77    YLR249W 14 -0.11 YLR029C 8 0.49 YLR354C 2 0.75    YKL062W 3 -0.11 YIL018W 10 0.48 YPL231W 9 0.75    YJL005W 14 -0.11 YPR080W 17 0.48 YDR171W 15 0.74    YLR276C 5 -0.11 YPL090C 6 0.48 YFL039C 10 0.74    YBL087C 2 -0.11 YDR326C 4 0.48 YLR361C-A 2 0.74    YOL127W 3 -0.12 YLL013C 17 0.47 YER151C 32 0.74    YPR035W 3 -0.13 YML016C 14 0.45 YDL025C 29 0.73    YHL023C 5 -0.13 YHR010W 6 0.45 YGL123W 7 0.73    YKR021W 2 -0.14 YBL072C 8 0.44 YDL055C 5 0.73    YGL123W 5 -0.14 YOL120C 6 0.44 YLL045C 5 0.72    YGR204W 2 -0.14 YOR134W 5 0.44 YNL298W 9 0.72    YDR475C 13 -0.14 YER091C 21 0.43 YOR133W 9 0.72    YGL234W 2 -0.14 YDR012W 7 0.43 YER043C 8 0.72    YBL072C 7 -0.14 YOL086C 11 0.42 YKL054C 5 0.71    YPL231W 5 -0.16 YAL038W 25 0.42 YER177W 3 0.71    YDR150W 11 -0.17 YDL055C 6 0.42 YKL035W 3 0.71      83 S2.2a S2.2c S2.2d ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  YKL060C 11 -0.17 YLR058C 12 0.42 YER081W 2 0.7    YBR140C 3 -0.17 YMR242C 5 0.42 YHR097C 22 0.7    YML016C 14 -0.17 YEL054C 3 0.42 YOR142W 2 0.7    YDR379W 2 -0.18 YJR017C 8 0.41 YPL135W 2 0.7    YLR371W 42 -0.18 YEL034W 4 0.41 YMR086W 2 0.7    YJR059W 13 -0.19 YDL014W 2 0.41 YML126C 2 0.69    YEL071W 7 -0.2 YMR273C 2 0.41 YOR070C 24 0.69    YBL085W 20 -0.21 YLR075W 8 0.4 YLR340W 2 0.69    YER125W 3 -0.21 YGR234W 7 0.39 YOR276W 4 0.69    YER082C 2 -0.21 YHR208W 5 0.38 YLR256W 4 0.69    YLR153C 3 -0.21 YJR009C 14 0.38 YOR134W 6 0.68    YDR293C 7 -0.21 YHR097C 19 0.38 YCR065W 2 0.68    YGR161C 2 -0.21 YCL030C 4 0.38 YAL041W 3 0.68    YGL045W 5 -0.23 YMR116C 4 0.37 YNR050C 2 0.68    YNL321W 7 -0.23 YDR477W 18 0.37 YPR080W 18 0.68    YOR070C 17 -0.23 YLR276C 8 0.36 YJR017C 5 0.67    YPL037C 2 -0.23 YJL005W 17 0.33 YOR267C 32 0.67    YDR326C 2 -0.24 YIR034C 3 0.33 YJL005W 31 0.66    YDR123C 2 -0.25 YLR303W 17 0.33 YIR034C 6 0.66    YKR039W 4 -0.25 YOR359W 11 0.33 YJL130C 5 0.66    YJL083W 2 -0.26 YOL059W 20 0.33 YML072C 8 0.64    YNL183C 2 -0.26 YER068W 9 0.33 YMR205C 4 0.64    YPL131W 4 -0.26 YDL185W 9 0.33 YNL154C 28 0.63    YLL013C 17 -0.27 YBL092W 7 0.3 YLL011W 3 0.62    YFR053C 2 -0.28 YLR388W 2 0.3 YML017W 26 0.62    YNL298W 6 -0.28 YJL026W 5 0.3 YOR201C 2 0.61    YLR403W 3 -0.28 YDL084W 2 0.29 YDR023W 4 0.61    YGL178W 14 -0.29 YDR171W 13 0.29 YLR276C 12 0.61    YOR276W 7 -0.29 YOR276W 6 0.29 YDL175C 7 0.6    YNL074C 2 -0.29 YGL031C 2 0.29 YGL014W 3 0.6    YBR086C 22 -0.29 YPR145W 4 0.29 YMR108W 3 0.6    YMR070W 6 -0.3 YBR140C 2 0.27 YDR353W 2 0.59    YCL037C 17 -0.32 YOL140W 9 0.24 YDR012W 6 0.59    YGR178C 25 -0.32 YNL154C 22 0.22 YOR042W 3 0.58    YDR524C 10 -0.34 YBL105C 3 0.22 YHR007C 5 0.58    YDR422C 3 -0.34 YBL085W 17 0.16 YDL182W 7 0.57    YPR065W 4 -0.35 YMR104C 13 0.16 YGL023C 2 0.57    YNL104C 2 -0.36 YBR249C 15 0.15 YDL224C 3 0.57    YDR348C 9 -0.36 YOR070C 16 0.15 YMR062C 2 0.56    YHR021C 3 -0.36 YDR150W 8 0.14 YLL013C 28 0.56    YHR073W 12 -0.37 YKL060C 8 0.1 YGL026C 4 0.56    YOL082W 2 -0.43 YNL308C 9 0.07 YGL135W 2 0.56    YGR097W 3 -0.45 YOR267C 24 0.06 YPL090C 4 0.56    YER155C 38 -0.46 YDR524C 7 0.02 YDR150W 15 0.55    YKL210W 18 -0.46 YER086W 6 -0.02 YKL198C 7 0.55    YBR238C 16 -0.47 YCL037C 15 -0.05 YBL027W 4 0.54    YER151C 6 -0.57 YBR011C 7 -0.1 YKL129C 23 0.54    YOR359W 10 -0.62 YGL178W 14 -0.1 YLR044C 19 0.54    YJR016C 2 -0.66 YDL025C 25 -0.11 YOR209C 5 0.53    YHL002W 2 -0.66 YDR348C 10 -0.11 YGL103W 12 0.53      84 S2.2a S2.2c S2.2d ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  ORF Pep Num. Log  YDL171C 2 -0.74 YEL071W 7 -0.13 YEL034W 4 0.52    YJL026W 4 -0.74 YNL321W 8 -0.24 YER155C 76 0.52    YER088C 12 -0.9 YDR234W 11 -0.29 YGR192C 8 0.52    YNR006W 3 -0.91 YNL298W 5 -0.33 YGL253W 3 0.51    YDR234W 10 -0.91 YDR293C 6 -0.36 YDR346C 2 0.51    YBL054W 7 -0.95 YJL042W 23 -0.37 YML026C 2 0.51       YBR086C 27 -0.5 YJL177W 3 0.51       YMR070W 6 -0.52 YMR116C 3 0.5       YBR238C 16 -0.56 YBL072C 7 0.49       YMR081C 7 -0.6 YDR025W 4 0.49       YDL171C 8 -0.62 YKL060C 10 0.49       YJL172W 14 -1.05 YKR075C 3 0.49    *S2.2a, S2.2c and S2.2d represent the figure panels that the data corresponding to in the supplemental data section. *ORF: yeast open reading frame; PepNum: number of peptide quantified; Log: Log2(14N/15N)    Supplementary Table 2.7 Quantitative mass spectrometry data for figure. S2.5b, d S2.5b S2.5d ORF Pep Num. Log ORF Pep Num. Log  ORF Pep Num. Log  YKR002W 3 5 YMR309C 3 5 YKL182W 11 0.02 YDL223C 74 1.13 YLR419W 3 5 YBL101C 18 0.02 YCL051W 2 1.09 YBR156C 3 5 YER088C 55 0.02 YHR183W 3 1.06 YKR101W 4 5 YKL060C 32 0.02 YER132C 2 0.93 YJL046W 4 5 YDR293C 34 0.02 YGL253W 2 0.86 YNR047W 2 5 YDR025W 39 0.02 YLR180W 8 0.85 YJL052W 2 5 YGR234W 11 0.02 YML057W 2 0.79 YPL009C 2 5 YMR205C 8 0.01 YDR394W 2 0.74 YDR172W 3 5 YOR063W 1145 0.01 YLR028C 2 0.73 YHR113W 2 5 YLR439W 4 0.01 YDL055C 6 0.71 YOL112W 3 5 YDR320C 3 0.01 YGL037C 3 0.69 YDR216W 2 5 YGL097W 2 0.01 YOL082W 10 0.68 YDL003W 2 5 YIL069C 2 0.01 YDR123C 6 0.64 YBR108W 15 5 YLR044C 82 0.01 YAL005C 4 0.64 YLR398C 3 5 YMR142C 72 0.01 YLR328W 3 0.63 YOL100W 2 5 YOR133W 39 0.01 YOR375C 7 0.6 YLL019C 2 5 YGL234W 4 0.01 YKL175W 2 0.59 YNL325C 2 5 YIL118W 2 0.01 YLR303W 13 0.58 YGR067C 2 5 YLR167W 1140 0.01 YLR342W 6 0.58 YOL019W 2 5 YGL014W 5 0 YCL061C 8 0.53 YLL054C 3 5 YPL188W 2 0 YEL060C 57 0.52 YOL081W 19 5 YNL055C 4 0 YDL066W 2 0.52 YJL201W 2 5 YPL131W 16 0 YOR133W 5 0.49 YBR215W 3 5 YHR064C 25 0 YHR174W 29 0.47 YOR073W 2 5 YHR010W 60 0   85 S2.5b S2.5d ORF Pep Num. Log ORF Pep Num. Log  ORF Pep Num. Log  YDL126C 4 0.46 YBR136W 2 5 YJR145C 262 0 YMR038C 3 0.46 YIL095W 2 5 YDR477W 79 0 YDR533C 2 0.46 YNL018C 2 5 YGL076C 2 0 YGR254W 3 0.44 YCL029C 2 5 YBR249C 39 0 YML028W 8 0.44 YML048W 2 5 YMR242C 108 0 YKL210W 2 0.42 YPL249C 2 5 YGR085C 24 -0.01 YJR009C 3 0.41 YIL136W 2 5 YOR335C 16 -0.01 YLR044C 45 0.41 YGR271W 3 5 YPL004C 3 -0.01 YCR031C 7 0.4 YBL003C 3 5 YGR086C 17 -0.01 YNR006W 6 0.4 YLR422W 2 5 YJL153C 3 -0.01 YDR171W 205 0.39 YNL012W 2 4.81 YMR108W 53 -0.01 YDR475C 4 0.38 YNL250W 4 4.73 YLR058C 84 -0.01 YJR017C 6 0.38 YLL029W 2 4.57 YDL060W 4 -0.02 YPL081W 2 0.38 YEL066W 3 4.27 YPL090C 36 -0.02 YHL015W 4 0.37 YOL098C 2 4.08 YKL129C 23 -0.02 YGR124W 69 0.36 YDL174C 2 4.05 YOR136W 4 -0.02 YBR059C 11 0.35 YNL027W 2 4.03 YCR012W 95 -0.02 YDR432W 18 0.35 YDR168W 3 4.03 YEL034W 45 -0.02 YER080W 29 0.34 YOR254C 2 3.88 YOR361C 6 -0.02 YDR477W 41 0.34 YGR042W 2 3.84 YBR127C 9 -0.02 YPR154W 3 0.33 YDR337W 2 3.47 YKL081W 14 -0.02 YOR063W 117 0.33 YGR185C 2 3.45 YLR075W 95 -0.02 YGR136W 5 0.32 YJL073W 2 3.45 YBR255W 12 -0.02 YAL041W 2 0.32 YDL143W 2 3.42 YGR027C 9 -0.02 YGR240C 5 0.32 YNL054W 2 3.33 YHR190W 2 -0.02 YAL038W 40 0.31 YDR430C 2 3.2 YLR185W 49 -0.02 YPL061W 4 0.31 YLR045C 3 3.1 YLR441C 22 -0.03 YGR192C 24 0.31 YBR140C 2 2.14 YML017W 19 -0.03 YBR255W 16 0.3 YCL014W 3 1.9 YDL022W 10 -0.03 YGL008C 20 0.29 YPL209C 2 1.89 YDL061C 46 -0.03 YML097C 2 0.29 YGR204W 14 1.8 YOR171C 6 -0.03 YCR012W 45 0.28 YLR310C 2 1.79 YPR035W 35 -0.03 YBR191W 3 0.27 YFR009W 3 1.75 YPL160W 2 -0.03 YOL121C 2 0.27 YLR106C 6 1.72 YOR341W 16 -0.03 YJL042W 39 0.27 YJL012C 3 1.59 YBL092W 349 -0.04 YKL152C 6 0.27 YMR037C 4 1.5 YFL039C 43 -0.04 YBR010W 4 0.27 YGR165W 4 1.46 YPR145W 19 -0.04 YKR094C 576 0.26 YDL058W 6 1.46 YER082C 14 -0.04 YOR209C 10 0.25 YIL149C 5 1.46 YLL011W 11 -0.04 YBL087C 2 0.25 YCR042C 2 1.44 YDL083C 18 -0.04 YOR207C 4 0.25 YOR207C 8 1.37 YNL067W 5 -0.05 YLR249W 14 0.24 YFL008W 2 1.21 YGL123W 20 -0.05 YOL086C 11 0.24 YEL046C 6 1.19 YHL002W 2 -0.05 YEL071W 6 0.24 YER003C 2 1.12 YIR034C 24 -0.05 YNL154C 38 0.23 YBL004W 5 1.11 YOR375C 11 -0.05 YMR242C 5 0.22 YGR155W 16 1.02 YBR172C 4 -0.05 YMR104C 24 0.21 YMR109W 2 1.01 YKR094C 3 -0.05 YML017W 174 0.21 YER013W 3 1.01 YBR025C 29 -0.05 YPR145W 34 0.2 YNL241C 2 0.97 YGL253W 21 -0.05 YPR080W 58 0.2 YLR431C 3 0.97 YOL121C 16 -0.05 YML008C 4 0.2 YCL030C 8 0.94 YJR016C 9 -0.05   86 S2.5b S2.5d ORF Pep Num. Log ORF Pep Num. Log  ORF Pep Num. Log  YBR011C 5 0.19 YLR304C 2 0.91 YGL045W 3 -0.05 YLR058C 65 0.19 YDR035W 8 0.87 YPL249C-A 22 -0.05 YLR027C 4 0.19 YOR158W 2 0.87 YOR185C 5 -0.05 YHR208W 2 0.19 YOR317W 9 0.86 YJL042W 66 -0.05 YBL024W 4 0.19 YDL171C 12 0.85 YCL009C 12 -0.05 YGL103W 584 0.17 YDR311W 2 0.85 YBL087C 18 -0.05 YIL051C 23 0.17 YGR173W 4 0.84 YLR298C 8 -0.06 YDR158W 2 0.17 YJL008C 2 0.82 YIL052C 40 -0.06 YIR034C 11 0.17 YOR227W 2 0.8 YGL147C 15 -0.06 YDL185W 19 0.15 YBR102C 2 0.78 YDR432W 2 -0.06 YER155C 145 0.15 YLR205C 2 0.78 YLL010C 2 -0.06 YKL129C 6 0.15 YGR240C 22 0.76 YOR201C 6 -0.06 YER151C 18 0.14 YLR355C 5 0.75 YDR234W 38 -0.06 YDR457W 2 0.13 YOR151C 4 0.66 YOL059W 93 -0.06 YBR084W 5 0.13 YDR389W 2 0.66 YBR029C 4 -0.06 YBL092W 11 0.12 YBR191W 4 0.66 YHR020W 2 -0.07 YDL182W 15 0.12 YJL083W 2 0.62 YHL001W 16 -0.07 YBR135W 13 0.12 YPR154W 3 0.6 YOR128C 2 -0.07 YEL046C 2 0.12 YML111W 3 0.6 YHR021C 23 -0.07 YLR340W 3 0.12 YML008C 7 0.57 YDL192W 2 -0.07 YDR524C 6 0.11 YBR072W 9 0.55 YCL037C 34 -0.07 YDL060W 9 0.11 YOR187W 3 0.53 YFL018C 5 -0.07 YBR238C 3 0.11 YHR216W 2 0.51 YER043C 36 -0.07 YGR178C 78 0.1 YDL136W 6 0.49 YPR041W 9 -0.08 YJL012C 2 0.1 YDR500C 12 0.49 YGL008C 57 -0.08 YOL019W 2 0.1 YGL129C 5 0.48 YLR303W 23 -0.08 YDL014W 3 0.1 YOR027W 7 0.48 YCL017C 4 -0.08 YCL037C 92 0.1 YDR475C 24 0.48 YHR108W 8 -0.08 YIL018W 70 0.1 YDL075W 25 0.47 YLR371W 55 -0.08 YOR042W 6 0.09 YKL035W 6 0.47 YIL018W 243 -0.08 YDR293C 23 0.09 YJL167W 8 0.46 YLR022C 3 -0.08 YOL040C 2 0.08 YMR031C 6 0.45 YGL245W 32 -0.08 YJR123W 3 0.08 YLR109W 6 0.45 YLR448W 6 -0.09 YER183C 3 0.08 YGR157W 2 0.44 YLL013C 63 -0.09 YEL026W 2 0.08 YML063W 10 0.44 YLR153C 20 -0.09 YMR142C 3 0.07 YMR012W 6 0.43 YDL229W 128 -0.09 YML026C 5 0.06 YDL122W 3 0.43 YER165W 74 -0.09 YOR267C 159 0.06 YLL026W 5 0.41 YHR027C 2 -0.09 YOL059W 115 0.06 YJL130C 9 0.41 YFR032C-A 23 -0.09 YMR108W 2 0.06 YER125W 13 0.4 YAR018C 2 -0.09 YDL131W 2 0.06 YMR120C 18 0.39 YBR086C 79 -0.09 YOR341W 3 0.06 YPL240C 4 0.39 YJL005W 4 -0.1 YOL087C 32 0.05 YCL061C 7 0.38 YPR080W 180 -0.1 YML016C 36 0.04 YOL086C 51 0.37 YER080W 94 -0.1 YKL060C 5 0.04 YJL014W 4 0.35 YGL048C 11 -0.1 YDR348C 28 0.03 YHR042W 8 0.33 YJL080C 62 -0.1 YJL005W 15 0.03 YML039W 9 0.32 YGR220C 15 -0.1 YMR012W 4 0.03 YFL045C 12 0.32 YDR379W 3 -0.1 YGR149W 11 0.02 YNL154C 36 0.32 YDR510W 10 -0.1 YER091C 28 0.01 YKL152C 17 0.31 YBR011C 7 -0.1 YMR189W 5 0 YKL198C 4 0.3 YLR180W 50 -0.1   87 S2.5b S2.5d ORF Pep Num. Log ORF Pep Num. Log  ORF Pep Num. Log  YLL011W 4 -0.01 YGR124W 24 0.3 YBR129C 9 -0.1 YOL007C 2 -0.01 YPL231W 13 0.3 YNL178W 40 -0.11 YLR075W 19 -0.01 YNL121C 25 0.29 YLR249W 117 -0.11 YOL140W 7 -0.01 YGL157W 8 0.29 YMR275C 6 -0.11 YDL025C 34 -0.01 YAL023C 5 0.28 YJR137C 25 -0.11 YPR074C 8 -0.02 YDR092W 3 0.28 YBR196C 2 -0.12 YHR021C 22 -0.02 YGL135W 15 0.28 YER052C 2 -0.12 YGL178W 10 -0.02 YHR208W 6 0.27 YJL084C 11 -0.12 YIL052C 5 -0.02 YDR292C 2 0.26 YNR050C 11 -0.12 YJR145C 7 -0.03 YBL072C 45 0.26 YGR237C 11 -0.12 YML126C 3 -0.03 YPL226W 9 0.26 YLR027C 5 -0.12 YBR249C 23 -0.06 YDR158W 5 0.25 YDR237W 4 -0.12 YMR116C 14 -0.07 YEL060C 69 0.25 YBR205W 7 -0.12 YER086W 20 -0.08 YER036C 9 0.25 YOR359W 23 -0.12 YBR129C 3 -0.08 YGR094W 5 0.24 YNR016C 53 -0.13 YPL231W 2 -0.08 YHR174W 12 0.24 YBL024W 2 -0.13 YDR037W 2 -0.08 YOL077C 2 0.24 YDR171W 77 -0.13 YBR225W 19 -0.09 YNR006W 10 0.22 YLR092W 2 -0.13 YOR276W 8 -0.1 YDR019C 10 0.21 YGR175C 4 -0.13 YOR201C 4 -0.1 YKL210W 12 0.21 YGR014W 2 -0.13 YLR371W 48 -0.11 YLR276C 6 0.21 YDL131W 3 -0.14 YOR096W 34 -0.11 YBR084W 13 0.21 YER126C 2 -0.14 YCL009C 17 -0.12 YBR143C 15 0.21 YDL126C 37 -0.14 YER088C 28 -0.12 YDR127W 10 0.21 YHR001W 2 -0.14 YBR086C 71 -0.12 YDR023W 4 0.18 YLR179C 2 -0.14 YBL027W 2 -0.13 YEL071W 21 0.18 YDR150W 15 -0.14 YBL085W 4 -0.14 YNL069C 13 0.18 YGR285C 34 -0.15 YDR237W 6 -0.15 YNL096C 125 0.18 YJR017C 23 -0.15 YGR234W 28 -0.16 YIL078W 17 0.17 YJL172W 6 -0.15 YDR150W 24 -0.16 YLR197W 4 0.17 YMR079W 7 -0.15 YHR010W 5 -0.16 YCR003W 11 0.17 YBR238C 3 -0.15 YHR097C 57 -0.2 YPL061W 34 0.17 YDR422C 2 -0.15 YOR359W 12 -0.2 YDL182W 27 0.16 YOL087C 18 -0.16 YLL013C 44 -0.24 YER086W 24 0.15 YDL055C 8 -0.16 YEL054C 5 -0.26 YLR244C 6 0.15 YNL183C 7 -0.16 YER068W 19 -0.27 YOR096W 61 0.14 YOR046C 2 -0.16 YMR120C 10 -0.28 YDR033W 3 0.14 YHL021C 2 -0.16 YBL081W 2 -0.31 YLR354C 6 0.14 YDR508C 2 -0.16 YBR039W 3 -0.33 YDL175C 6 0.13 YDR502C 5 -0.17 YBL054W 4 -0.35 YIL094C 2 0.13 YGL009C 24 -0.17 YEL034W 12 -0.36 YBL027W 104 0.13 YER090W 6 -0.17 YDR232W 2 -0.36 YOR116C 4 0.12 YHL034C 3 -0.17 YNL096C 12 -0.38 YGL195W 5 0.12 YKR039W 40 -0.17 YJL045W 2 -0.44 YBR009C 6 0.12 YDR205W 4 -0.18 YHR148W 5 -0.6 YBR079C 11 0.12 YCL012C 2 -0.18 YNL103W 8 -0.64 YIL053W 16 0.12 YKL150W 2 -0.18 YDR234W 20 -0.65 YOR204W 33 0.12 YHR072W-A 2 -0.18 YBL002W 17 -0.68 YNL308C 27 0.11 YDL013W 8 -0.19 YDR155C 18 -1.07 YBR010W 4 0.1 YBR179C 2 -0.19 YHR073W 4 -1.24 YGL031C 15 0.1 YLR028C 4 -0.19 YOL083W 3 -2.17 YKL175W 2 0.1 YBR177C 4 -0.19   88 S2.5b S2.5d ORF Pep Num. Log ORF Pep Num. Log  ORF Pep Num. Log  YJL172W 6 -3.07 YHL015W 52 0.1 YOR018W 4 -0.19    YER081W 4 0.1 YNL180C 4 -0.2    YNL061W 7 0.1 YBL054W 8 -0.2    YOL120C 99 0.1 YDL025C 88 -0.2    YDR224C 9 0.09 YPR163C 3 -0.21    YFR053C 2 0.09 YML016C 24 -0.21    YNL112W 5 0.09 YER068W 40 -0.21    YBL085W 30 0.09 YCR030C 26 -0.21    YLR406C 4 0.09 YNL104C 5 -0.22    YGR118W 28 0.08 YJR059W 20 -0.22    YPL117C 3 0.07 YOR267C 116 -0.22    YJL190C 28 0.07 YML072C 2 -0.22    YGR034W 11 0.07 YGL125W 3 -0.23    YDL185W 23 0.07 YGL178W 44 -0.23    YHR141C 40 0.07 YDL223C 18 -0.23    YOL040C 30 0.07 YMR104C 24 -0.23    YCR031C 27 0.07 YBR225W 51 -0.23    YBL076C 3 0.07 YDR388W 15 -0.24    YBL030C 20 0.07 YCR009C 11 -0.24    YER091C 32 0.07 YML097C 7 -0.24    YKL180W 37 0.07 YDR524C 5 -0.25    YKR021W 3 0.06 YDR348C 3 -0.25    YML024W 47 0.06 YOR023C 9 -0.25    YIL117C 4 0.06 YER155C 91 -0.25    YER177W 3 0.06 YER151C 63 -0.26    YPR165W 8 0.06 YPL221W 2 -0.27    YMR189W 8 0.06 YLR248W 18 -0.27    YPR074C 4 0.06 YKL126W 7 -0.28    YNL010W 2 0.06 YBR218C 2 -0.28    YOR276W 18 0.06 YNL173C 2 -0.28    YLR048W 6 0.06 YBR026C 2 -0.29    YPL143W 39 0.05 YMR022W 2 -0.3    YHR148W 22 0.05 YBR294W 9 -0.3    YDR064W 23 0.05 YDR266C 10 -0.3    YML126C 3 0.05 YOR065W 3 -0.31    YBR039W 2 0.05 YOR125C 2 -0.31    YNL103W 8 0.05 YIL033C 4 -0.31    YJR123W 25 0.04 YLL024C 72 -0.32    YDR164C 5 0.04 YOR117W 2 -0.33    YER131W 41 0.04 YOR070C 30 -0.33    YPL079W 191 0.04 YLR342W 29 -0.33    YGR192C 59 0.04 YGL105W 17 -0.34    YPR043W 27 0.04 YPR183W 2 -0.35    YOL140W 3 0.04 YJL026W 21 -0.36    YOR168W 2 0.04 YDR003W 2 -0.36    YBR189W 38 0.04 YGR149W 2 -0.36    YOL127W 11 0.04 YOR198C 31 -0.37    YDL198C 2 0.04 YBR170C 2 -0.38    YDR050C 9 0.04 YER143W 7 -0.38    YKL056C 23 0.03 YAL005C 8 -0.38    YLR029C 61 0.03 YDR155C 20 -0.4   89 S2.5b S2.5d ORF Pep Num. Log ORF Pep Num. Log  ORF Pep Num. Log     YEL054C 27 0.03 YPL106C 73 -0.42    YHR183W 10 0.03 YDL124W 9 -0.42    YDR012W 2 0.03 YGR097W 25 -0.43    YBR031W 51 0.03 YGL254W 4 -0.43    YDR037W 65 0.03 YDR497C 8 -0.43    YLR388W 15 0.03 YHR097C 49 -0.44    YLR328W 11 0.03 YPL145C 3 -0.45    YOR182C 13 0.03 YMR070W 3 -0.45    YML026C 137 0.03 YOR134W 8 -0.46    YKL127W 4 0.03 YHR205W 3 -0.46    YGR254W 73 0.03 YGR178C 580 -0.55    YGL103W 378 0.03 YPR065W 2 -0.56    YPL048W 5 0.03 YGR032W 2 -0.79    YLL045C 66 0.03 YHR007C 3 -1.29    YAL038W 166 0.03 YPR091C 3 -3.37    YEL013W 3 0.02 YLR340W 7 -5 *S2.5b and S2.5d represent the figure panels that the data corresponding to in the supplemental data section. *ORF: yeast open reading frame; PepNum: number of peptide quantified; Log: Log2(14N/15N)    90 Chapter 3: The HECT ubiquitin ligase Rsp5 leads a major PQC degradation pathway that targets cytosolic proteins misfolded due to HS  3.1 Introduction PQC pathways have evolved in eukaryotic cells to either refold or eliminate misfolded polypeptides that threaten cell integrity due to their high propensity to aggregate[6, 8, 146]. The importance of these PQC pathways is underscored by the numerous conformational pathologies associated with protein misfolding or aggregation like Parkinson’s disease and the transmissible spongiform encephalopathies caused by the prion protein. Several compartmentalized degradation PQC pathways have been identified in which E3 ubiquitin ligases selectively target misfolded proteins for degradation by the proteasome, often with the help of chaperones to mediate substrate recognition[147, 148]. These degradation pathways, especially in the cytosol, are often directly competing with other components of the folding machinery. The HS response is a major system that protects the cell from perturbations causing protein misfolding[149, 150]. In addition to upregulating HS proteins (Hsp), pioneering work showed that HS also causes higher ubiquitylation levels and increased proteasomal degradation in eukaryotic cells[151-153]. We showed in the previous chapter that the Hul5 proteasome-associated ubiquitin ligase is in part responsible for this   91 response; however, the major PQC pathway in this case remained elusive. More importantly, it is unclear how misfolded proteins that are destined for proteolysis are recognized and triaged under stress conditions, while most chaperone proteins are presumably sequestered by other misfolded polypeptides.  In this chapter, we present a novel PQC pathway in which the Rsp5 HECT ligase targets cytosolic misfolded proteins for proteasomal degradation upon HS. Rsp5 is the only yeast member of the Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) family of E3 ubiquitin ligases, which are characterized by the presence of a C-terminal catalytic HECT domain and several WW domains that mediate substrate recognition or recruitment of substrate-adaptor proteins containing PY motifs[41]. Rsp5 regulates several key cellular processes including endocytosis, unsaturated fatty acid and sterol synthesis, nuclear export of mRNAs related to the HS response, and transcription[154-157]. Rsp5 has also recently been shown to target misfolded plasma membrane proteins for lysosome degradation[74, 158]. In addition, the mammalian Nedd4 targets for degradation α-synuclein, which is the main constituent of Lewy bodies associated with Parkinson’s disease[159]. Correspondingly, rsp5 mutant yeast cells display reduced fitness upon overexpression of α-synuclein. Recently, a small molecule reducing α-synuclein toxicity in both yeast and higher eukaryotes was found to target Rsp5, indicating that Rsp5/Nedd4 ubiquitin ligase has a great potential for therapeutics[160]. However, the exact mechanism of the compound remains elusive and a broader function of Rsp5 in PQC has not yet been assessed. We found that Rsp5 targets, together with the deubiquitinases Ubp2 and Ubp3, cytosolic misfolded proteins for proteasomal degradation. Our work shows that Rsp5 has a broad role in PQC and   92 provides insights into the recognition mechanism of misfolded proteins that involves both Rsp5 PY-binding motifs and the association of the Ydj1 Hsp40 co-chaperone protein.              93 3.2 Methods 3.2.1 Yeast strains, plasmids and reagents All Saccharomyces cerevisiae strains (S288C background) and plasmids are listed in Supplementary Table 3.1 and 3.2 respectively. Temperature sensitive (ts) mutant strains rsp5-1, rsp5-sm1 and rsp5-3 were generously provided by Dr. Charlie Boone from the yeast ts conditional mutant collection. Mutant strains from this collection were generated by integrating the ts alleles into their endogenous loci in the BY4741 background strain (MATa, his3-1; leu2-0; ura3-0; met15-0)[161]. The strain in which Rsp5 expression is controlled by the Tet-Promoter (Tetp::RSP5) was purchased from the Yeast Tet-Promoter Hughes Collection (yTHC; MATa, URA3::CMV-tTA, his3-1, leu2-0, met15-0) distributed by Open Biosystems (Thermo Scientific)[162]. All the single deletion strains were from the Yeast Knockout (YKO) Collection (Open Biosystems, Thermo Scientific) and kindly provided by Dr. Michel Roberge with the exception of ubp2Δ and ubp10∆, which was generated by PCR-based homologous recombination strategy[163]. The art1-10∆ and art1-8,10∆ deletions strains in the BY4741 background were kindly provided by Dr. Pelham[164]. All the double mutant strains were generated by mating and tetrad dissection.  All SILAC mass spectrometry experiments were done in BY4742 background cells (MATα, his3Δ-1, leu2Δ-0, lys2Δ-0, ura3Δ-0). YDJ1-TAP was kindly provided by Dr. Elizabeth Conibear from the Yeast TAP-Tagged ORFs Collection (Open Biosystems, Thermo Scientific). CDC19-3HA, PDC1-3HA and SUP45-3HA were generated by inserting the 3xHA tag at the 3′ end of the endogenous genes by homologous recombination[163]. The guk1-7 and rpt6-20 ts mutants were generously   94 provided by Dr. Phil Hieter and were generated by random PCR mutagenesis[165]. The rsp5-1, guk1-7-13MYC strain was generated by mating the Guk1-7-13MYC strain which was previously described[166] with rsp5-1. MUP1-GFP and CAN1-GFP strains are from Yeast-GFP Clone Collection (Invitrogen) and kindly provided by Dr. Phil Hieter. Plasmids expressing RSP5 (BPM587) and rsp5-C777A (BPM588) were obtained by first subcloning the GALp into pRS426 (URA3, 2µ) using EcoRI and NaeI, and then subcloning HA-RSP5 and HA-rsp5-C777A that were generously provided by Dr. Jon Huibregtse using NaeI and NotI. The GST-Rsp5 (BPM98) in pGEX-6P-1 was previously described[167]. GST-rsp5-C777A (BPM514) was generated by subcloning rsp5-C777A to replace a fragment of RSP5 in BPM98 using NotI and AfeI restriction sites. Rsp5-WW1,2,3* (in which all three WW domains are mutated)[168] was kindly provided by Dr. Teresa Zodalek and subcloned with primers containing EcoRI and SalI restriction sites to replace RSP5 in BPM98 to generate GST-rsp5-WW1,2,3* (BPM518). The Cdc19-13MYC (BPM563) and Pdc1-13MYC (BPM565) plasmids were generated by PCR amplifying CDC19 or PDC1 from genomic DNA and inserting them into a pRS313 plasmid with GPD promoter, 13xMYC tag and PGK terminator sequence (BPM173) using BamHI and XmaI restriction sites. The Cdc19-PY/AA-13MYC plasmid (BPM564) was obtained by site-directed mutagenesis of BPM563 so that P363 and Y365 were mutated to alanine residues. Pdc1-PY1-13MYC (BPM566), Pdc1-PY2-13MYC (BPM567) and Pdc1-PY1/2-13MYC (BPM568) were similarly generated to mutate P500, Y502 (PY1) and P541, Y543 (PY2) to alanine residues. The GFP-Sup45 (BPM580) was obtained by PCR amplifying SUP45 from genomic DNA and insert into pRS313-GPDp-EGFP-PGKt (BPM519). guk1-7-GFP was produced by cloning a guk1-7 fragment   95 amplified from genomic DNA into pRS313-GPDp-GFP(S65T)-PGKt (BPM241) using BamHI and XbaI. pRS316-RSP5 (BPM573) was constructed by inserting a PCR product of the RSP5 gene including 5’ and 3’ regions into pRS316 using XbaI and SmaI. The resulting plasmid was then subject to site directed mutagenesis to produce rsp5-C777A (BPM575). The K48-only ubiquitin (BPM591) and K63-only ubiquitin (BPM592) plasmids were generated by PCR amplifying K0-Ub from the plasmid LHP306-Ubiquitin-noLys (BPM435; in which all 7 lysine residues are replaced by arginine) purchased from Addgene with forward primer that contains the MYC tag sequence, followed by site-directed mutagenesis to revert R48 to K48 and R63 to K63, respectively and then by subcloning in a pRS313 plasmid with GPD promoter and PGK terminator (BPM171). The 13MYC-MUP1 (BPM596) and 13MYC-CAN1 (BPM595) plasmids were generated by PCR amplification from MUP1 and CAN1 containing plasmids kindly provided by Dr. Elizabeth Conibear and inserted into BPM173 (pRS313 with GPD promoter, 13xMYC tag and PGK terminator sequences) using the NotI site. The YDJ1 plasmid (BPM390) was obtained from Elizabeth Craig, which was then modified by site-directed mutagenesis to mutate P317 and P319 to glycine residues to generate ydj1-PY/GG (BPM569). The magaprimer method used to generate HA-tagged YDJ1 and ydi1-PY/GG was adapted from a previously descripted method[169].  Briefly, the 3xHA sequence was PCR amplified using pFA6a-3xHA-His3MX6 to generate the megaprimer, used to PCR amplify BPM390, BPM569 prior to DpnI digestion and bacteria transformation. The LSM7-TAP, H8-ubiquitin plasmid (BPM297) was generated by PCR amplifying LSM7 with its endogenous promoter (+570bp) and C-terminal TAP sequence and inserted into pRS316-GPDp-H8-ubiquitin-PGKt (BPM30) using NotI site.    96 Mouse monoclonal anti-ubiquitin antibody MAB1510 (Millipore) was used for assessing ubiquitin levels by western blot and quantitative dot-blot assays. The house-keeping gene PGK1 (3-phosphoglycerate kinase) was used as a loading control and was detected using rabbit polyclonal anti-Pgk1 antibodies (AP21371AF-N) from Acris Antibodies. For detection of MYC-, HA-, GFP-tagged and TAP-tagged proteins, mouse monoclonal anti-MYC (1:5,000 9E10), anti-HA (1:2,000 12CA5) antibodies from the AbLab UBC in-house facility, GFP mouse monoclonal antibody from Roche, and polyclonal anti-TAP (CAB1001) from Pierce (Thermo Scientific) were used. Goat anti-Rsp5 antibody (1:1000, sc-26193) from Santa Cruz was used. Anti-mouse-800, anti-rabbit-700 and anti-goat-800 fluorescent secondary antibodies (1:10,000; LI-COR) were detected with an Odyssey Infrared Imaging System (LiCor). Protease inhibitor cocktail was purchased from Roche and all other reagents were purchased from Sigma unless specified.  3.2.2 HS assays For direct HS experiments, overnight-saturated cultures were diluted and grown to exponential phase in YPD at 25 °C to an A600 of 1-1.5, about 2 ml of cells were then incubated at 45 °C in a thermomixer for the indicated times (typically 15 min). For some temperature sensitive strains, a pre-incubation at 37 °C was included when indicated. Overnight-saturated cultures were diluted and grown to exponential phase in YPD at 25 °C to an A600 of 1-1.5 before pre-incubation at 37 °C for 30 min followed by HS at 45 °C for 15 min. For add-back experiments using the GALp-controlled expression   97 plasmids, cells were grown in SD-URA media with 2% raffinose and 0.05% dextrose until the A600 reached 1, and 2% galactose was added for 1 hr at 37 °C to induce expression of Rsp5 or Rsp5-C777A prior to HS at 45 °C for 15 min. For the HS experiment with ectopic ubiquitin expression, cells carried out a plasmid with H8-ubiquitin expressed from the GPD promoter (BPM30). For western and dot blots, cell pellets were snap frozen right away after treatment in liquid nitrogen and washed twice with cold 1×TBS (50 mM Tris 150 mM, NaCl pH 7.5) before lysis. Lysis was carried out with glass beads in the Precellys 24 tissue homogenizer (Precellys) in pre-warmed 1 × SDS-PAGE Laemmli sample buffer without reducing agent and dye. All samples were normalized using a Bradford assay (BioRad). For dot the blot assay, 3 μl of the normalized samples (5–10 μg proteins) was spotted and dried overnight on a nitrocellulose membrane. Membranes were rehydrated with 1 × TBS and processed as other western blots. All quantitative dot blots were performed by measuring signals from three biological replicates. For each sample, the ubiquitin signal was normalized using the Pgk1 signal. When reporting the normalized ubiquitylation levels (e.g., 25 °C versus 45 °C), the signals were normalized to the averaged ubiquitylation level in the reference sample (typically WT at 25 °C) that was set to the arbitrary value of 1 and then averaged. When reporting the increase in ubiquitylation, the difference of normalized signal between two temperatures for each sample (e.g., 45 °C - 25 °C) was calculated, then averaged across the three replicates and normalized to the averaged difference in the reference sample (set to the arbitrary value of 1).     98 3.2.3 Down-regulation of Tet-Rsp5 expression by doxycycline Tetp::RSP5 and the corresponding parental background strains (R1158) were grown in YPD supplemented with 0.5% Tween-80 and 100 µg/ml doxycycline at 25 ºC until saturated and then diluted into the same media. This process was repeated three times before the last dilution, cells were then grown to mid-log phase for a direct heat-shock assay as above.   3.2.4 In vitro HS ubiquitylation assay in cell extracts MYC-ubiquitin (U-115) was purchased from BostonBiochem. Recombinant Rsp5, Rsp5-C777A and Rsp5-ww1,2,3* were purified from BL21 (DE3)  bacteria cells. After a three-hour induction with 1 mM IPTG at 25 °C, cells were lysed in cold lysis buffer (1 x PBS pH 7.3, 1 mM EDTA, 10% glycerol, 1 mM DTT, 0.5% Triton-X100, 1 mM PMSF, 1 x Protease inhibitor cocktail) by sonication. Purification procedures were following the manufacturer’s protocol. Lysate was twice passed through a 500 µl bed volume of pre-conditioned Glutathione Sepharose 4B resin (GE healthcare) by gravity at 4 °C. Resin was then washed once with lysis buffer with inhibitors, twice with lysis buffer without inhibitors and with 0.1% Triton X-100, and once with cleavage buffer (50 mM Tris-HCl pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.01% Triton-X100). Rsp5, Rsp5-C777A or Rsp5-ww1,2,3* were cleaved from the GST tag by addition of PreScission Protease (GE healthcare) to the resin in cleavage buffer at 4 °C for 5 hrs. Cleaved proteins were obtained from the flow-through and stored at -80 °C. Mid-log phase cells of both WT and rsp5-1 grown in YPD were subjected to 37 °C pre-  99 incubation for 30 min to inactivate rsp5-1. Frozen cell pellets were lysed in native lysis buffer (30 mM HEPES pH 7.4, 150 mM KOAc, 250 mM Sorbitol, 7.5 mM MgCl2, 1 mM DTT, 1mM EDTA, 1 mM PMSF, 1 x protease inhibitors cocktail, 1 mM sodium orthovandate, 2.5mM sodium pyrophosphate and 1 mM β-glycerophosphate) using mortar and pestle. Clarified lysates were aliquoted and stored in liquid nitrogen. For the HS ubiquitylation assay, cell lysates were warmed up to room temperature. 2 mM ATP and if indicated around 0.05 µg (in 0.2 µl) of Rsp5, Rsp5-C777A, Rsp5-ww1,2,3* and 2 µl of 1 µg/µl MYC-ubiquitin were added to 20 µl of cell lysate right before incubating the samples at the indicated temperatures for 10 min. Samples were mixed with 3 x SDS-PAGE Laemmli sample buffer to stop the reaction and processed for western blot analysis.  3.2.5 In vivo cross-linking and co-immunoprecipitation experiments Cells were grown at 25 °C to and A600 of 1 and then heat shocked at 45 °C for 10min before adding 1% formaldehyde and incubating for a further 10 min. The cross-linking reaction was quenched with an excess of glycine (250 mM) for 5 min at 4 °C. The samples were centrifuged, washed twice with 1 x TBS, and then frozen in liquid nitrogen. Cells were lysed with modified RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% SDS, 1 mM PMSF, 10  mM chloroacetamide, 1 mM Phenanthroline, and 1 x protease inhibitors cocktail)  using glass beads. For the TAP tagged proteins IP, Tris-HCl was replaced by 50 mM HEPES at pH 8. Lysates were then slowly diluted to 0.1% SDS final for IP. Cell pellets were also resolubilized in lysis   100 buffer containing 0.1% SDS at 4 ºC for thirty minutes and mixed with corresponding lysates, as some additional precipitations occurred in presence of 0.5% SDS. Sample concentrations were measured by Bradford assay and normalized before incubated with beads overnight at 4 °C. Beads were washed six times in lysis buffer with 500 mM NaCl and eluted with 1 x SDS buffer without reducing agent to avoid elution of cross-linked IgG. Reducing agent was added and samples were boiled prior to SDS-PAGE.  3.2.6 Yeast growth assays following HS For plate assays, exponentially growing cells were diluted to an A600 of 0.05 and 3 µl were plated in a 1/5 dilution series on YPD or SC plates. For culture fitness assay, exponentially growing cells in YPD media were diluted to an A600 of ~0.2 and incubated for 30 min at 25 °C or 45 °C and then transferred to a 96-well plate placed in an Infinite 200 PRO plate reader system (Tecan) with constant shaking at 25 °C. Three biological replicates were analyzed in the same experiments. For each well, four measurements were taken every 30min and averaged. This experiment was repeated once and we obtained similar results (data not shown).  3.2.7 35S-labeling and protein turnover assays   Yeast cells grown in SC medium with additional tyrosine (20 µg/ml) to an A600 of 0.8-1, washed and incubated in SD-Met media with additional tyrosine (20 µg/ml) for 50  min[170]. EXPRE35S35S protein labeling mix (50µCi/m1, PerkinElmer) was added for 5 min or 90 min prior to washing cells with ice-cold SC-chase media containing cysteine   101 (0.5 mg/ml), methionine (6 mg/ml) and cycloheximide (0.5 mg/ml). Cells were then incubated at either 25 °C or 38 °C and collected at the indicated times to be then mixed to a final concentration of 10% TCA. After an overnight incubation at 4 °C, radioactivity in both TCA-soluble and -insoluble fractions was measured in a MicroBeta2 radiometric detector (PerkinElmer). The percentage of protein degradation was calculated by subtracting the signal from TCA-soluble at time 0 from indicated time that was then divided by the signal in the TCA-insoluble fraction at time 0. To assess turnover of soluble proteins, around 5 A600 of radiolabeled cells were lyzed in 100 µl native buffer (50 mM Tris-HCL pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM PMSF, 10 mM chloroacetamide, 1 mM Phenanthroline, and 1 x protease inhibitors cocktail) by glass beating. After a 15 min centrifugation at 16,000 rpm in a microfuge, samples were normalized to same concentration in 90 µl. One fraction of soluble proteins (70 µl) was precipitated by TCA, while the other fraction (20 µl) was analyzed by SDS-PAGE. Radioactivity for each time point was measured in the TCA-soluble fraction and normalized to the amount of proteins detected by Coomassie on the protein gel. All experiments were performed in three independent replicates.   3.2.8 Fluorescence based degradation assays Protein stability was assessed by cycloheximide chase assays using flow cytometry. Briefly, strains were grown at the permissive temperature (25 ˚C) in supplemented minimal media to exponential phase (A600 between 0.8 and 1.0). Cells were then incubated at the permissive or non-permissive temperature (37 ˚C) in the presence of cycloheximide (100 µg/ml). Samples were removed at the indicated time   102 points and a total of 50,000 cell events were analyzed by flow cytometry using a Becton Dickinson FACSCalibur instrument. Data was analyzed using FlowJo. To evaluate protein levels, we used the median intensity values in which we subtracted median background signal from control cells (no GFP). All experiments were repeated independently three times.  3.2.9 Microscopy Cells were grown to an A600 of 1 at 25 °C and subjected or not to a 40 °C HS for 2 hrs. In the last 30min incubation, 2.5 µg/ml of Hoechst 33342 (Invitrogen) was added to each culture for DNA staining. Live cells were mounted on a slide with 1 x PBS and immediately imaged with an inverted Zeiss Axio Observer microscope. One image plan per field (225 µm2) was acquired with a 63 x oil objective and processed with Zeiss Zen software.   3.2.10 DiGly peptide enrichment for triple SILAC mass spectrometry analysis Only lysine (K) labeling was used for the diGly SILAC experiments. Cells were grown in small volume of SD-URA supplemented with the indicated isotopically labeled lysines (0.3 mg/L) at 25 °C until saturation, and diluted in larger volume of the same media to an A600 of 0.2. Cells were then grown to an A600 of 1. RSP5 cells labeled with light-lysine (K0) and rsp5-1 cells labeled with heavy-lysine (K8) were subjected to 20 min HS at 45 °C prior to lysis, while medium-labeled RSP5 cells (K4) were kept at 25 °C for control. Cells were lysed in 8 M urea lysis buffer (8 M urea, 50 mM Tris-HCl   103 and 150 mM NaCl at pH 7.5, 10 mM chloroacetamide, 1 x protease inhibitors cocktail, 1 mM sodium orthovandate, 2.5 mM sodium pyrophosphate and 1 mM β-glycerophosphate). Equal amount of lysate from differentially labeled cells were mixed together to obtain ~30 mg of proteins in total. The lysate was reduced in 3 mM TCEP (3,3′,3′′-Phosphanetriyltripropanoic acid) at 25 °C for 30 min and then was alkylated in 55mM chloroacetamide at 25 °C for 45 min in the dark. 50 mM Tris-HCl pH 7.6 was then used to slowly dilute lysate to final concentration of ~2 M urea with addition of 1 mM CaCl2 prior to trypsin digestion. 300 µg of trypsin (1/100) was used to digest the lysate at 30 °C for 36 hrs. Digestion was stopped by adding 1.5% formic acid and incubated at 25 °C for 10 min prior to 10 min 15,000g centrifugation at 15 °C. Acetified peptides were cleaned up by two high capacity C18 columns (Thermo). Peptides were eluted from each column using three times 2 ml 50% acetonitrile elution buffer. The PTMScan Ubiquitin Remnant Motif Kit (Cell Signaling Technology) was used to immunoprecipitate (IP) diGly peptides. Speed-vac dried peptides were resuspended in 1.4 ml PTMScan IP buffer (50 mM MOPS [NaOH] pH 7.2, 10 mM Na2HPO4, 50 mM NaCl) for 2hrs at 4 °C after brief sonication.  IP was performed according to the manufactured protocol except beads were cross-linked prior to the IP[171] and 1/8 of recommended amount of beads  was used per IP instead. Briefly, antibody-bound beads were washed three times with wash buffer (100 mM sodium borate, pH 9.0) prior to cross-linking for 30 min at 25 °C in cross-linking buffer (20 mM DMP in 100 mM sodium borate, pH 9.0). Cross-linking was stopped by washing twice with blocking buffer (200mM ethanolamine pH 8.0) and by a 2 hrs incubation in blocking buffer at 4 °C. Prior to adding lysate, beads were washed three times in IAP buffer. IP was done at   104 4 °C for 1 hr. Beads were washed twice with IP buffer and 3 times with PBS by gently inverting tubes 5 times. Bound peptides were eluted with 50 µl 0.15 % (vol/vol) Trifluoroacetic acid at 25 °C for 5 min before being cleaned up by C18 stage tips without fractionation[172].  3.2.11 IMAC-purification of ubiquitylated proteins In all experiments, about 1 μl of MagneHis (Promega) was used per 200 μg of protein extract. For HA-tagged substrates validation and the SRM mass spectrometry analysis, 150ml of cells carrying H8-ubiquitin plasmid (BPM30)[173] or corresponding empty plasmid for control were grown in SD-URA media at 25 °C. Cells with or without 20 min heat-shock treatment at 45 °C were washed twice with ice-cold 1 x TBS and cell pellets snap frozen in liquid nitrogen. Thawed cells were lysed in HU buffer (8 M urea, 100 mM HEPES at pH 8, 0.05% SDS, 10 mM chloroacetamide, 1 mM PMSF, 10 mM imidazole and protease inhibitors cocktail) by glass beads. Following 90 min incubation with cell extracts at ambient temperature, nickel beads were washed three times in HU buffer with 1% SDS. Bound proteins were eluted by incubating the beads in one volume of 8 M HU and one volume of 2 M imidazole for 10 min at ambient temperature with shaking. One volume of 3 x SDS-PAGE Laemmli sample buffer was added and samples were heated at 70 °C for 10 min before western blot analysis.    105 3.2.12 In-gel digestion of IMAC samples for ubiquitin linkage quantification by AQUA-SRM For SRM analysis, IMAC eluted samples were subjected to in-gel digestion using procedures modified from previous described method[174]. Gel bands that were above 75 kDa (poly-ubiquitylation fraction) from each sample were cut out for further processing and trypsin digestion. Briefly, gel was fixed and stained in coomassie staining solution for 30 min before washing in ddH2O for 3 hrs. Gel bands above 75 kDa were then excited and cut into small cubes for distaining 3 times in 50 mM NH4HCO3/ethanol (1:1) for 20 min. Ethanol-dehydrated gel pieces were incubated with 10 mM DTT in H2O at 56 °C for 45 min following by 30 min in 55 mM chloroacetamide (in 50 mM NH4HCO3) at ambient temperature. Ethanol-dehydrated gel pieces were incubated with trypsin solution (12.5 ng/µl in 50 mM NH4HCO3) on ice for 60 min before starting digestion at 37 °C for 18-22 hrs. Digested peptides were extracted by extraction buffer (0.5% acetic acid) with increase amount of acetonitrile till the gel pieces were completely dehydrated. Combined extracted liquid from each sample was dried and resuspended in Buffer A (0.5% acetic acid) for purification with C18 stage tips without fractionation. Equally amount of ubiquitin AQUA peptides (2.5 µl of 10 pmol/µl) 48GG, 48N, 63GG, and 63N (“N” designating the peptide containing the indicated unmodified lysine) were digested in 100 mM Tris-HCl pH 8.5 with 0.2 µl of 0.2 µg/µl trypsin at 37 °C overnight. Following C18 stage tip purification[172], equal volume of AQUA peptides-mixture was spiked in all in-gel digested samples for analysis on a G6460 triple quadrupole (QQQ) mass spectrometer (Agilent Technologies).   106 3.2.13 Mass spectrometry analysis For diGly sites mass spectrometry analysis, purified peptides were analyzed using a LTQ-Obitrap Velos (ThermoFisher Scientific) coupled to an Agilent 1290 Series HPLC using a nanospray ionization source (ThermoFisher Scientific), with a 2 cm-long (100 μm-inner diameter) fused silica trap column packed with Aqua C18 beads (5 μm-diameter; Phenomenex), 50 μm-inner diameter fused silica fritted analytical column packed with 3 μm-diameter Reprosil-Pur C18-AQ beads (Dr. Maisch, www.Dr-Maisch.com), and a 20 μm-inner diameter fused silica gold coated spray tip (6 μm-diameter opening, pulled on a P2000 laser puller from Sutter Instruments, coated on Leica EM SCD005 Super Cool Sputtering Device).  Samples were run with a 120 min or 180 min HPLC gradient. 120 min gradient ran from 100% buffer A (0.5% acetic acid), 0% B (0.5% acetic acid and 80% acetonitrile) to 32% B over 81 min (141 min for 180 min gradient), then to 40% B in the next 5 min, increased to 100% B over 2 min period, held for 2.5 min, and then dropped to 0% B for another 20 min. The HPLC system consisted of an Agilent 1290 series Pump and Autosampler with Thermostat set at 6 °C.  The sample was loaded on the trap column at 5 μl/min and analyzed at 0.1 μl/min.  The LTQ-Orbitrap Velos was set with the following parameters: full-range scan at 60,000 resolution from 350 to 1600 Th in the Orbitrap, fragmentation of top 5 ions by HCD in each cycle in the LTQ (minimum intensity 1000 counts), exclusion of singly charged ions and previously analyzed for 30 sec.  The Orbitrap was continuously recalibrated using lock-mass function[175]. Mass accuracy: error of mass measurement is typically within 5ppm and is not allowed to exceed 10 ppm.     107 For SRM analysis, peptides (K48GG, K48N, K63GG, K63N) were purchased from JPT Peptide Technology and each peptide was reconstituted in 0.1% Acetic Acid to yield 10 pmol/ul for storage at -80 ºC. C-terminal JPT-tag can be removed by trypsin digestion and peptides are labeled with terminal heavy residue (Lysine: +8; Arginine: +10). SRM analysis was done using a triple quadrupole mass spectrometer G6460 (Agilent Technologies) on-line coupled to an Agilent 1200 Series HPLC using an Agilent ChipCube nanospray ionization source.  A large capacity protein chip (Agilent Technologies) 150 mm 300 A C18 column with 160nl trap column (both with C18) was used.  Buffer A consisted of 0.1% formic acid and 3% acetonitrile in water, and buffer B consisted of 0.1% formic acid and 30% acetonitrile. 34 min gradient method adapted from a previous study was used[176], which run at 95% buffer A and 5% buffer B for 3 min, increased to 13% B in 15 min and to 25% B in 12 min, increased to and held at 80% B for 1 min, dropped to 3% B in 30 sec, and held at 3% B for another 2.5 min. The samples were loaded with an Agilent 1200 Cap Pump at 4 μl/min and the analysts were eluted with a 1200 nano-pump at 300 nl/min. The HPLC system included Agilent 1200 series Capillary and Nano Pump, two 1200 series Degassesr and an Autosampler with a thermostat set at 7 °C. The most abundant charge states of heavy and light versions of each peptide were determined empirically and used for SRM transition development as previously described and SRM transitions (fragmented ions) with the top 3 highest intensities were selected to monitor the parental ion[176]. Collision energies for each transition were optimized empirically to maximize signal and improve limits of detection and retention time for each peptide was determined by monitoring all transitions of all peptides in mixture samples during the whole 34 min gradient run (see Supplementary   108 Table 11). Peptides with defined retention times were then monitored using dynamic MRM mode with 1000 ms cycle time and delta retention time for each transition was set to 5 min (+/- 2.5 min).  3.2.14 Analysis of mass spectrometry data For data generated by the Velos instrument, centroided fragment peak lists were processed to Mascot generic format using Proteome Discoverer (Version 1.2). Fragment spectra were searched using the Mascot algorithm (2.3.0) against the Saccharomyces Genome Database (SGD-05Jan2012 with 6147 protein sequences and 6147 randomized sequences) using the following parameters: peptide mass accuracy 10 ppm, fragment mass accuracy 0.6 Da, trypsin (two miscleavages), two13C option, carbamidomethyl fixed modification (C), ESI-TRAP fragment characteristics. Each quantification channel (K0, K4 and K8) was searched individually with variable modifications for GlyGly (K), GlyGly (K) and phosphorylation (STY) or GlyGly (K) and oxidation (M) (totally 9 mascot searches for each raw file and results were merged by Proteome Discoverer and the redundant peptides were removed). Isotopic peak intensities were quantified by Proteome Discoverer. The cut-off FDR of glygly peptides was set below 1% (0.01). Similar to previous studies, we removed peptides with a C-terminus GlyGly and considered peptides with log2 (ratios) ≥ 1[177, 178]. For each dataset, peptide ratios were normalized to the median protein ratio obtained from an aliquot of the input whole cell lysate sample. The ratios of diGly peptides independently quantified several times were averaged.   109 For data generated by QQQ Peak integration was performed using MassHunter Quantitative Analysis software (Agilent Technologies, version B04.00, build 4.0.479.0), and area under the curve (AUC) was used to determine the abundance of each light peptide in the sample relative to its corresponding heavy internal standard[176].  3.2.15 Computational analyses of Rsp5 candidate substrates Localization of Rsp5, Ubp2 and Ubp3 candidate substrates was assigned according to a previous systems-wide localization analysis[179]. Candidate Rsp5 substrates induced by heat-shock (combining both proteomic experiments; 108 proteins) and high-confidence Rsp5 substrates identified in a protein array (40 proteins)[180] were used to search for PY motifs (PPXY, LPXY. PPXF, SPXF, VPXY, VPXF, PPPP and PPR) or PXY using an in-house python script (X: any amino acid). Enrichment of a given motif among Rsp5 candidates relative to its prevalence in the whole proteome was tested using Fisher’s exact test. We determined whether PY motifs  (PPXY, LPXY. PPXF, SPXF, VPXY, VPXF, PPPP and PPR) are located on disordered regions using Disopred2[181]. Solvent accessibility of motifs was predicted using Sable[182, 183] for the four individual residues in the PY motifs and was then averaged. The Mann-Whitney U test was performed to determine if the distributions of the average solvent accessibility of PY motifs in both datasets is significantly different. The Ydj1 binding motif was previously defined as GX[LMQ]{P}X{P}{CIMPVW}[184]. Fisher’s exact test was used to determine if proteins containing at least one, two or three Ydj1 motifs were enriched in the HS dataset compared to the genome.    110 3.3 Results 3.3.1 The RSP5 E3 ligase is required for the HS induced ubiquitylation response  We sought to identify the main ubiquitin ligase that mediates the increased ubiquitylation observed upon HS. We showed in Chapter 2 that acute HS leads to the ubiquitylation of mainly cytosolic proteins in yeast. As Rsp5 targets misfolded plasma membrane proteins[74] and its overexpression increases the thermotolerance of S. cerevisiae cells[185], we tested whether Rsp5 could also target cytosolic misfolded proteins upon HS. We first assessed the rsp5-1 thermo-sensitive (ts) mutant allele, which has been widely used in the past[186], by comparing levels of total ubiquitin conjugates before and after HS (45 °C, 15 min). We found that the HS-induced ubiquitylation response was markedly reduced in rsp5-1 cells in comparison to WT cells when analyzed by western blot and by a quantitative dot-blot assay (Fig. 3.1a and S3.1a). We confirmed these data using two additional ts alleles of the essential RSP5 gene (rsp5-sm1 and rsp5-3) for which the HS-induced ubiquitylation response was also significantly decreased (Fig. S3.1b). To further verify that RSP5 is involved in the HS ubiquitylation response, we repeated our experiments by reducing RSP5 expression using a doxycycline-titratable promoter. As RSP5 is essential for its regulatory role in unsaturated fatty acid synthesis[155], we supplemented cells with the oleic acid precursor TWEEN-80 to maintain cell viability. In these conditions, the increased ubiquitylation upon HS was also largely impaired in the absence of Rsp5 (Fig. 3.1b, c).  To determine whether the ubiquitin ligase activity of Rsp5 is important, we performed an add-back experiment with WT RSP5 and the catalytic-dead mutant rsp5-  111 C777A. We found that the expression of RSP5 from a plasmid rescued the HS ubiquitylation response in rsp5-1 cells, but not of rsp5-C777A (Fig. 3.1c). While lower free ubiquitin levels in rsp5-1 cells were reported[187], which could affect the increased ubiquitylation upon HS, we did not observe any change in levels of free ubiquitin in RSP5-deficient cells (Fig. 3.1a). Moreover, the ectopic expression of ubiquitin under the GPD promoter was not sufficient to restore the increased ubiquitylation in rsp5-1 cells upon HS (Fig. S3.1d). These results indicate that a functional RSP5 ubiquitin ligase is required for the ubiquitylation of proteins upon HS.      112 Figure 3.1 Functional Rsp5 is required for the HS ubiquitylation response    (a). Ubiquitylation levels in both WT and rsp5-1 cells before and after a direct HS analyzed by western blots (top) and dot blot (bellow). Free mono-ubiquitin detected on a separate protein gel and Pgk1-loading control are also shown. a.u. denotes arbitrary units (each value is relative to the averaged value of the reference sample). (b). Normalized ubiquitylation levels quantified by dot-blot in Tetp::RSP5 cells before and after HS. (c). Normalized ubiquitylation levels before and after HS in WT and rsp5-1 cells containing the indicated plasmids (- denotes presence of a control empty plasmid). Both RSP5 and rsp5-C777A were expressed under a Gal-promoter induced for 60 min with 2% Galactose at 37 °C. All experiments were done with three biological replicates and the averaged values are shown with standard deviations.     113 3.3.2 Rsp5 directly ubiquitylates heat-induced misfolded proteins We next sought to demonstrate that Rsp5 directly ubiquitylates proteins upon HS. One concern is that Rsp5 may indirectly affect ubiquitylation levels, as it regulates the nuclear export of Hsf1 and Msn2/4 mRNAs, two major transcription factors of the HS response[188]. Because the deubiquitinase Ubp2 was previously shown to specifically process Rsp5 substrates[189], we reasoned that if Rsp5 directly targets misfolded proteins, then deletion of UBP2 should lead to a more pronounced increase in ubiquitylation upon HS. Indeed, we found that deletion of UBP2 lead to a 5-fold increase in the measured accumulation of ubiquitin conjugate levels of upon HS (Fig. 3.2a). To determine how general this effect is, we assessed ubiquitylation levels in a panel of single gene deletions of 17 yeast deubiquitinases. We found that only cells lacking UBP2 or UBP3 had markedly more poly-ubiquitylation upon HS (45 °C, 5 min) compared to WT cells (Fig. S3.2a). Similar to Ubp2, the deubiquitinase Ubp3 can also antagonize Rsp5 activity, which regulates cellular level of Sec23[190]. Upon deletion of RUP1 that facilitates the tethering of Ubp2 to Rsp5[191] and BRE5 that is a co-factor of Ubp3[192], a similar increase of ubiquitin conjugate levels upon HS was observed (Fig. S3.2b). The increased ubiquitylation upon HS was also observed earlier in ubp2∆ cells in comparison to WT cells (Fig. S3.2c), in agreement with a direct role of Rsp5 in the ubiquitylation of misfolded proteins upon HS. Importantly, the increased ubiquitylation upon the deletion of the two deubiquitinases was dependent on RSP5 (Fig. 3.2a, b). In contrast, deletion of HUL5, another ubiquitin ligase involved in the HS response had only a marginal effect. These results indicate that, besides Hul5 pathway, Rsp5 is the   114 major E3 ligase that ubiquitylates proteins upon HS, and suggest that conjugated proteins are further processed by the deubiquitinases Ubp2 and Ubp3.   To further demonstrate a direct role of Rsp5, we developed an in vitro HS ubiquitylation assay in cell extracts using recombinant MYC-ubiquitin to monitor newly-catalyzed ubiquitylation events. In these conditions, we found that there was an RSP5-dependent increase in poly- ubiquitylation upon a short HS (10 min) (Fig. S3.2d). Importantly, the addition of WT recombinant Rsp5, but not the ligase-dead mutant Rsp5-C777A, restored the increased poly-ubiquitylation upon HS in extracts derived from rsp5-1 cells (Fig. 3.2c). Rsp5 possesses three WW-domains of approximately 35 amino acids each, which include two conserved tryptophan residues that bind predominately to substrates or substrate-adaptor proteins containing proline-rich (PY) motifs[193]. Addition of the triple WW-domain mutant Rsp5-WW1,2,3* failed to complement the lack of RSP5 activity in rsp5-1 cell extracts (Fig. 3.2c). Our results indicate that Rsp5 ubiquitylates heat-induced misfolded proteins and that the recognition of these misfolded proteins is mediated by Rsp5-WW domains.           115 Figure 3.2 Rsp5 directly ubiquitylates heat-induced misfolded proteins   (a)-(b). Increased ubiquitylation levels after a 15 min HS at 45 ºC in the indicated cells quantified by dot-blots. Experiments were done with three biological replicates and averaged values are shown with standard deviations. (c). In vitro ubiquitylation performed with the indicated recombinant proteins in WT and rsp5-1 cell extracts incubated at 25 °C or 42 °C (HS) for 10 min and analyzed by western blot. The asterisks denote unspecific bands. Rsp5 (with relative quantified revels) and Pgk1 loading control are also shown.   116 3.3.3 Rsp5 ubiquitylates heat-induced cytosolic misfolded proteins  To identify which proteins are ubiquitylated by Rsp5 upon HS, we used a proteomic approach in which we combined triple-SILAC (stable isotope labeling with amino acids in cell culture) analysis with antibody-based enrichment of diGly peptides (that correspond to ubiquitylated peptides)[194]. In this experiment, we compared ubiquitylated proteins in WT cells that were or not subjected to a HS treatment (light and medium SILAC-labeled, respectively) and HS treated rsp5-1 cells (heavy-labeled) to unequivocally distinguish which proteins are ubiquitylated upon HS in an RSP5-dependent manner (Fig. 3.3a). We found that about 80% (130/163 sites and Sup. Table 3.3) of the diGly sites that were further enriched upon HS were affected by the rsp5 mutation (≥2 fold; Fig. 3.3b and S3.3a). These results confirm that Rsp5 plays a major role in the heat-induced ubiquitylation of proteins. To validate the proteomic analysis, we selected three Rsp5-candidate substrates: the Cdc19 pyruvate kinase, the Pdc1 pyruvate decarboxylase and the Sup45 translation release factor 1. We used an orthogonal approach, in which conjugated proteins were enriched from cells expressing the H8-ubiquitin by IMAC for western blot analysis. We found that for all three substrates tested there was a marked increase in poly-ubiquitylation upon HS in WT cells, which was readily reduced in rsp5-1 cells (Fig. 3.3c). We found that the majority of the 82 proteins ubiquitylated upon HS in an RSP5-dependent manner were cytosolic (Fig. 3.3d).  We confirmed this trend in a second independent experiment (Fig. S3.3a, b). Our results indicate that Rsp5 is the main E3 ligase that ubiquitylates misfolded cytosolic proteins upon HS.    117 Consistent with a role in targeting cytosolic proteins, we found that the HS ubiquitylation response mediated by Rsp5 is distinct from the plasma membrane surveillance system that targets misfolded plasma membrane proteins for lysosome degradation. This pathway relies on a network of arrestin-related trafficking adaptor (ART) proteins to mediate the ubiquitin-dependent endocytosis of misfolded plasma membrane proteins[74]. Deletion of all yeast ART proteins (art1-10∆) does not inhibit the increase in levels of ubiquitin conjugates upon HS (Fig. S3.3c). Furthermore, deletion of the N-terminal C2 domain of Rsp5, which is required for Rsp5’s function in sorting cargo into MVB vesicles[195], does not impair RSP5 function in the HS ubiquitylation response in an add-back experiment (Fig. S3.3d). These data indicate that the role of Rsp5 in the increased ubiquitylation induced by HS is a novel function of this ubiquitin ligase distinct from its role at the plasma membrane.   We previously found in chapter two that the HECT ligase Hul5 ubiquitylates a fraction of cytosolic misfolded proteins, including the prion-like protein Pin3 that was also identified as an Rsp5 substrate[196, 197]. In this case, our data suggests that Rsp5 primarily adds the first ubiquitin moiety that is then further conjugated by Hul5, which acts as an E4-elongating enzyme (data not shown). One possibility is that both ubiquitin ligases together target the same pool of proteins. However, we found that the defect of the HS-response in the double hul5 and rsp5 mutant (hul5∆, rsp5-1) was additive compared to the single mutants, and strikingly caused a complete abolition of the accumulation of ubiquitin conjugates upon HS (Fig. 3.3e and S3.3e). If Hul5 was solely an E4 targeting Rsp5 substrates, its deletion should not have aggravated the rsp5-1 phenotype. To confirm these data, we assessed the ubiquitylation levels of the three   118 Rsp5 substrates in hul5∆ cells. While ubiquitylation of Pdc1 was also HUL5 dependent, ubiquitylation of both Cdc19 and Sup45 was not affected by HUL5 deletion (Fig. 3.3f). Moreover, ubiquitylation of Lsm7, another Hul5 substrate, was not impaired in rsp5-1 cells (Fig. S3.3f). These results first indicate that Rsp5 and Hul5 are the two main ubiquitin ligases responsible for the accumulation of ubiquitin conjugates upon HS. In addition, these data imply that each ubiquitin ligase targets, in part, two different pools of proteins while they also share some substrates (e.g., Pdc1 and Pin3).   119 Figure 3.3 Rsp5 targets cytosolic proteins upon HS   120 (a). Schematic representation of the triple SILAC experiment to identify HS-induced Rsp5 substrates (L:light; M:medium; and H: high labels). (b). Plot of the log2 ratios of each quantified ubiquitylated peptide. In the y-axis, ratios of L versus M are reported to display sites affected by HS, and ratios of L versus H are reported in the x-axis to indicate sites affected by the absence of Rsp5 activity. RSP5-dependent ubiquitylation sites are indicated in green. The are 69 overlapping sites with log2 (ratios) ≥ 5.64. (c) and (f). IMAC-enriched ubiquitin conjugates were analyzed by western blot. Samples were derived from indicated cells (- RSP5 using rsp5-1 cells) was assed expressing endogenously tagged candidates (3xHA) and H8-Ubiquitin. HS was applied to all samples in (f). (d). Localization of HS-induced Rsp5 substrates identified in C (C: Cytoplasm; N: Nuclear; M: Membrane; Mit: Mitochondria). (e). Increased ubiquitylation levels quantified by dot-blot after a 15 min HS at 45 ºC in the indicated cells. Experiments were done with three biological replicates and averaged values are shown with standard deviations.   121 3.3.4 Rsp5 is required for the degradation of cytosolic misfolded proteins We next sought to identify the role of Rsp5-mediated ubiquitylation of cytosolic misfolded proteins. Degradation of short-lived proteins by the proteasome is markedly augmented upon increased misfolding due to a mild HS[153]. We found that the increased degradation rate of short-lived proteins induced in these mild HS conditions was abolished in rsp5-1 cells (Fig. 3.4a). In contrast, degradation of short-lived proteins at 25 °C was not markedly affected. Interestingly, degradation of 35S-labeled misfolded proteins is only partially affected by hul5∆ in the same conditions, indicating that Rsp5 is the major ubiquitin ligase targeting misfolded proteins for degradation upon HS. We confirmed that, in our experimental conditions, the increased degradation of 35S pulsed-labeled proteins upon HS was proteasome-dependent in RSP5 cells and not affected by deletion of the two major lysosome proteases (Fig. S3.4a, b). We also observed that the increased degradation of long-lived proteins (90 min labeling) upon HS was abrogated in rsp5-1 cells (Fig. 3.4b). Consistent with a role in targeting long-lived proteins, the increased ubiquitylation upon HS in cells pre-treated with cycloheximide (that blocks translation, thereby allowing the depletion of short-lived proteins) was also affected in rsp5-1 cells (Fig. S3.4c).  To further demonstrate a role for Rsp5 in targeting cytosolic misfolded proteins for degradation, we isolated a cytosolic fraction prior to quantitation of radio-labeled proteins using a lysis buffer lacking detergent to deplete membrane associated-proteins (Fig. S3.4d). In these conditions, the half-life of the pulse-labeled proteins was distinctly lower upon HS in WT but not in rsp5-1 cells (Fig. 3.4c).  These   122 results indicate that RSP5 is essential for the HS-induced degradation of cytosolic proteins.  A large portion of candidate substrates identified in the proteomic approach are abundant proteins, and typically only a small portion of any given protein is found ubiquitylated upon HS. Therefore, the half-lives of these proteins are not expectedly dramatically altered upon HS. To further confirm the role of Rsp5 in cytosolic PQC, we instead determined its role in the degradation of a model substrate. We previously identified nine ts alleles of genes encoding for cytosolic proteins that are degraded by the proteasome upon misfolding[84]. While degradation of three of these mutants is affected by the E3 Ubr1, which is involved in cytosolic PQC[198], turnover of other model substrates remained uncharacterized. We found that the degradation of a mutant of Guk1 guanylate kinase (Guk1-7) was reduced in rsp5-1 cells (Fig. 3.4d). To better quantify Guk1-7 degradation, we adapted a fluorescence-based assay to directly monitor levels of Guk1-7-GFP in the cells (Fig. S3.4e). We confirmed that the degradation of the mutant protein was affected in the three different rsp5 mutants (Fig. S3.4f), and importantly, that the rapid turnover of Guk1-7 in rsp5-1 cells can be re-established by adding back a plasmid encoding for the WT RSP5 but not the rsp5-C777A inactive mutant (Fig. 3.4e). These data strongly support a broad role of Rsp5 in the degradation of cytosolic misfolded proteins.   123 Figure 3.4 Rsp5 is required for the degradation of cytosolic misfolded proteins   124 (a)-(c). Degradation of 35S labeled proteins in WT (grey) and rsp5-1 (green) cells at 25 °C (dotted lines) or 38 °C (solid lines). The portion of proteins degraded after the chase at the indicated times was measured for short-lived ‘a’ and long-lived ‘b’ proteins. The remaining percentages of soluble short-lived proteins that mainly correspond to cytosolic proteins was measured at the indicated times ‘c’. (d). The turnover of Guk1-7 expressed from its endogenous locus and fused to a C-terminal 13xMYC tag was assessed by western blot upon the addition cycloheximide in cells shifted to 37 ºC. Normalized protein levels measured with the infrared scanner are shown below. (e). Fluorescent intensities of Guk1-7 fused to GFP that is expressed from a first HIS3 plasmid in RSP5 (grey) and rsp5-1 (green) cells that also contain a second empty URA3 control plasmid (dotted), or in rsp5-1 cells (straight) containing the URA3 plasmid expressing RSP5 (grey) or rsp5-C777A (green). Fluorescent intensities were measured by flow cytometry after shifting the cells to 37 °C with cycloheximide at the indicated time points. All experiments were done in three independent experiments and averaged values are shown with standard deviations.   125 3.3.5 The deubiquitinases Ubp2 and Ubp3 are required for the degradation of misfolded protein and their absence reduces cell fitness upon HS We reasoned that deletion of UBP2 or UBP3 could accelerate the degradation of HS induced Rsp5 substrates, based on the observed increased ubiquitylation in these mutant cells (Fig.3. 2a, b). To our surprise, deletion of either UBP2 or UBP3 abrogated the heat-induced degradation of short-lived proteins (Fig. 3.5a). One possibility is that deletion of these deubiquitinases could lead to a change of Rsp5-substrate specificity, e.g., by allowing more ubiquitylation of membrane proteins (that would then not be rapidly degraded by the proteasome). To test this possibility, we used a triple SILAC mass spectrometry approach to identify which ubiquitylated proteins are affected by the absence of Ubp2 or Ubp3 (Fig. S3.5a). After sub-categorizing the quantified ubiquitylated sites, we found 154 sites (48%) and 182 sites (55%) further conjugated in ubp2∆ and ubp3∆ cells, respectively (Fig. S3.5a and Sup. Table 3.5 and 3.6). Sup45, Pdc1 and Cdc19 were found to be affected by the deletion of UBP2 in our proteomic study. We therefore went on to confirm our results for Sup45, Pdc1 and Cdc19 using the IMAC-based orthogonal approach (Fig. S3.5b). Notably, we did not observe a marked difference in the localization of proteins ubiquitylated upon HS in ubp2Δ or ubp3Δ cells compared to WT (Fig. S3.5c). These results indicate that the deubiquitinases Ubp2 and Ubp3 also mainly target cytosolic proteins in HS conditions.  The proteomic data also suggest that Ubp2 and Ubp3 are unlikely to be involved in modulating Rsp5-substrate specificity. As the Ubp2 activity is more specific for K63 chains in vitro[189] and K63 chains are not thought to efficiently mediate proteasomal   126 degradation[199], we next hypothesized that Ubp2 and Ubp3 could edit ubiquitin chains to promote proteasomal degradation of the heat-induced cytosolic misfolded proteins. We first expressed MYC-tagged ubiquitin mutants containing only K48 or K63 (while the other six K are mutated to R) to estimate the levels of K48- and K63-linked poly-ubiquitin chains, respectively. In this assay, K48-chains were not distinctly increased in ubp2∆ and ubp3∆ cells upon HS, while they were more abundant in WT cells compared to K63-chains (Fig. 3.5b). In contrast, there were more K63-linked chains in ubp2∆ (2.6-fold) and ubp3∆ (2.4-fold) cells in comparison to WT cells (Fig. 3.5b). To confirm our results, we used a selected reaction monitoring (SRM) approach to quantify the levels of K48 and K63 chains in the cell by mass spectrometry (Sup. Table 3.4). We first enriched proteins conjugated to H8-ubiquitin upon HS by IMAC in both WT and ubp2∆ cells and isolated high-molecular weight species on a protein gel. After spiking in labeled synthetic ubiquitin peptides, we found that there was a significantly higher increase in K63-chains in ubp2∆ versus WT cells than K48-linkages (Fig. 3.5c). Using SRM we also confirmed that the Rsp5 substrates Sup45, Pdc1 and Cdc19 were distinctly further conjugated to K63-linked ubiquitin chains in ubp2∆ cells (Fig. S3.5d). These results strongly support a model in which the deubiquitinases Ubp2 and Ubp3 further process the HS induced cytosolic Rsp5 substrates by removing K63 linkages to promote their conjugation to K48-linked ubiquitin chains and proteasomal degradation.  We next determined whether the loss of UBP2 or UBP3 also impairs the plasma membrane surveillance pathway by evaluating the turnover of Can1 and Mup1, two membrane proteins degraded upon misfolding[74]. Endocytosis of both Can1 and Mup1 was not affected after shifting the cells to a higher temperature in ubp2∆ and ubp3∆   127 cells (Fig. S3.5e). Similarly, degradation of both proteins upon HS was not inhibited in ubp2∆ and ubp3∆ cells (Fig. S3.5f). These observations indicate that Ubp2 and Ubp3 are most likely solely implicated in the cytosolic Rsp5-PQC pathway. To assess the importance of the Rsp5 PQC pathway, we assessed cell fitness in stress conditions in the absence of UBP2 or UBP3. We first monitored cell growth following a 30 min HS at 45 °C and found that there was a longer lagging recovery time in ubp2∆ and ubp3∆ cells in comparison to WT cells (Fig. 3.5d), while cell viability was not noticeably different between these cells (data not shown). We also observed both a reduced viability and slower growth of ubp2∆ and ubp3∆ cells in presence of canavanine, an arginine analog causing protein misfolding (Fig. 3.5e). These data indicate that Ubp2 and Ubp3 are both required for cells to adequately respond to protein misfolding stresses, consistent with their role in the cytosolic Rsp5-PQC pathway.               128 Figure 3.5 Ubp2 and Ubp3 are required for the degradation of cytosolic misfolded proteins   129 (a). Degradation of 35S labeled proteins (5 min) in indicated cells and temperatures in three independent experiments (with standard deviations). The same values for the WT samples are shown in the two graphs (all strains were analyzed together). (b). Indicated cells expressing the MYC tagged ubiquitin mutants either containing only K48 or K63 were subjected to HS (45 ºC, 1 5 min) and were analyzed by western blot. The relative values of MYC signal (>75 kDa, normalized to Pgk1 and compared to WT) is shown below. (c). Relative ratios of K48- and K63-linkages of poly-ubiquitylated proteins (>75 kDa) isolated from ubp2∆ and UBP2 cells subjected to HS (45 °C, 15 min) were measured by SRM. Averaged from three independent experiments with standard deviation are indicated and assessed using a two-tail student t-test. (d). A600 of indicated cells grown at 25 °C following a HS (solid lines) or no stress (dotted lines). Averaged values from three biological replicates are plotted with standard deviations. Values indicate the averaged time required to complete one A600 doubling after HS. (e). Serial dilutions (1/5) of indicated cells plated on synthetic complete (SC) agar-containing media with or without canavanine and grown at 30 ºC.   130 3.3.6 Heat-induced Rsp5 substrates contain PY motifs preferably embedded in structured regions and Hsp40 co-chaperone Ydj1 assists Rsp5 to target heat-induced misfolded proteins To reveal how Rsp5 recognizes misfolded proteins, we first performed a series of computational analyses to identify communality between Rsp5 candidate-substrates induced by HS. Similarly to Rsp5 candidate substrates identified using a protein array[200], we found that PY motifs (defined by PxY) were significantly enriched among HS induced substrates (Fig. S3.6a). Interestingly, the canonical PPxY motif was more prevalent among candidate substrates identified in the protein array while slight variations of the PxY motif such as, SPxF, VPxF and PPR were significantly enriched among HS induced candidate substrates. We also found that PY motifs among HS-induced Rsp5 candidate-substrates were significantly more often localized in regions predicted to form secondary structures in comparison to other Rsp5 candidate substrates (Fig. 3.6a).  The higher prevalence of PY motifs in disordered regions among substrates identified in none-HS conditions is consistent with the required accessibility of the motif to mediate binding with one of the Rsp5 WW domains in normal conditions (i.e., no HS). One possibility is that the HS-induced substrates are mainly recognized by Rsp5 upon misfolding, as their motifs might otherwise be shielded in the native protein structure. In agreement with this idea, PY motifs of Rsp5 candidate-substrates induced by HS were predicted to be less accessible in comparison to the other candidate substrates, when considering PY motifs in structured regions (Fig. S3.6b). These analyses indicate that   131 PY motifs, and possibly newly exposed PY motifs due to misfolding, may play an important role in mediating substrate recognition by Rsp5. To assess the role of these motifs, we mutated the putative PY motif of the Cdc19 Rsp5-substrate (LPNY to LANA). This putative PY motif is located, based on a crystal structure, in the interaction interface between two monomers and is thereby not accessible for Rsp5 binding upon the assembly of a functional Cdc19 homo-tetramer. We found that the ubiquitylation of the Cdc19 (PY/AA) mutant upon HS was reduced in comparison to the WT Cdc19. (Fig. 3.6b). These data confirm the importance of the PY motif in mediating substrate recognition. However, the ubiquitylation of the Cdc19 (PY/AA) mutant was not fully abolished in comparison to conjugation levels in rsp5-1 cells. In addition, ubiquitylation of Pdc1 was not affected upon the deletion of two putative PY motifs in this protein (Fig. S3.6c). These results suggest that there may be a second mechanism to mediate recognition of misfolded proteins. We next hypothesized that a chaperone could mediate the recognition of a wide-range of heat-induced misfolded client proteins. We observed that several chaperone proteins also contain PY motifs including Ydj1, which contains a putative PY motif sequence (PIPKY) in its C-terminal region. Ydj1 is a type I Hsp40 co-chaperone of the DnaJ family, which both stimulates Hsp70 ATPase activity and mediates selectivity of Hsp70 client proteins[201]. Interestingly, YDJ1 was also shown to be required for the ubiquitylation and degradation of certain misfolded proteins in a previous study[202]. However, the putative role of Ydj1 in proteolysis remained unclear. We posit that Ydj1 may associate with Rsp5 to facilitate substrate recognition. Indeed, we found that there was a significant enrichment of proteins containing short sequences   132 (GX[LMQ]{P}X{P}{CIMPVW}, where [XY] denotes either X or Y and {XY} denotes neither X nor Y[184]) predicted to mediate binding with Ydj1 upon misfolding among HS-induced Rsp5 substrates (Fig. S3.6d). To determine whether Ydj1 and Rsp5 interact in HS conditions, we performed co-immunoprecipitations from cells crossed-linked prior to lysis. In these conditions, we observed an increased interaction between Rsp5 and Ydj1 upon HS (Fig. 3.6c). We confirmed that the deletion of YDJ1 impaired the overall increased ubiquitylation upon HS (Fig. 3.6d). The increased ubiquitylation was restored upon the expression of the WT YDJ1 but not after the expression of the ydj1 (PP/GG) mutant in which the two proline residues of the putative PY motif were mutated to glycine residues (Fig. 3.6d). We confirmed that the mutation of the putative PY motif impaired the interaction between Ydj1 and Rsp5 upon HS (Fig. 3.6e). Finally, we examined ubiquitylation levels of Sup45, a heat-induced substrate of Rsp5 that does not contain any obvious PY motif. Deletion of YDJ1 lead to the abolition of Sup45 ubiquitylation that can be fully restored upon the expression of YDJ1 but not of the ydj1 (PP/GG) mutant (Fig. 3.6f). Our results indicate that the association of Ydj1 with Rsp5 may play a major role in the HS-induced ubiquitylation of Rsp5 substrates that lack of PY motifs.  Similar to the deletion of substrate-PY motifs, the deletion of YDJ1 was not sufficient to abolish the HS-induced ubiquitylation of all Rsp5 substrates. We reasoned that a bipartite mechanism could mediate recognition of PY-containing misfolded proteins by Rsp5. Consistent with this idea, we found that the Cdc19 (PY/AA) mutant was no longer ubiquitylated upon HS in ydj1∆ cells (Fig. 3.6g). Similarly, mutation of either the LPTF motif (to LATA) or LPVF motif (to LAVA) of Pdc1 reduced ubiquitylation   133 of this Rsp5 substrate in ydj1∆ cells (Fig. S3.6e). These results indicate that both Ydj1-association and substrate PY motifs are important to mediate ubiquitylation of Rsp5 substrates upon misfolding.   134 Figure 3.6 Rsp5 directly and with Ydj1-adaptor ubiquitylates misfolded proteins upon HS     135 (a). The pie chart (left) indicates which portion of HS induced Rsp5 candidate substrates identified in the two mass spectrometry experiments contains the following proline-containing motifs ([PLV]PxY, [PSV]PxF, PPPP or PPR; dark green), additional PxY motifs (light green) or no obvious PY motif (grey). x denotes any residue. The histogram (right) indicates whether [PLV]PxY, [PSV]PxF, PPPP or PPR motifs are located in regions predicted disordered among Rsp5 candidate substrates induced by HS identified in the protein array  (Array). (b) and (g). IMAC analyzed by western blot. Samples were derived from cells expressing H8-ubiquitin (from a first URA3 plasmid) and either Cdc19, the Cdc19 (PY/AA) mutant (both fused C-terminally with 13xMYC) or no open reading frame from a second HIS3 plasmid. YDJ1 cells (B) and ydj1∆ cells (g) were assessed; as well as rsp5-1 cells (- RSP5 lane in B). (c). TAP immunoprecipitation (IP) was performed after in vivo cross-linking with 1% formaldehyde in cells grown at 25 °C (- HS lane) or during a HS (45 ºC, 20 min). The endogenous YDJ1 was C-terminally TAP-tagged in the indicated lanes. The IP samples were analyzed by western blot (the - TAP control was analyzed on the same membrane but not adjacent to the other two lanes). (d). Increased ubiquitylation levels quantified by dot-blot after HS (45 °C, 15 min) in the indicated cells that carried a LEU2 plasmid that was empty (-) or with YDJ1 or ydj1 (PP/GG) expressed from the YDJ1 promoter. Three biological replicates were assessed and averaged values are shown with standard deviations. (e). HA IP was performed after in vivo cross-linking with 1% formaldehyde in ydj1∆ cells subjected to a HS (45 ºC, 20 min) and that carried a LEU2 plasmid that was empty (-) or contained C-terminally tagged (3xHA) YDJ1 or ydj1 (PP/GG) expressed from the YDJ1 promoter. (f). IMAC from YDJ1 or ydj1∆ cells expressing when indicated H8-Ubiquitin from a first URA3 plasmid, Sup45 fused N-terminally to GFP or GFP alone from a second HIS3 plasmid, and YDJ1 or ydj1 (PP/GG) under the YDJ1 promoter from third LEU2 plasmid.   136 3.4 Discussion Here, we discovered a novel PQC degradation pathway in which the Rsp5 ubiquitin ligase targets misfolded cytosolic proteins for degradation. Using both genetic and biochemical approaches, we show that Rsp5 ubiquitylates cytosolic misfolded proteins upon HS. While Hul5 ubiquitin ligase also targets certain Rsp5 substrates (e.g., Pdc1), our data suggest that both ligases also target different pools of proteins. Importantly, Rsp5 and Hul5 are the main two ubiquitin ligases involved in the HS ubiquitylation response.  Rsp5 recognizes misfolded proteins via PY motifs that are preferably located in structured domains and through the association of the PY-containing Ydj1 co-chaperone protein. Canonical and atypical PY motifs that interact with WW domains are key mediators of substrate recognition in the Nedd4 E3 family, either directly or via adaptor proteins[41]. The importance of Rsp5 WW domains in mediating substrate recognition upon HS is underscored in the in vitro ubiquitylation assay, in which no ubiquitylation is observed in the presence of a triple mutant of the three Rsp5 WW domains. We propose that Rsp5 recognizes misfolded proteins using a bipartite mechanism (Fig. 3.7).          137 Figure 3.7 Model for Rsp5 recognition of misfolded proteins    Schematic of the proposed bipartite model for the recognition of cytosolic misfolded proteins by Rsp5.    We found that a large portion of proteins ubiquitylated by Rsp5 upon HS contain PY motifs (typical and atypical) that are more often located in structured regions and have the tendency to be less accessible in comparison to PY-motifs on other Rsp5 substrates. We propose that buried PY motifs in proteins could signal misfolding by mediating Rsp5 recognition when exposed (Fig. 3.7). Consistent with this inducible degron model, PY motifs that are in the structured regions of the Rsp5-substrates induced by HS were predicted to be less accessible than the ones identified by protein array[203] under no stress conditions (Fig. S3.6b). This mechanism, based on the presence of a protein-protein interaction motif, is distinct from other known PQC pathways, e.g., the nuclear E3 ligase San1 recognizes less defined exposed   138 hydrophobic stretches that are normally buried in the native conformation of the substrates[204].  In addition to direct substrate recognition, we found that the Ydj1 Rsp5-association promotes the ubiquitylation of misfolded proteins (Fig. 3.7). We found that a non-canonical PY motif (PIPKY) located in the C-terminal of the Hsp40 co-chaperone Ydj1 mediates Rsp5 interaction upon HS. We propose that Ydj1 acts as a substrate-adaptor protein to promote the ubiquitylation of its misfolded client proteins either alone or in conjunction of exposed PY-motifs on the substrate (Fig. 3.7). It will be interesting to determine how the Rsp5-Ydj1 association is upregulated upon HS, e.g., the Ydj1-PY motif may be more accessible upon a stress that increases protein misfolding similar to the proposed activation mechanism of Hsp33 upon oxidative stress[205]. Interestingly, Sis1, another Hsp40 co-chaperone, was shown to cooperate with the Ubr1 ubiquitin ligase to target a misfolded model substrate for degradation[86]. Hart and colleagues recently established that Sis1 but not Ydj1 is sequestered in assessed protein aggregates, and Sis1 sequestration inhibits the degradation of misfolded proteins[206]. One possibility is that Ydj1 and Rsp5 participate in a major PQC pathway that is activated upon major misfolding stresses when other PQC components like Sis1 are no longer able to function properly. Surprisingly, Ubp2 and Ubp3 induce proteasomal degradation of cytosolic misfolded proteins. Other deubiquitinases typically limit the degradation of proteins targeted by PQC pathways[207, 208]. The unusual activity of Ubp2 and 3 may be explained by the fact that both deubiquitinases mainly suppress K63- but not K48-linked chains assembled by Rsp5 upon HS. One possibility is that both the deubiquitinases and Rsp5   139 are active concurrently, so that K48 chains are gradually built upon stochastic K48 conjugations by Rsp5, which is a K63-specific ligase (Fig. S3.7). Alternatively, Ubp2 and Ubp3 might preferably leave the proximal ubiquitin moiety on the substrate that is then further elongated by other ubiquitin ligases like Hul5 (Fig. S3.7). This step may be required for substrate-processing by Cdc48, an ubiquitin selective segregase that is required for the degradation of misfolded proteins[153]. Consistent with the importance of the Rsp5 PQC pathway, deletion of UBP2 or UBP3 affect cell fitness in stress conditions.   140 3.5 Supplemental Data Figure S3.1 Functional Rsp5 is required for the HS ubiquitylation response  (a). Dot-blot images used for the quantification in Fig. 3.1a. (b). Normalized ubiquitylation levels quantified by dot-blot in WT, rsp5-1, rsp5-sm1 and rps5-3 cells under the indicated conditions. Quantification and images from infrared scanner are shown on the top or bottom, respectively. Increase in ubiquitylation from 37 ºC to 45 ºC   141 was compared to WT with a two-tail student t-test (**: p<0.01; *: p<0.05).  (c). ubiquitylation levels of Tetp::Rsp5 cells treated with (OFF) or without (ON) 100µg/ml doxycycline before and after heat-shock were assessed by both dot-blot assay (bottom; quantified in Fig. 3.1c) and western-blot (top). (d). ubiquitylation levels in indicated cells over-expressing H8-Ubiquitin before and after HS were quantified using dot-blot assay. All experiments were done with three biological replicates and the averaged values are shown with standard deviations; a.u. denotes arbitrary units (each value is relative to the reference sample).   142 Figure S3.2 Rsp5 directly ubiquitylates heat-induced misfolded proteins    (a). HS screening of a panel of deubiquitinase single deletions. Normalized ubiquitylation levels of indicated strains at 25 ºC and 45 ºC are shown. We reasoned that the increase in ubiquitylation upon HS should occur earlier in absence of a specific deubiquitinase, and HS was therefore performed for 5 min. (b). Normalized ubiquitylation levels in WT, rup1∆, and bre5∆ cells before and after HS quantified by dot-bot assay. Experiment was done with three biological replicates and the averaged values are shown with standard deviations.  For A and B, a.u.: denotes arbitrary units (each value is relative to the reference sample). (c). Heat-induced ubiquitylation in WT and ubp2∆ cells at indicated times were analyzed by western blot with anti-ubiquitin antibodies. The Pgk1 loading control is also shown. (d). In vitro ubiquitylation performed with MYC-ubiquitin when indicated in WT and rsp5-1 cell extracts incubated at 25°C, 42 °C or 45 °C for 10 min and analyzed by western blot using anti-MYC antibodies. Pgk1 loading control is also shown. Lower increased in poly-ubiquitylation was observed at 45 °C, presumably due to an increased protein precipitation in these conditions (data not shown).    143 Figure S3.3 Rsp5 targets cytosolic proteins upon HS    144 (a). Increased ubiquitylation levels in WT, art1-10∆, and art1-8,10∆ cells from 25 ºC to 45 ºC were assessed by dot-blot. ubiquitylation levels were normalized to Pgk1 level. (b). Normalized ubiquitylation levels before and after heat-shock in WT and rsp5-1 cells containing the indicated plasmids (- denotes presence of a control empty plasmid) were quantified by dot-blot. Both RSP5 and rsp5-C2∆ were expressed under a Gal-promoter induced for 60 min with 2% Galactose at 37 °C. (c). Summary table of ubiquitylation sites (GG peptides) and proteins in two independent SILAC experiments. Experiment 1 (Exp1) analyses are also shown in Fig. 3.3c, e. (d). Localization of HS-induced Rsp5 candidate substrates identified in the second SILAC-diGly mass spectrometry experiment shown in (d). Distribution of proteins in each compartment is shown in percentage. C: Cytoplasm; N: Nuclear; M: Membrane; Mit: Mitochondria. (e). Increased ubiquitylation levels quantified by dot-blot after a 15 min HS at 45 ºC in the indicated cells. Cells were pre-incubated at 37 °C for 60 min prior to the HS. Experiments were done with three biological replicates and averaged values are shown with standard deviations. (f). IMAC of samples from WT, hul5∆ and rsp5-1 cells that expressed H8-ubiquitin and Lsm7TAP from the same plasmid after a HS at 45 °C for 15  min were analyzed by western-blot using anti-TAP antibodies. Relative levels of the poly-ubiquitin signal are indicated below. Experiments in A, B and E were done with three biological replicates and the averaged values are shown with standard deviations; a.u. denotes arbitrary units (each value is relative to the reference sample).   145 Figure S3.4 Rsp5 is required for the degradation of cytosolic misfolded proteins                          146 (a)-(b). Degradation of 35S labeled proteins in WT (grey) and indicated mutant or deletion strains (green) cells at 25 ºC (dotted lines) or 38 ºC (straight lines). The portion of proteins degraded at the indicated times was measured for short-lived proteins (5 min labeling). Three independent experiments were analyzed and averaged values are shown with standard deviations. The same values for WT samples are shown in (a) and (b) as all samples were analyzed together. (c). Increased ubiquitylation levels from 25 ºC to 45 ºC in WT and rsp5-1 cells treated with or without 100 µg/ml cycloheximide were analyzed by dot-blot assay. Three biological replicates were analyzed and average values are shown with standard deviation. a.u. denotes arbitrary units (each value is relative to the reference sample). (d). Protein localization of the cytosolic fraction sample obtained by native lysis and analyzed by mass spectrometry. 348 proteins were identified in this experiment. The same lysis method was used for samples analyzed in Fig. 3.4c. C: Cytoplasm; N: Nuclear; M: Membrane; Mit: Mitochondria. (e). Fluorescent intensities of Guk1-7 fused to GFP that is expressed from an URA3 plasmid in RSP5 (grey) and rsp5-1 (green) cells were measured by flow cytometry after incubating the cells at 25 °C (dotted lines) or 37 °C (straight lines) with 100 µg/ml cycloheximide at the indicated time points. (f). Loss of fluorescent intensities of plasmid-encoded Guk1-7GFP in WT, rsp5-1, rsp5-sm1, and rsp5-3 cells at 37 ºC for 2 hrs. Experiments in (e) and (f) were done in three independent experiments and averaged values are shown with standard deviations.   147 Figure S3.5 Ubp2 and Ubp3 are required for the degradation cytosolic misfolded proteins   148 (a). Schematic representation (left panel) of the triple SILAC experiments to identify HS-induced Ubp2 or Ubp3 substrates. L, M and H denote the three different SILAC-labels (light, medium and heavy, respectively). Number of GG sites in each category in both ubp2∆ and ubp3∆ experiments are shown on the right panel. Others denote ubiquitylation sites not affected by HS, more ubiquitylated in WT after HS but less in the deletion strain, and less ubiquitylated after HS, respectively. (b). IMAC from samples derived from WT and ubp2∆ cells that expressed H8-ubiquitin and the indicated endogenously tagged candidate proteins (C-terminal, 3xHA) subjected to HS (45 °C, 20 min). The samples were analyzed by western-blot using anti-HA antibodies. Inputs and Pgk1 control are also shown. (c). Localization analysis of HS-dependent candidate substrates of the indicated deubiquitinases. C: Cytoplasm; N: Nuclear; M: Membrane; Mit: Mitochondria. (d). Endogenously expressed Sup45, Pdc1 and Cdc19 that were tagged with C-terminal 3xHA-tag in WT and ubp2∆ cells that were subjected to HS (45 °C, 20 min) were immunoprecipitated (anti-HA) and ubiquitylated species (>75 kDa) were quantified by SRM. Ratios from ubp2∆ vs. WT cells are shown for each conjugated (GG) or unconjugated (N) K48 and K63 peptide associated to the indicated proteins. (e). Fluorescent images of WT, ubp2∆ and ubp3∆ cells that expressed endogenously tagged Can1GFP or Mup1GFP at 25 ºC or 40 ºC for 2 hrs. Both the GFP and DAPI channels are shown. (f). Protein levels of 13MYCCan1 or 13MYCMup1 that were expressed from a plasmid in WT, ubp2∆ and ubp3∆ cells incubated 40 ºC with 100 µg/ml cycloheximide at indicated times. Pgk1 loading control and relative levels of quantified MYC signal (grey) are also shown.   149 Figure S3.6 Rsp5 directly and with Ydj1-adaptor ubiquitylates misfolded proteins upon HS   150 (a). The number of proteins in each dataset containing a given PY motif is shown (x denotes any residue). Enrichment of each motif in each dataset relative to the genome was tested using Fisher’s exact test, with significant p-values shown in brackets. Only PY motifs significantly enriched in one of the two datasets were used in the subsequent analyses. (b). The predicted percentage relative solvent accessibility were plotted for the PY motifs within structured regions of Rsp5 candidate substrates that are HS-induced or identified in a protein array. The two datasets were tested for difference with the Whitney-Manning U test and found to have a p-value just above significance. The median solvent accessibility of PY motifs in disordered regions in both datasets combined (presumably accessible) is marked with a dotted line for comparison. A box plot of the data is also shown above the plot.  (c) and (e). IMAC from samples derived from cells expressing H8-ubiquitin from a first plasmid and the indicated Pdc1 constructs (with C-terminal 13xMYC) in a second plasmid. YDJ1 (c) and ydj1∆ (e) cells were HS (45 °C, 20 min) prior to lysis, as well as rsp5-1 cells (in - RSP5 lane in C). IMAC were analyzed by western blot using anti-MYC antibodies. Inputs and Pgk1 are also shown. (d). Histograms of proteins with at least 1, 2 or 3 Ydj1 binding motif among HS-induced Rsp5 candidate substrates and in the proteome. Significant of differences are shown.     151 Figure S3.7 Model for Rsp5 proteasome targeting with deubiquitinases Ubp2 and Ubp3    Rsp5 substrates may be first ubiquitylated by Rsp5 then deubiquitylated by Ubp2 and Ubp3 before a second ubiquitin ligase further conjugate them (I). Alternatively, Ubp2 and Ubp3 may remove K63-linked ubiquitin during conjugation while leaving K48-linked ubiquitin stochastically added by Rsp5 (II).    152 Supplementary Table Table 3.1 Yeast strains used in chapter 3 Strain ID Alias Genotype Background Mating Type Source YTM408 BY4741 his3∆1, leu2∆0, met15∆0, ura3∆0 S288C a Open Biosystems Collection, parental strain YTM409 BY4742 his3∆1, leu2∆0, met15∆0, ura3∆0 S288C alpha Open Biosystems Collection, parental strain YTM450 YDJ1-TAP his3∆1, leu2∆0, met15∆0, ura3∆0, ydj1∆::YDJ1-TAP-HIS3MX S288C a Open Biosystems Collection YTM639 rsp5-1 his3Δ1, leu2Δ, ura3Δ0, met15Δ0, RSP5::rsp5-1-KanR S288C a C. Boone ts collection, ts at 37C YTM640 rsp5-sm1 his3Δ1, keu2Δ, ura3Δ0, met15Δ0, RSP5::rsp5-sm1-KanR S288C a C. Boone ts collection, ts at 37C YTM641 rsp5-3 his3Δ1, keu2Δ, ura3Δ0, met15Δ0, RSP5::rsp5-3-KanR S288C a C. Boone ts collection, ts at 37C YTM660 ydj1∆ his3∆1, leu2∆0, lys2∆0, ura3∆0, ydj1∆::KanMX S288C alpha  Open Biosystems Collection YTM710 rsp5-1, hul5∆ his3Δ1, leu2∆0, LYS2, ura3∆0, hul5∆::KanMX6, RSP5::rsp5-1-KanR S288C a This paper YTM736 guk1-7-13myc his3Δ1, leu2Δ0, LYS2, met15Δ0, ura3Δ0, guk1-7- 13myc::KanMX6::URA3,  CAN1 S288C a Khosrow-Khavar et al. (2012) G3 2(5):619-28 YTM882 ubp2∆ his3∆1, leu2∆0, met15∆0, ura3∆0, ubp2∆::HIS3MX6 S288C a This paper YTM884 ubp2∆ his3∆1, leu2∆0, lys2∆0, ura3∆0 ubp2∆::HIS3MX6 S288C alpha This paper YTM959 hul5∆ his3∆0, leu2∆0, met15∆0, ura3∆0, hul5∆::kanMX S288C a Open Biosystems Collection YTM1002 rsp5-1 his3Δ1, leu2∆0,met15∆0?, lys2∆0, ura3∆0, RSP5::rsp5-1-KanR S288C a This paper YTM1063 ubp2∆hul5∆ his3∆1, leu2∆0, met15∆0, lys2∆0, ura3∆0 ubp2∆::His3MX6, hul5∆::KanMX6 S288C alpha This paper YTM1067 ubp2∆rsp5-1 his3∆1, leu2∆0, met15∆0?, lys2∆0?, ura3∆0 ubp2∆::His3MX6, RSP5::rsp5-1-KanR S288C alpha  This paper YTM1076 ubp1Δ his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp1∆::KanMX6 S288C alpha Open Biosystems Collection YTM1077 ubp2Δ his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp2∆::KanMX6 S288C alpha Open Biosystems Collection YTM1078 ubp3Δ his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp3∆::KanMX6 S288C alpha Open Biosystems Collection YTM1079 ubp4Δ(doa4Δ) his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp4∆::KanMX6 S288C alpha Open Biosystems Collection YTM1080 ubp5Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp5∆::KanMX6 S288C alpha Open Biosystems Collection   153 Strain ID Alias Genotype Background Mating Type Source YTM1081 ubp6Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp6∆::KanMX6 S288C alpha Open Biosystems Collection YTM1082 ubp7Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp7∆::KanMX6 S288C alpha Open Biosystems Collection YTM1083 ubp8Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp8∆::KanMX6 S288C alpha Open Biosystems Collection YTM1084 ubp9Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp9∆::KanMX6 S288C alpha Open Biosystems Collection YTM1085 ubp10Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp10∆::KanMX6 S288C alpha Open Biosystems Collection YTM1086 ubp11Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp11∆::KanMX6 S288C alpha Open Biosystems Collection YTM1087 ubp12Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp12∆::KanMX6 S288C alpha Open Biosystems Collection YTM1088 ubp13Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp13∆::KanMX6 S288C alpha Open Biosystems Collection YTM1089 ubp14Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp14∆::KanMX6 S288C alpha Open Biosystems Collection YTM1090 ubp15Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp15∆::KanMX6 S288C alpha Open Biosystems Collection YTM1091 ubp16Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, ubp16∆::KanMX6 S288C alpha Open Biosystems Collection YTM1092 yuh1Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, yuh1∆::KanMX6 S288C alpha Open Biosystems Collection YTM1096 rup1Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, rup1∆::KanMX6 S288C alpha Open Biosystems Collection YTM1098 bre5Δ  his3∆1, leu2∆0, lys2∆0, ura3∆0, bre5∆::KanMX6 S288C alpha Open Biosystems Collection YTM1123 ubp3Δhul5Δ his3∆1, leu2∆0, lys2∆0, ura3∆0, MET15 ubp3∆::KanMX6, hul5∆::KanMX6 S288C alpha This paper YTM1125 rsp5-1ubp3∆ his3Δ1, leu2∆0,met15∆0?, lys2∆0, ura3∆0, RSP5::rsp5-1-KanR, ubp3∆::KanMX S288C alpha This paper YTM1132 SUP45-3HA/rsp5-1 his3Δ1, leu2Δ0, MET15, ura3Δ0, SUP45::SUP45-3HA-HIS3MX6, RSP5::rsp5-1-KanR S288C a This paper YTM1134 SUP45-3HA his3Δ1, leu2Δ0, ura3Δ0, MET15, SUP45::SUP45-3HA-HIS3MX6 S288C a This paper YTM1249 guk1-7-13myc/rsp5-1 his3Δ1, leu2Δ0, met15Δ0, ura3∆0, LYS2, RSP5::rsp5-1-KanR, guk1-7- 13myc::KanMX6::URA3, CAN1 S288C a This paper YTM1295 PDC1-3HA his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0, MET15, PDC1::PDC1-3HA-HIS3MX6 S288C a This paper   154 Strain ID Alias Genotype Background Mating Type Source YTM1296 PDC1-3HA/rsp5-1 his3Δ1, leu2Δ0, met15Δ0?,lys2Δ0?, , ura3Δ0, PDC1::PDC1-3HA-HIS3MX6, RSP5::rsp5-1::KanR S288C a This paper YTM1308 CDC19-3HA his3Δ1, leu2Δ0, met15Δ0, LYS2, ura3Δ0, CDC19::CDC19-3HA-HIS3MX6 S288C a This paper YTM1312 EN59 ecm21Δ::G418 csr2Δ::G418 bsd2Δ rog3Δ::natMX rod1Δ ygr068cΔ aly2Δ aly1Δ ldb19Δ rim8Δ ylr392cΔ::HIS his3Δ, ura3Δ, leu2 Δ S288C a Hugh R. B. Pelham YTM1313 EN60 ecm21Δ::G418 csr2Δ::G418 bsd2Δ rog3Δ::natMX rod1Δ ygr068cΔ aly2Δ aly1Δ ldb19Δ ylr392cΔ::HIS his3Δ, ura3Δ, leu2 Δ S288C a Hugh R. B. Pelham YTM1356 rpt6-20 his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, RPT6::rpt6-20-KanR S288C a C. Boone ts collection, ts at 37C YTM1357 pep4Δprb1Δ his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, lys2Δ0, prb1Δ::KanMAX6, pep4Δ::His3MX6 S288C a This paper YTM1513 Can1-GFP his3∆1, leu2∆0, met15∆0, ura3∆0, CAN1::CAN1-GFP-His3MX6 S288C a Invitrogen GFP Collection YTM1514 Can1-GFP/ubp2∆ his3∆1, leu2∆0, met15∆0, ura3∆0, CAN1::CAN1-GFP-His3MX6, ubp2∆::KanMX S288C a This paper YTM1515 Can1-GFP/ubp3∆ his3∆1, leu2∆0, met15∆0, ura3∆0, CAN1::GFP::His3MX6, ubp3∆::KanMX S288C a This paper YTM1516 Mup1-GFP his3∆1, leu2∆0, met15∆0, ura3∆0, MUP1::MUP1-GFP-His3MX6 S288C a Invitrogen GFP Collection YTM1517 Mup1-GFP/ubp2∆ his3∆1, leu2∆0, met15∆0, ura3∆0, MUP1::MUP1-GFP-His3MX6, ubp2∆::KanMX S288C a This paper YTM1518 Mup1--GFP/ubp3∆ his3∆1, leu2∆0, met15∆0, ura3∆0, MUP1::MUP1-GFP-His3MX6, ubp3∆::KanMX S288C a This paper YTM1520 Tetp-RSP5 RSP5p::CMV-tTA, his3∆1, leu2∆0, met15∆0 S288C a Open Biosystems Collection    Supplementary Table 3.2 Plasmids used in chapter 3 Strain ID Name Source & Note Reference BPM30 pRS316-H8-ubiquitin H8-ubiquitin with GPD promotor and PGK terminator is insert into pRS316  yeast URA3, CEN, ARS vector in the EcoRI and SmaI sites Mayor, T. et al. (2007) MCP 6(11):1885-95 BPM42 pRS316 yeast URA3, CEN, ARS vector with MCS. Sikorski, R. S. and Hieter, P. (1989) Genetics 122:19-27 BPM45 pRS313 yeast HIS3, CEN, ARS vector with MCS. Sikorski, R. S. and Hieter, P. (1989) Genetics 122:19-27 BPM49 pRS315 yeast LUE2, CEN, ARS vector with MCS. Sikorski, R. S. and Hieter, P. (1989) Genetics 122:19-27 BPM59 pFA6a-GFP(S65T)-His3MX6 Plasmid for PCR-based endogenous gene tagging in yeast. Longtine et al. (1998) Yeast 14:953-961.   155 Strain ID Name Source & Note Reference BPM98 GST-Rsp5 N-terminal GST tag. Ampicillin and chloramphenicol resistance markers. In bacterial strain BL21.  R. Deashies BPM99 GST-Rsp5 N-terminal GST tag. Ampicillin resistance marker. In bacterial strain DH5a Saeki, Y. (2005) Meth Enz 399:215-27 BPM173 pRS313-GPD-13myc-PGK yeast HIS3, CEN/ARS vector. Ampicillin resistance. Khosrow-Khavar et al. (2012) G3 2(5):619-28 BPM241 pRS313-GPD-GFP(S65T)-PGK Yeast HIS3, CEN/ARS vector with GFP (S65T) under the control of the GPD promoter and PGK1 terminator.  Khosrow-Khavar et al. (2012) G3 2(5):619-28 BPM297 LSM7-TAP C-terminal TAP tagged LSM7 was PCR amplified from genomic DNA and insterted into BPM30 using NotI. This paper BPM390 YDJ1 pRS315 LEU2, CEN/ARS plasmid. Ydj1 gene is inserted using SacI and KpnI sites.  Johnson, J. and Craig E. (2001) J Cell Biol 152(4):851-6 BPM453 pRS313-GPD-GUK1-GFP(S65T)-PGK Yeast HIS3, CEN/ARS vector with GUK1 inserted between a GPD promoter and GFP using BamHI and XbaI This paper BPM458 pRS313-GPD-GUK1-7-GFP(S65T)-PGK Yeast HIS3, CEN/ARS vector with GUK1-7 inserted between a GPD promoter and GFP using BamHI and XbaI This paper BPM470 pRS426-GAL1p The GAL1 promoter was inserted into the yeast pRS426 URA, 2µ vector using NaeI and EcoRI This paper BPM484 pRS424-GALp-HA-RSP5-C777A HA-Rsp5 was inserted into the yeast pRS424 TRP1, 2µ vector using EcoRI and NotI This paper BPM514 pGEX-6P1-GST-RSP5-C777A Rsp5-C777A was swapped into GST-Rsp5 (BPM98) using NotI and AfeI This paper BPM517 pRS316-GPD-PGK Yeast URA, CEN/ARS vector with GPD promoter and PGK1 terminator inserted using SacII and XhoI This paper BPM518 pGEX-6P1_GST-RSP5-ww1*ww2*ww3* Used for expressing recombinant Rsp5-ww123*. Rsp5-ww123* was PCR amplified and inserted into pGEX-6P1 using EcoRI and SalI This paper BPM519 pRS313-GPD-eGFP-PGK Yeast HIS3, CEN/ARS vector to be used for N-terminal GFP tagging. GFP was inserted between the GPD promoter and PGK1 terminator using BamHI and SalI This paper BPM563 pRS313-GPD-CDC19-13MYC-PGK CDC19 was amplyfied from genomic DNA and insert into BPM173 with BamHI and XmaI sites This paper BPM564 pRS313-GPD-CDC19-PY*-13 MYC -PGK Plasmid was created by site directed mutagenesis using BPM563 as a template. Mutations created the following changes: P363A and Y365A. This paper BPM565 pRS313-GPD-PDC1-13 MYC -PGK PDC1 was amplyfied from genomic DNA and insert into BPM173 with BamHI and XmaI sites This paper BPM566 pRS313-GPD-PDC1-PY1*-13 MYC -PGK Plasmid was created by site directed mutagenesis using BPM565 as a template. Mutations created the following changes in the PY1 motif: P500A and F502A. This paper BPM567 pRS313-GPD-PDC1-PY2*-13 MYC -PGK Plasmid was created by site directed mutagenesis using BPM565 as a template. Mutations created the following changes in the PY2 motif: P541A and F543A. This paper BPM568 pRS313-GPD-PDC1-PY1/2*-13 MYC -PGK Plasmid was created by site directed mutagenesis using BPM565 as a template. Mutations created changes in the PY1 motif (P500A, F502A) and the PY2 motif (P541A, F543A). This paper   156 Strain ID Name Source & Note Reference BPM569 pRS315-YDJ1-PY* Plasmid was created by site directed mutagenesis using BPM390 as a template. Mutations created the following changes: P317G and P319G. This paper BPM573 pRS316-Rsp5 A PCR amplicon containing the Rsp5 gene and 5' and 3' regions was inserted into the yeast URA3, CEN/ARS vector using XbaI and SmaI sites This paper BPM575 pRS316-Rsp5 (C777A) Plasmid was created by site directed mutagenesis using BPM573 as a template. This paper BPM580 pRS313-GPD-EGFP-SUP45-PGK SUP45 was amplyfied from genomic DNA and insert into BPM519 with NotI and SalI sites This paper BPM583 pRS315-YDJ1-WT-3HA Generated using magaprimer method. See method section   This paper BPM584 pRS315-YDJ1-PY*-3HA Plasmid was created by site directed mutagenesis using BPM583 as a template. Mutations created the following changes: P317G and P319G. This paper BPM587 pRS426-GALp-HA-RSP5 HA-Rsp5 was inserted into the pRS426-GAL1p vector using EcoRI and NotI sites This paper BPM588 pRS426-GALp-HA-Rsp5-C777A HA-Rsp5 (C777A) was inserted into the pRS426-GAL1p vector using EcoRI and NotI sites This paper BPM589 pRS426-Galp-HA-rsp5-C2Δ HA-Rsp5-C2Δ was inserted into the pRS426-GAL1p vector using EcoRI and NotI sites This paper BPM591 pRS313-GPD-MycUb-K48-PGK MycUb-K48 was inserted between the GPD promoter and PGK terminator using BamHI of a yeast HIS3, CEN/ARS vector This paper BPM592 pRS313-GPD-MycUb-K63-PGK MycUb-K63 was inserted between the GPD promoter and PGK terminator using BamHI of a yeast HIS3, CEN/ARS vector This paper BPM595 pRS313-GPD-13MYC-Can1-PGK CAN1 was inserted into BPM173 using NotI This paper BPM596 pRS313-GPD-13MYC-Mup1-PGK MUP1 was inserted into BPM173 using NotI This paper    Supplementary Table 3.3 DiGly peptides quantified in figure. 3.3b ORF Peptide Sequence Modifications IonScore  Log(L/M) Log(L/H) YGL245W APAAkLDDATEDVFNK K5(GlyGl) 60 5.64 5.64 YGL105W APAGGAADAAAkADEDVSK K12(GlyGl) 62 5.64 5.64 YJL080C APIEIPLEkVcGSTEGENAEK K9(GlyGl), C11(Carbamidomethyl) 46 5.64 5.64 YKL081W DEAkPADDAAPAK K4(GlyGl) 59 5.64 5.64 YNL055C DGPLSTNVEAkLNDK K11(GlyGl) 52 5.64 5.64 YBR245C DIISPLLLNPTkR K12(GlyGl) 27 5.64 5.64 YKL060C DSkSPIILQTSNGGAAYFAGK K3(GlyGl) 71 5.64 5.64 YLR249W DSLGALSkALK K8(GlyGl) 41 5.64 5.64 YCL037C EGDNVTGEAkEPSPLDK K10(GlyGl) 72 5.64 5.64   157 ORF Peptide Sequence Modifications IonScore  Log(L/M) Log(L/H) YNL121C EkFGDIDTATATPTELSTQPAK K2(GlyGl) 50 5.64 5.64 YEL037C EkVPLDLEPSNTILETK K2(GlyGl) 37 5.64 5.64 YLR087C ESSkDPEFLK K4(Label:2H(4)+GlyGly) 29 5.64 5.64 YLR249W EVkAAATAAMTK K3(GlyGl) 45 5.64 5.64 YDR003W GEFHPNGkTEYLAPPPLSEEQASSTDK K8(GlyGl) 61 5.64 5.64 YKL081W GQDFAPAFDVAPDWESYEYTkLDPTK K21(GlyGl) 77 5.64 5.64 YER165W HEDAVkAVEALNDSELNGEK K6(GlyGl) 49 5.64 5.64 YDR172W IkGVEEEDISPGFVLTSPK K2(GlyGl) 45 5.64 5.64 YPL106C IPTLkQSISEAFGKPLSTTLNQDEAIAK K5(GlyGl) 50 5.64 5.64 YER165W kAIEQLNYTPIK K1(GlyGl) 34 5.64 5.64 YJR123W kAQcPIIER K1(Label:2H(4)+GlyGly), C4(Carbamidomethyl) 33 5.64 5.64 YDR409W kAVLQDLIR K1(Label:13C(6)15N(2)+GlyGly) 41 5.64 5.64 YBR215W kEPtAkSPK K1(GlyGl), T4(Phosp), K6(GlyGl) 27 5.64 5.64 YGR066C kFILER K1(GlyGl) 26 5.64 5.64 YJL130C kGETLQDTIR K1(GlyGl) 56 5.64 5.64 YDR341C kGTVVFLDNILEETK K1(GlyGl) 62 5.64 5.64 YER072W kIALPTR K1(GlyGl) 30 5.64 5.64 YKL081W kKDEAKPADDAAPAK K1(GlyGl) 34 5.64 5.64 YLR249W KkELGDAYVSSDEEF K2(GlyGl) 34 5.64 5.64 YJL080C KPTPLPSLkDLPSLGSNAAFANVK K9(GlyGl) 55 5.64 5.64 YDR508C kSSYITVDGIK K1(GlyGl) 49 5.64 5.64 YNL121C kSTAPSNPPIYPVSSNGEPDFSNK K1(GlyGl) 72 5.64 5.64 YOR198C kVVADDLVLVTPK K1(GlyGl) 32 5.64 5.64 YPL106C LEEEYAPFASDAEkTK K14(GlyGl) 70 5.64 5.64 YDL229W LSSEEIEkMVNQAEEFK K8(GlyGl) 81 5.64 5.64 YPL265W QNPDNFPVkEQEIYNIDLEENNVSSR K9(Label:2H(4)+GlyGly) 59 5.64 5.64 YGL009C SDDTPAKPSSSGMkPFLTLEGISAPLDK K14(GlyGl) 51 5.64 5.64 YNL121C SPESYDkADESFTK K7(GlyGl) 39 5.64 5.64 YGR191W TDSAFPLSSkDSPGINQTTNDITSSDR K10(Label:2H(4)+GlyGly) 100 5.64 5.64 YNL088W TEEEENAPSSTSSSSIFDIkK K20(GlyGl) 86 5.64 5.64 YDR497C TSQSNVGDAVGNADSVEFNSEHDSPSkR K27(GlyGl) 79 5.64 5.64 YOR275C TVVESSLkEAQR K8(GlyGl) 29 5.64 5.64 YPR163C VDAAVEkLQDK K7(GlyGl) 31 5.64 5.64 YJL212C ESDSLESEPSPTPTTIPIQINMEEEkK K26(GlyGl) 53 5.64 5.64 YIL053W EkGcDIIVK K2(GlyGl), C4(Carbamidomethyl) 44 5.64 5.64 YOR042W DSSLLEkSNVPESINEDISK K7(GlyGl) 63 5.64 5.64 YNL268W kkHGSLQGGAIADVNSITNSLTR K1(Label:2H(4)+GlyGly), K2(Label:2H(4)+GlyGly) 29 5.64 5.64 YLR044C kLIDLTQFPAFVTPMGK K1(GlyGl) 35 5.64 5.64 YJL130C EkQELLSSVQK K2(GlyGl) 33 5.64 5.64 YDR365W-B EVHTNQDPLDVSASkTEEcEK K15(GlyGl), C19(Carbamidomethyl) 49 5.64 5.64 YIL053W LEGEIPEkYGEHSIEVPGAVK K8(GlyGl) 39 5.64 5.64 YBR025C DIEFAQkALEGAEK K7(GlyGl) 54 5.64 5.64 YOL086C LPLVGGHEGAGVVVGMGENVkGWK K21(GlyGl) 64 5.64 5.64 YDR038C DGVkITPLTDcDVETIR K4(Label:2H(4)+GlyGly), C11(Carbamidomethyl) 80 5.64 5.64 YDL229W kTGLDISDDAR K1(GlyGl) 41 5.64 5.64 YPL265W NSSSLDSDHDAYYSkQNPDNFPVK K15(GlyGl) 65 5.64 5.64 YOL139C kFEENVSVDDTTATPK K1(GlyGl) 42 5.64 5.64   158 ORF Peptide Sequence Modifications IonScore  Log(L/M) Log(L/H) YBL047C AEPTkVATPSIPQQPIPLK K5(GlyGl) 37 5.64 5.64 YGL245W KAPAAkLDDATEDVFNK K6(GlyGl) 35 5.64 5.64 YBR025C DDSAIkAAGK K6(GlyGl) 37 5.64 5.64 YDL171C LDNkLIDEAEVTLDR K4(GlyGl) 58 5.64 5.64 YCR088W TPSPAPAAkISSR K9(Label:13C(6)15N(2)+GlyGly) 38 5.64 5.64 YPL004C APTAAELQAPPPPPSSTkSK K18(GlyGl) 52 5.64 5.64 YNL121C STAPSNPPIYPVSSNGEPDFSNkANFTAEEK K23(GlyGl) 46 5.64 5.64 YGR178C TPSAkTVSPTTQISAGK K5(GlyGl) 53 5.64 5.64 YBR143C DAEDNEVIkFAEPEAK K9(GlyGl) 41 5.64 5.64 YBL007C DDELQNDVVGSAAGkR K15(Label:2H(4)+GlyGly) 57 5.64 5.64 YDL229W FDDESVQkDmK K8(GlyGl), M10(Oxidation) 30 5.64 5.64 YFL004W KPLNLLkNAGPVNVEAK K7(GlyGl) 48 5.64 3.93 YDL229W STGkSSNITISNAVGR K4(GlyGl) 42 5.64 2.62 YLR249W FPEPGYLEGVkTK K11(GlyGl) 52 5.64 1.63 YIL078W DVmEQQGkNATVSVEEVLK M3(Oxidation), K8(GlyGl) 64 5.62 5.64 YPL106C LNELIEkENEMLAQDK K7(GlyGl) 60 5.6 4.81 YIL078W DGAVkEATSWETTPMDIAK K5(GlyGl) 33 5.44 5.64 YLR044C DAkNPVILADAccSR K3(GlyGl), C12(Carbamidomethyl), C13(Carbamidomethyl) 44 5.43 1.01 YPL106C kDTEGDVDMD K1(GlyGl) 42 5.38 5.64 YOR198C QEEQTkQNIESVEK K6(GlyGl) 35 5.35 5.61 YOR089C IPLkTAEEQNSASNER K4(GlyGl) 45 5.33 5.58 YGL009C DQDQSSPkVEVTSEDEK K8(GlyGl) 57 5.26 5.52 YPL106C KLNELIEkENEMLAQDK K8(GlyGl) 28 5.21 5.47 YLR441C NVGkTLVNK K4(GlyGl) 30 5.2 5.46 YNR016C TPSPGkLVK K6(GlyGl) 25 5.2 5.45 YDR155C kVESLGSPSGATK K1(GlyGl) 55 5.18 1.69 YGL105W KAPAGGAADAAAkADEDVSK K13(GlyGl) 40 5.12 5.38 YOR335C ETLGNDVDQkGSLVAPEK K10(GlyGl) 37 5.01 5.27 YKL178C ETDDILDEIDLkESR K12(Label:2H(4)+GlyGly) 51 4.99 5.24 YLR342W kAMEEANPEDTEETLNK K1(GlyGl) 49 4.96 5.22 YGR180C DTEDFQkLTDDQK K7(GlyGl) 28 4.95 5.2 YKL060C EDLYTkPEQVYNVYK K6(GlyGl) 42 4.84 2.17 YFL004W kPLNLLKNAGPVNVEAK K1(GlyGl) 37 4.82 1.22 YGL206C ETkDGTNSVAIVDLAK K3(GlyGl) 31 4.65 2.3 YNL268W kHGSLQGGAIADVNSITNSLTR K1(Label:2H(4)+GlyGly) 58 4.60 4.73 YBR025C kLQTISALPK K1(GlyGl) 31 4.57 4.83 YPL226W ASNLAkPSVDDDDSPANIK K6(GlyGl) 58 4.5 4.76 YDL171C EPkVVDLEDAVPDSK K3(GlyGl) 55 4.43 2.62 YKL060C SPIILQTSNGGAAYFAGkGISNEGQNASIK K18(GlyGl) 109 4.31 1.97 YDR497C VHELkYEPTQEIIEDI K5(Label:2H(4)+GlyGly) 48 4.2 4.46 YDL229W KVEkAVITVPAYFNDAQR K4(GlyGl) 40 4.19 1.47 YMR079W kLLEDAGFIER K1(GlyGl) 49 4.14 5.64 YPL106C ENEMLAQDkLVAETEDR K9(GlyGl) 64 4.13 1.61 YNR016C NPEYNPDkLLGAVVEPLADIAHK K8(GlyGl) 35 4.03 1.9 YML123C kIHDTSDEDMAINGLER K1(Label:2H(4)+GlyGly) 35 3.69 3.45 YJL080C kPTPLPSLK K1(GlyGl) 26 3.68 3.94 YER056C SPVIGSSLENEkK K12(GlyGl) 62 3.51 4.73 YLR249W MPELIPVLSETMWDTkK K16(GlyGl) 72 3.28 2.44   159 ORF Peptide Sequence Modifications IonScore  Log(L/M) Log(L/H) YBR118W VETGVIkPGMVVTFAPAGVTTEVK K7(GlyGl) 55 3.26 3.71 YER068W ELHNkQQAQQQSGGTAFTR K5(Label:13C(6)15N(2)+GlyGly) 27 3.22 4.7 YDR266C SPSASYDPFATTVkK K14(GlyGl) 43 3.22 1.12 YDL126C EVkVEGEDVEMTDEGAK K3(GlyGl) 39 3.02 1.6 YGL008C kGLDAIDK K1(GlyGl) 46 2.93 2.16 YBR118W LPLQDVYkIGGIGTVPVGR K8(GlyGl) 47 2.8 2.89 YDL229W VEkAVITVPAYFNDAQR K3(GlyGl) 42 2.75 2.1 YLL039C LIFAGkQLEDGR K6(GlyGl) 60 2.74 1.02 YDR385W DLEGkALLK K5(GlyGl) 43 2.57 2.56 YDL171C DGGYkHVNEPTAIASLQDTVR K5(GlyGl) 32 2.57 2.3 YDR098C-B;YDR365W-B kNIADVMTKPLPIK K1(GlyGl) 26 2.42 2.71 YOL109W EEQNIADGVEQkK K12(GlyGl) 66 2.4 2.4 YDR098C-B;YDR365W-B AHNVSTSNNSPSTDNDSISkSTTEPIQLNNK K20(GlyGl) 40 2.31 1.32 YKR039W EVDLDLLkQEIAEEK K8(GlyGl) 41 2.26 3.34 YGL206C DGTNSVAIVDLAkGNEVTR K13(GlyGl) 53 2.23 2.82 YCR012W VLENTEIGDSIFDkAGAEIVPK K14(GlyGl) 71 2.07 1.31 YBR068C kETSPDSISIR K1(GlyGl) 42 2.04 5.64 YOL109W EQAEASIDNLkNEATPEAEQVK K11(GlyGl) 85 2.03 2.25 YOR007C kVLDIEGDNATEAMK K1(GlyGl) 56 1.98 2.18 YPL106C EELEELVkPLLER K8(GlyGl) 26 1.93 2.11 YOL086C EkDIVGAVLK K2(GlyGl) 46 1.89 2.73 YOL109W LEETkESLQNK K5(GlyGl) 37 1.81 2.56 YLL039C TLTGkTITLEVESSDTIDNVK K5(GlyGl) 102 1.41 1.15 YGR254W IEEELGDNAVFAGENFHHGDkL K21(GlyGl) 30 1.31 1.66 YAL038W SEELYPGRPLAIALDTkGPEIR K17(GlyGl) 35 1.3 2.2 YGL137W GEIEEAIENVLPNVEGkDSLTK K17(GlyGl) 33 1.13 1.27 YHR064C DLkTGNAVK K3(GlyGl) 28 5.64 -0.01 YKL081W EIVDGkVLK K6(GlyGl) 34 5.44 0.45 YER143W GVGTGkIIGR K6(GlyGl) 32 5.22 0.85 YML048W DLTSPSANGAkNSGGNNNTTDLK K11(GlyGl) 49 5.13 0.7 YPR062W EGGVPIGGcLINNkDGSVLGR C9(Carbamidomethyl), K14(GlyGl) 37 4.98 0.41 YGR157W IPQNkDANSNFSTNSNSFSEK K5(GlyGl) 66 4.96 0.62 YJR105W ENDAILVDAkSGDAK K10(GlyGl) 31 4.96 0.34 YDR266C NLPTLkSPSASYDPFATTVK K6(GlyGl) 55 4.81 0.54 YDR098C-B EVHTNQDPLDVSASkIQEYDK K15(GlyGl) 36 4.8 -0.95 YKL081W TPFAEVkLAEK K7(GlyGl) 33 4.56 0.95 YDR127W DVLkPMGcK K4(GlyGl), C8(Carbamidomethyl) 27 4.37 0.4 YPR041W NPETEIIITkDNDLVR K10(GlyGl) 51 4.36 0.76 YLR249W AYEELSNTDLEFkFPEPGYLEGVK K13(GlyGl) 62 4.31 0.91 YGR254W DQkAVDDFLISLDGTANK K3(GlyGl) 62 4.26 -1.42 YHL033C kVAPAPFGAK K1(GlyGl) 32 3.99 0.73 YGR103W kLQVSLADFR K1(Label:13C(6)15N(2)+GlyGly) 34 3.58 -1.25 YOR316C ASkILLQATPSTLSGDQVEGDLLK K3(GlyGl) 83 3.47 -0.48 YGL245W DVVPVDLVDFDHLITkDR K16(GlyGl) 36 3.08 0.21 YLR249W kSAVIIDNMcK K1(GlyGl), C10(Carbamidomethyl) 28 3.05 0.55 YAL003W IETLkQLNASLADK K5(GlyGl) 64 2.77 -0.36 YBL032W kLEAAEGDATVVTER K1(GlyGl) 28 2.69 0.43 YAL005C NTISEAGDkLEQADK K9(GlyGl) 61 2.59 0.59   160 ORF Peptide Sequence Modifications IonScore  Log(L/M) Log(L/H) YMR229C GGASALTPLELkQVANEAASDVLFGNESVK K12(GlyGl) 89 2.52 0.58 YNL064C kATVDEcVLADFDPAK K1(GlyGl), C7(Carbamidomethyl) 28 2.51 -0.25 YNL121C NPTVENFIEATNLLEkASK K16(GlyGl) 65 2 0.14 YOR369C VPLIkVADAK K5(GlyGl) 32 1.85 -0.16 YGR264C DSSSFkNIGAVK K6(GlyGl) 49 1.8 0.51 YLL024C NTISEAGDkLEQADKDAVTK K9(GlyGl) 28 1.78 0.58 YLR249W cPAAkAYEELSNTDLEFK C1(Carbamidomethyl), K5(GlyGl) 58 1.6 0.76 YBR177C DNkEQVDFDEFANK K3(GlyGl) 50 1.51 -0.39 YNL178W TGPkALPDAVTIIEPK K4(GlyGl) 45 1.5 0.87 YDR385W DTDAEGkPLER K7(GlyGl) 44 1.21 0.57 YKL210W KPLLESGTLGTkGNTQVIIPR K12(GlyGl) 58 1.2 -0.3 YLR249W VGNVGEDDAIPEVSHAGDVSTTLQVVNELLkDETVAPR K31(GlyGl) 45 0.97 0.91 YGR192C ELDTAQkHIDAGAK K7(GlyGl) 26 0.95 2.15 YCL009C TPLkTSTEEAADEDEK K4(GlyGl) 66 0.88 -1.2 YLR150W IETAEkEAYVPATK K6(GlyGl) 49 0.83 0.36 YDR508C EkQIGSIEPENEVEYFEK K2(GlyGl) 51 0.8 3.96 YMR079W EFLESYPQNcPPDALPGTPGNLDSAQEkALAELR C10(Carbamidomethyl), K28(GlyGl) 29 0.8 2 YLL039C TLSDYNIQkESTLHLVLR K9(GlyGl) 66 0.76 1.12 YFL010C GPPPGVNNEkSSR K10(GlyGl) 45 0.72 5.26 YOR375C VDQELkR K6(GlyGl) 37 0.7 1.06 YHL016C EQEEETNSLVSDSEkNDVR K15(GlyGl) 65 0.65 4.63 YMR230W KDFNQAkHEEIDTK K7(GlyGl) 33 0.56 0.14 YGL147C DGAkFIEVR K4(GlyGl) 31 0.55 -0.09 YER023W EQKPLVNELISQVGkYVELPEK K15(GlyGl) 36 0.44 0.43 YML028W kTAVVDGVFDEVSLDK K1(GlyGl) 74 0.32 -0.3 YML123C NkNNDIESSSPSQLQHEA K2(GlyGl) 54 0.23 2.07 YGL008C kADTGIAVEGATDAAR K1(GlyGl) 38 0.06 1.78 YDR418W VGEDIAkATK K7(GlyGl) 30 0.05 0.82 YJL177W SVkFVQGLLQNAAANAEAK K3(GlyGl) 65 0.05 0.21 YLL039C TITLEVESSDTIDNVkSK K16(GlyGl) 76 0.03 0.69 YBR181C kGEQELEGLTDTTVPK K1(GlyGl) 57 0.03 0.39 YLR109W DTTHIkFASDPGcAFTK K6(GlyGl), C13(Carbamidomethyl) 67 -0.22 0.06 YOL058W kAHVDLEGLTLDK K1(GlyGl) 42 -0.29 0.08 YER009W DIVEkLVSLPFQK K5(GlyGl) 44 -0.31 0.65 YGL077C VEEEIkPLDDmDSK K6(GlyGl), M11(Oxidation) 31 -0.43 1.34 YNL178W ALPDAVTIIEPkEEEPILAPSVK K12(GlyGl) 51 -0.64 -3.33 YML123C LELAAAAQEQDGEkK K14(GlyGl) 52 -1 4.56 YMR242C DLkFPLPHR K3(GlyGl) 32 -1.1 -0.21 YOL119C EGPSSGYNPNFNAADAILkK K19(GlyGl) 42 -1.16 2.97 YHL015W EkVEEQEQQQQQIIK K2(GlyGl) 85 -1.22 -0.42 YOL119C kNSDQVDLDVNK K1(GlyGl) 42 -1.35 0.2 *ORF: yeast open reading frame; Peptide Sequence: peptides that contain di-gly (ubiquitylation) sites on lysine (lowercase); Modifications: modifications on the peptide in the left column with modified a.a. indicated; IonScore: Mascot ion score reported by Proteome Discoverer. For peptides that were identified more than once, the average ion   161 scores are shown; Log: Log2 ratio; L(light): WT HS, M(medium): WT noHS, H(heavy): rsp5-1 HS.   Supplementary Table 3.4 AQUA ubiquitin peptides used in SRM analysis Name Squence with tag (* residuces are labeled) Charge AQUA Precursor (m/z) Transition T1 (m/z) Transition T2 (m/z) Transition T3 (m/z) Fragmentor Voltage  T1 Collision Energy  T2 Collision Energy  T3 Collision Energy  ∆ Retention Time (min) Retention Time (min) 48GG LIFAGK(GlyGly)QLEDGR*-JPTtag 3+ 490.94 199.18 242.15 599.3 140 10 15 15 5 12 48N LIFAGK*-JPTtag 2+ 328.72 199.18 212.14 430.25 110 5 5 10 5 9.6 63GG TLSDYNIQK(GlyGly)ESTLHLVLR*-JPTtag 4+ 564.31 647.42 510.37 136.07 90 19 19 18 5 17.8 63N TLSDYNIQK*-JPTtag 2+ 545.29 187.14 875.43 673.37 110 15 14 15 5 7.8    Supplementary Table 3.5 DiGly peptides comparison between ubp2Δ and WT upon HS  ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YLR413W IIEEHESPIDAEkNFAR K13(Label:2H(4)+GlyGly) 40 0.8 5.64 YLL045C AEDEAALAkLVSTIDANFADK K9(GlyGl) 92 0.59 5.64 YPL106C AEEWLYDEGFDSIkAK K14(GlyGl) 73 0.59 5.64 YGL105W AGDkVFFEGFGDEAPMK K4(GlyGl) 54 0.59 5.64 YKL056C AkLQETNPEEVPK K2(GlyGl) 42 0.59 5.64 YOL086C ANGTTVLVGMPAGAkccSDVFNQVVK K15(GlyGl), C16(Carbamidomethyl), C17(Carbamidomethyl) 72 0.59 5.64 YGL105W APAGGAADAAAkADEDVSK K12(GlyGl) 59 0.59 5.64 YMR295C ASDVkISEDDK K5(GlyGl) 43 0.59 5.64 YPL226W ASNLAkPSVDDDDSPANIK K6(GlyGl) 72 0.59 5.64 YER165W AVEALNDSELNGEkLYVGR K14(Label:2H(4)+GlyGly) 51 0.59 5.64 YBR025C cEDVFEYkDDSAIK C1(Carbamidomethyl), K8(GlyGl) 45 0.59 5.64 YJL130C cIVNPSPASITASAELQSTSAkR C1(Carbamidomethyl), K22(Label:13C(6)15N(2)+GlyGly) 79 0.59 5.64 YBR143C DAEDNEVIkFAEPEAK K9(GlyGl) 38 0.59 5.64 YJL080C DAENSVTkTIDIPAER K8(Label:2H(4)+GlyGly) 36 0.59 5.64 YBL007C DDELQNDVVGSAAGkR K15(Label:13C(6)15N(2)+GlyGly) 67 0.59 5.64 YBR069C DFTITEkQDEVSGQTAEPR K7(Label:13C(6)15N(2)+GlyGly) 56 0.59 5.64 YLR249W DkEIQSVASETLISIVNAVNPVAIK K2(GlyGl) 102 0.59 5.64 YOL122C DkkDSTVVIEGEAPVR K2(Label:13C(6)15N(2)+GlyGly), K3(Label:13C(6)15N(2)+GlyGly) 33 0.59 5.64 YBR143C DkSFAIDK K2(GlyGl) 40 0.59 5.64   162 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YDL190C DPVILPASkMNIDR K9(GlyGl) 41 0.59 5.64 YKL060C DSkSPIILQTSNGGAAYFAGK K3(GlyGl) 75 0.59 5.64 YGR264C DSSSFkNIGAVK K6(GlyGl) 51 0.59 5.64 YPL226W DVLVEHGFEkLVQK K10(GlyGl) 30 0.59 5.64 YIL078W DVMEQQGkNATVSVEEVLK K8(GlyGl) 69 0.59 5.64 YLR354C DYkGEADPGVISVK K3(GlyGl) 51 0.59 5.64 YBL042C EAFEEYGGVSTGYEkIR K15(Label:13C(6)15N(2)+GlyGly) 61 0.59 5.64 YHR108W EDNAVQAkQAISSELNK K8(GlyGl) 66 0.59 5.64 YLR314C EEQVSIkQDPEQEER K7(Label:2H(4)+GlyGly) 52 0.59 5.64 YPR062W EGGVPIGGcLINNkDGSVLGR C9(Carbamidomethyl), K14(Label:2H(4)+GlyGly) 39 0.59 5.64 YDL229W;YNL209W EIAEAkIGK K6(GlyGl) 34 0.59 5.64 YDL225W EIkQENENLIR K3(Label:2H(4)+GlyGly) 33 0.59 5.64 YGR116W EIQkGSLYEDIK K4(GlyGl) 31 0.59 5.64 YJL145W EkcAGYDELYGYK K2(GlyGl), C3(Carbamidomethyl) 42 0.59 5.64 YNL121C EkFGDIDTATATPTELSTQPAK K2(GlyGl) 64 0.59 5.64 YER068W ELHNkQQAQQQSGGTAFTR K5(Label:2H(4)+GlyGly) 48 0.59 5.64 YMR079W ELLkQIPAENLPVK K4(GlyGl) 26 0.59 5.64 YGR162W ELNPDITDETNEGkTGPK K14(GlyGl) 36 0.59 5.64 YPL226W EMMTkEIDIDDGR K5(Label:2H(4)+GlyGly) 27 0.59 5.64 YNL121C ENkFDDcETLFSEAK K3(GlyGl), C7(Carbamidomethyl) 32 0.59 5.64 YPR163C ENkVDAAVEK K3(GlyGl) 43 0.59 5.64 YCR051W EQMLGQGSMAGSGDEPDSkR K19(Label:2H(4)+GlyGly) 61 0.59 5.64 YIL121W ETADkLALTR K5(Label:13C(6)15N(2)+GlyGly) 75 0.59 5.64 YDR261C-D EVHTNQDPLDVSASkTEEcEK K15(GlyGl), C19(Carbamidomethyl) 81 0.59 5.64 YLR044C EVIDTILALVkDAK K11(GlyGl) 41 0.59 5.64 YLR249W EVkAAATAAmTK K3(GlyGl), M10(Oxidation) 56 0.59 5.64 YNL121C FDDcETLFSEAkR C4(Carbamidomethyl), K12(Label:2H(4)+GlyGly) 63 0.59 5.64 YGR155W FDENSkLSDLNR K6(Label:2H(4)+GlyGly) 32 0.59 5.64 YNL121C FGDIDTATATPTELSTQPAkER K20(Label:2H(4)+GlyGly) 53 0.59 5.64 YMR079W FGGkSEVDESK K4(GlyGl) 36 0.59 5.64 YER165W FGPIVSASLEkDADGK K11(GlyGl) 53 0.59 5.64 YFR010W FNDDkVSVVEK K5(GlyGl) 50 0.59 5.64 YPL274W FTkIETESTTIPNDSDR K3(Label:13C(6)15N(2)+GlyGly) 97 0.59 5.64 YPL106C GkLEEEYAPFASDAEK K2(GlyGl) 42 0.59 5.64 YOR335C IDGPGFEkAK K8(GlyGl) 36 0.59 5.64 YOR323C IDLAVAkETGLADSLLK K7(GlyGl) 82 0.59 5.64 YCR088W IDLQkVIAEEK K5(GlyGl) 32 0.59 5.64 YHR174W IGLDcASSEFFkDGK C5(Carbamidomethyl), K12(GlyGl) 40 0.59 5.64 YGR234W IIVHTDTEPLINAAFLkEK K17(GlyGl) 44 0.59 5.64 YPL106C ILTAAEkLK K7(GlyGl) 31 0.59 5.64 YJL012C IPkGTTFDTQIR K3(Label:13C(6)15N(2)+GlyGly) 37 0.59 5.64 YGR157W IPQNkDANSNFSTNSNSFSEK K5(GlyGl) 62 0.59 5.64 YER125W IPVNGFkDLQGSDGPR K7(Label:2H(4)+GlyGly) 32 0.59 5.64 YJL080C IVkDAENSVTK K3(GlyGl) 52 0.59 5.64 YDR155C IVVAkSGEL K5(Label:2H(4)+GlyGly) 31 0.59 5.64 YER165W kAIEQLNYTPIK K1(GlyGl) 59 0.59 5.64 YML123C kIHDTSDEDMAINGLER K1(Label:13C(6)15N(2)+GlyGly) 32 0.59 5.64 YJL080C kSGDIVILGPR K1(GlyGl) 56 0.59 5.64   163 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YNL121C kSTAPSNPPIYPVSSNGEPDFSNK K1(GlyGl) 61 0.59 5.64 YKL060C kTGVIVGEDVHNLFTYAK K1(GlyGl) 39 0.59 5.64 YAL005C;YLL024C LDkSQVDEIVLVGGSTR K3(Label:2H(4)+GlyGly) 54 0.59 5.64 YGL009C LDQQIIIDkLIPIANK K9(GlyGl) 42 0.59 5.64 YPL106C LEEEYAPFASDAEkTK K14(GlyGl) 62 0.59 5.64 YNL121C LFEEQLDkNNEDEK K8(GlyGl) 49 0.59 5.64 YHR042W LGkLGEADDGAGTTDEDYMAWK K3(GlyGl) 76 0.59 5.64 YGL195W LLDTLSDESkSGDR K10(Label:2H(4)+GlyGly) 46 0.59 5.64 YJR105W LLNENEkAGVK K7(GlyGl) 36 0.59 5.64 YER124C LNDDQEDIVFTkR K12(Label:13C(6)15N(2)+GlyGly) 73 0.59 5.64 YNR016C LPAkLDEQMEELVAR K4(Label:2H(4)+GlyGly) 51 0.59 5.64 YOL086C LPLVGGHEGAGVVVGMGENVkGWK K21(GlyGl) 50 0.59 5.64 YNL243W LPVDAPDVFLINDVDESkEIK K18(GlyGl) 48 0.59 5.64 YGR264C NLkPEVDNDNAAMELR K3(Label:2H(4)+GlyGly) 61 0.59 5.64 YGL195W NPEIQkLVPVLLQAIGDPTK K6(GlyGl) 74 0.59 5.64 YPR041W NPETEIIITkDNDLVR K10(Label:2H(4)+GlyGly) 42 0.59 5.64 YNL121C NPTVENFIEATNLLEkASK K16(GlyGl) 61 0.59 5.64 YER165W NQQIVAGkPLYVAIAQR K8(Label:2H(4)+GlyGly) 71 0.59 5.64 YLR220W NSAQDLENSPMSVGkDNR K15(Label:13C(6)15N(2)+GlyGly) 75 0.59 5.64 YIL053W PLTTkPLSLK K5(GlyGl) 49 0.59 5.64 YDL229W;YNL209W QATkDAGAISGLNVLR K4(GlyGl) 84 0.59 5.64 YPL106C QSISEAFGkPLSTTLNQDEAIAK K9(GlyGl) 82 0.59 5.64 YMR319C QYEEFGDSNDYkNDDVVR K12(Label:13C(6)15N(2)+GlyGly) 41 0.59 5.64 YGR180C SATPSkEINFDDDF K6(Label:2H(4)+GlyGly) 45 0.59 5.64 YLR249W SAVIIDNMckLVEDPQVIAPFLGK C9(Carbamidomethyl), K10(GlyGl) 61 0.59 5.64 YCR030C SDLkSSTEYYNTLDQQVVR K4(GlyGl) 92 0.59 5.64 YNL173C SEGYVTDGLGkTQSSESR K11(Label:13C(6)15N(2)+GlyGly) 47 0.59 5.64 YPL106C SkQEASQMAAMAEK K2(GlyGl) 44 0.59 5.64 YIL078W SkQQSLYLDPEPTFIEER K2(Label:2H(4)+GlyGly) 45 0.59 5.64 YDR330W SNQEEVPSTGEEQkR K14(GlyGl) 38 0.59 5.64 YNL112W SNYNQPQELIkPNWDEELPK K11(GlyGl) 44 0.59 5.64 YLR414C SRPTVIYANAPIEEkPLI K15(Label:13C(6)15N(2)+GlyGly) 50 0.59 5.64 YJL012C TDIGVDWPFkQLDDK K10(GlyGl) 56 0.59 5.64 YBR143C TDLAkSELFDPR K5(GlyGl) 29 0.59 5.64 YNR016C TEQIEHILkSSVVK K9(GlyGl) 35 0.59 5.64 YGL195W TFVkSLSDATNETLR K4(GlyGl) 50 0.59 5.64 YHR064C TGNAVkGEL K6(Label:2H(4)+GlyGly) 53 0.59 5.64 YCR088W TPSPAPAAkISSR K9(Label:2H(4)+GlyGly) 55 0.59 5.64 YKR093W TQAVTLkDSYVSDDVANSTER K7(Label:2H(4)+GlyGly) 73 0.59 5.64 YGL167C TSQTIEkSSFNDQPNSIVPISER K7(Label:2H(4)+GlyGly) 51 0.59 5.64 YAL035W TTQENASEAIkSDSK K11(Label:13C(6)15N(2)+GlyGly) 55 0.59 5.64 YJR070C TVAEEFATkPEEAK K9(GlyGl) 35 0.59 5.64 YIL053W TYDAIAkFAPDFADEEYVNK K7(GlyGl) 60 0.59 5.64 YMR186W VDEGGAQDkTVK K9(GlyGl) 31 0.59 5.64 YER125W VDLPQYVDYDSMkQK K13(GlyGl) 43 0.59 5.64 YOR375C VDQELkR K6(Label:13C(6)15N(2)+GlyGly) 39 0.59 5.64 YOL086C VLGIDGGEGkEELFR K10(Label:2H(4)+GlyGly) 44 0.59 5.64 YPL106C VTEPVTkALAQAK K7(GlyGl) 60 0.59 5.64   164 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YIL053W VVVFEDAPAGIAAGkAAGcK K15(GlyGl), C19(Carbamidomethyl) 72 0.59 5.64 YER166W WNEkYDIAAASLANR K4(Label:2H(4)+GlyGly) 56 0.59 5.64 YDR429C YGSEkGSPAGPSAVTAR K5(Label:2H(4)+GlyGly) 56 0.59 5.64 YIL078W YVGkIETWDAAESK K4(GlyGl) 61 0.59 5.64 YLR342W LVGDESEkAAGDASR K8(Label:13C(6)15N(2)+GlyGly) 52 -0.21 5.64 YNL268W kHGSLQGGAIADVNSITNSLTR K1(Label:13C(6)15N(2)+GlyGly) 87 0.39 5.22 YIL078W DSMkDISESPER K4(Label:2H(4)+GlyGly) 31 2.13 4.91 YDR497C VHELkYEPTQEIIEDI K5(Label:13C(6)15N(2)+GlyGly) 53 3.87 4.57 YDL126C ETVEYPVLHPDQYTkFGLSPSK K15(GlyGl) 31 1.73 4.48 YLR044C DAkNPVILADAccSR K3(GlyGl), C12(Carbamidomethyl), C13(Carbamidomethyl) 49 2.35 4.33 YDL229W;YNL209W STGkSSNITISNAVGR K4(GlyGl) 62 2.33 4.24 YAL005C;YLL024C DNNLLGkFELSGIPPAPR K7(Label:2H(4)+GlyGly) 50 2.3 4.22 YDL171C LDNkLIDEAEVTLDR K4(Label:2H(4)+GlyGly) 59 -0.81 4.08 YML123C NkNNDIESSSPSQLQHEA K2(Label:2H(4)+GlyGly) 47 1.85 3.84 YDR508C kSSYITVDGIK K1(GlyGl) 74 -0.15 3.46 YDR038C DGVkITPLTDcDVETIR K4(GlyGl), C11(Carbamidomethyl) 58 -1.475 3.26 YHR174W YDLDFkNPESDK K6(GlyGl) 43 2.42 3.25 YGL195W NYATkAVDLLLPELER K5(GlyGl) 47 1.91 3.23 YJL012C APPGkTIcVPVR K5(GlyGl), C8(Carbamidomethyl) 30 1.42 3.23 YDL229W;YNL209W FDDESVQkDMK K8(GlyGl) 46 3.615 3.15 YDL229W;YNL209W kTGLDISDDAR K1(GlyGl) 59 3.04 3.15 YDL229W;YNL209W MVNQAEEFkAADEAFAK K9(GlyGl) 62 3.62 3.12 YPL106C QEASQMAAMAEkLAAQR K12(Label:2H(4)+GlyGly) 65 2.27 3.12 YNL287W SETTLDTTPEAESVPEkR K17(Label:2H(4)+GlyGly) 51 0.56 3.12 YDL229W;YNL209W LIGDAAkNQAALNPR K7(GlyGl) 52 2.54 3.11 YLR249W NLTEEVWAVkDGR K10(GlyGl) 46 3.07 3.09 YAL030W;YOR327C DNINkVAER K5(GlyGl) 35 0.96 3.08 YLR043C VVGANPAAIkQAIAANA K10(GlyGl) 34 2.25 2.98 YLR249W VLEELFQkLSVATADNR K8(Label:2H(4)+GlyGly) 60 2.58 2.9 YDR210C-D SLEDNETEIkVSR K10(Label:2H(4)+GlyGly) 39 1.73 2.81 YLR249W LLPGLkSNFATIADPEAR K6(Label:2H(4)+GlyGly) 36 1.11 2.63 YDR508C QIGSIEPENEVEYFEkTVEK K16(GlyGl) 57 -2.76 2.52 YLR167W TLSDYNIQkESTLHLVLR K9(GlyGl) 43 1.85 2.44 YLR167W LIFAGkQLEDGR K6(GlyGl) 48 1.54 2.4 YBR118W TLLEAIDAIEQPSRPTDkPLR K18(Label:2H(4)+GlyGly) 50 1.58 2.23 YDL229W;YNL209W VNckENTLLGEFDLK C3(Carbamidomethyl), K4(GlyGl) 57 4.62 1.99 YOR042W VVAETTYIDTPDTETkK K16(GlyGl) 51 1.8 1.94 YNL096C VLLDSkDVQQIDYK K6(GlyGl) 45 0.91 1.4 YOR375C STATGPSEAVWYGPPkAANLGGVAVSGLEMAQNSQR K16(Label:13C(6)15N(2)+GlyGly) 113 3.5 1.01 YBR118W FLkSGDAALVK K3(GlyGl) 35 2.95 0.91 YDL126C kTPLEPGLELTAIAK K1(GlyGl) 45 3.29 0.88 YPR159W LDLSQNkGVSDYK K7(GlyGl) 29 3.17 0.86 YBL047C NDPIVDASLSkGPIVNR K11(GlyGl) 99 0.7 0.74 YDL126C EHFAEAMkTAK K8(GlyGl) 41 -0.09 0.65   165 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YLR167W TITLEVESSDTIDNVkSK K16(GlyGl) 84 -0.31 0.63 YLR167W QIFVkTLTGK K5(GlyGl) 63 -0.13 0.62 YEL063C MTNSkEDADIEEK K5(GlyGl) 70 -0.83 0.58 YEL063C TNSkEDADIEEK K4(GlyGl) 56 -0.84 0.48 YNL178W TGPkALPDAVTIIEPK K4(GlyGl) 36 0.64 0.44 YEL037C TkVTEPPIAPESATTPGR K2(GlyGl) 48 4.86 0.4 YER133W GSkPGQQVDLEENEIR K3(GlyGl) 52 4.02 0.4 YHL015W YIDLEAPVQIVkR K12(GlyGl) 53 2.66 0.31 YHR039C-A EFEQkNAGGVGELEK K5(GlyGl) 29 4.32 0.25 YBR023C ETDSYLLQDMNTTDkK K15(GlyGl) 59 1.13 0.25 YML063W NVGkTLVNK K4(GlyGl) 38 1.24 0.19 YDR090C IEQDISkSDR K7(GlyGl) 31 -0.19 0.18 YGR082W ELEAASkFYK K7(GlyGl) 43 4.02 0.16 YGR136W TGDkIQVLEK K4(GlyGl) 87 1.24 0.15 YKL210W DPPEkSIPLcTLR K5(GlyGl), C10(Carbamidomethyl) 32 1.02 0.14 YEL037C TkLAQSIScEESQIK K2(GlyGl), C9(Carbamidomethyl) 46 4.19 0.12 YJR105W IVkDSPVEK K3(GlyGl) 40 3.61 0.12 YER043C VPAINVNDSVTkSK K12(GlyGl) 65 1.26 0.12 YBR031W;YDR012W GPLVVYAEDNGIVkALR K14(GlyGl) 43 2.44 0.1 YER009W DIVEkLVSLPFQK K5(GlyGl) 61 -0.31 0.1 YAL038W EVLGEQGkDVK K8(GlyGl) 33 1.33 0.09 YNL178W ALPDAVTIIEPkEEEPILAPSVK K12(GlyGl) 62 -0.55 0.09 YEL037C LAQSIScEESQIkLIYSGK C7(Carbamidomethyl), K13(GlyGl) 58 3.42 0.07 YLR044C kLIDLTQFPAFVTPMGK K1(GlyGl) 42 2.57 0.07 YGL195W EEQELVNEQLAkESAVR K12(GlyGl) 79 4.41 0.05 YGL008C TDTSSSSSSSSASSVSAHQPTQEkPAK K24(GlyGl) 75 5.14 0.03 YGR240C IPQQEATNkAQSQGALLK K9(GlyGl) 49 4.71 0.01 YHR108W ISESDLAVLkPSNQLK K10(GlyGl) 46 4 0.01 YHR027C kQQTIDEQSQISPEK K1(GlyGl) 56 -0.12 0 YOL086C SPIkVVGLSTLPEIYEK K4(GlyGl) 51 5.04 -0.01 YML097C DLTNDDTLLEkIR K11(GlyGl) 53 4.9 -0.01 YHR203C ITDEEASYkLGK K9(GlyGl) 51 4.9 -0.01 YDR139C TLTGkEISVELK K5(GlyGl) 39 2.31 -0.01 YOR063W VGkGDDEANGATSFDR K3(GlyGl) 31 1.66 -0.01 YDL136W EQLASQLVDLkK K11(GlyGl) 61 0.14 -0.02 YBL057C cPDkFTMDELYAK C1(Carbamidomethyl), K4(GlyGl) 35 -0.46 -0.03 YGL245W APAAkLDDATEDVFNK K5(GlyGl) 61 4.5 -0.04 YOL086C EkDIVGAVLK K2(GlyGl) 49 3.26 -0.04 YLR249W FPEPGYLEGVkTK K11(GlyGl) 50 0.41 -0.05 YML097C EEDVSSLIkK K9(GlyGl) 60 -0.34 -0.05 YKL081W kKDEAKPADDAAPAK K1(GlyGl) 36 5.64 -0.07 YDR385W DLEGkALLK K5(GlyGl) 44 2.69 -0.07 YNL287W NkDDVIAQNLIESK K2(GlyGl) 41 5.58 -0.08 YPL226W IPEcQSITDckNQIK C4(Carbamidomethyl), C10(Carbamidomethyl), K11(GlyGl) 35 4.67 -0.08 YKR100C DSkDEAALASSELK K3(GlyGl) 91 2.33 -0.08 YBL042C ESEmGDATkITSK M4(Oxidation), K9(GlyGl) 37 0.74 -0.08 YLR044C VATTGEWDkLTQDK K9(GlyGl) 42 5.64 -0.09 YGR234W VGDEIkLSAPAGDFAINK K6(GlyGl) 36 2.86 -0.09   166 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YHR108W TASEIQkEQEIAQAAK K7(GlyGl) 67 5.38 -0.1 YPL143W IEGVATPQDAQFYLGkR K16(GlyGl) 47 0.11 -0.11 YLR249W DSLGALSkALK K8(GlyGl) 60 3.62 -0.12 YOR046C IDNEkEDTSEVSTK K5(GlyGl) 76 4.2 -0.13 YLR167W TLTGkTITLEVESSDTIDNVK K5(GlyGl) 110 1.77 -0.14 YGL195W LSkEEQELVNEQLAK K3(GlyGl) 51 4.82 -0.18 YDR385W TGTLTTSETAHNMkVMK K14(GlyGl) 37 3.77 -0.18 YGL030W APVkSQESINQK K4(GlyGl) 42 1.36 -0.18 YHR203C YPDPNIkVNDTVK K7(GlyGl) 29 0.55 -0.18 YOL127W VIEQPITSETAMkK K13(GlyGl) 66 -0.04 -0.18 YML097C SEQSNKEEDVSSLIkK K15(GlyGl) 31 -0.23 -0.18 YGR135W TLSkTTDSSALTYDR K4(GlyGl) 73 -0.65 -0.18 YOR369C LVEGLANDPENkVPLIK K12(GlyGl) 71 1.68 -0.19 YLL024C NTISEAGDkLEQADK K9(GlyGl) 51 1.61 -0.19 YPL265W NSSSLDSDHDAYYSkQNPDNFPVK K15(GlyGl) 63 1.36 -0.2 YLL024C NTISEAGDkLEQADKDAVTK K9(GlyGl) 28 1.01 -0.21 YML097C SEQSNkEEDVSSLIK K6(GlyGl) 57 0.12 -0.21 YOL004W DFkSQAIDTPGVIER K3(GlyGl) 55 2.91 -0.22 YJR105W ENDAILVDAkSGDAK K10(GlyGl) 64 5.64 -0.23 YGR065C SSkTDVTAETTAVEPHPHNLR K3(GlyGl) 30 0.46 -0.23 YGR060W IPSAkEQLYcLK K5(GlyGl), C10(Carbamidomethyl) 87 -0.94 -0.24 YDR129C QQALEAVSkDGDATYDEAR K9(GlyGl) 67 3.88 -0.25 YGL147C DGAkFIEVR K4(GlyGl) 45 0.58 -0.25 YGR285C TPIPSLGNkDSSK K9(GlyGl) 51 5.64 -0.26 YPL226W EGNSTGEkIEEWK K8(GlyGl) 43 3.33 -0.26 YDR385W EEVPGWQEYYDkL K12(GlyGl) 32 3.02 -0.26 YER043C EcINIkPQVDR C2(Carbamidomethyl), K6(GlyGl) 35 2.24 -0.26 YML097C FNELFSPIGEPTQEEALkSEQSNK K18(GlyGl) 47 1.42 -0.26 YDL229W;YNL209W LSSEEIEkMVNQAEEFK K8(GlyGl) 74 4.6 -0.27 YOL022C NVDSLSEkMASTSLEK K8(GlyGl) 42 3.67 -0.27 YER145C EVLHVkADSL K6(GlyGl) 54 1.71 -0.27 YGL008C kADTGIAVEGATDAAR K1(GlyGl) 29 0.21 -0.28 YLR249W AAATAAMTkATETVDNK K9(GlyGl) 66 5.64 -0.31 YOR204W VGSTSENITQkVLYVENQDK K11(GlyGl) 67 5.64 -0.32 YLR249W LVEDPQVIAPFLGkLLPGLK K14(GlyGl) 53 4.38 -0.32 YDL229W;YNL209W FEDLNAALFkSTLEPVEQVLK K10(GlyGl) 87 4.34 -0.32 YDR155C VESLGSPSGATkAR K12(GlyGl) 52 5.19 -0.33 YHR174W DGkYDLDFK K3(GlyGl) 43 3.73 -0.33 YOR104W NDAkNDTFYDEVK K4(GlyGl) 48 1.33 -0.33 YFL010C GPPPGVNNEkSSR K10(GlyGl) 52 1.17 -0.33 YHR108W FQkIIEEEQEDDALVQDLLK K3(GlyGl) 100 0.32 -0.33 YKL180W ETAQAINGWELTkAQK K13(GlyGl) 54 0.3 -0.33 YKL182W cIAAcTGVPDDkWEQTYK C1(Carbamidomethyl), C5(Carbamidomethyl), K12(GlyGl) 60 0.04 -0.33 YER056C LEEGNNVYEIQDLEkR K15(GlyGl) 45 0.92 -0.34 YLR109W DTTHIkFASDPGcAFTK K6(GlyGl), C13(Carbamidomethyl) 55 0.15 -0.34 YPL106C TDLPEGEEkPR K9(GlyGl) 32 2.6 -0.35 YOR316C ASkILLQATPSTLSGDQVEGDLLK K3(GlyGl) 64 1.74 -0.35 YMR246W DILAAVkPDVER K7(GlyGl) 29 5.64 -0.36   167 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YLL024C EDIEkMVAEAEK K5(GlyGl) 40 3.58 -0.36 YHR203C VNDTVkIDLASGK K6(GlyGl) 79 0.77 -0.36 YHR203C IDLASGkITDFIK K7(GlyGl) 51 0.55 -0.36 YJR109C ELESVkSLSDLSK K6(GlyGl) 33 -0.86 -0.36 YDR054C GFIMPTSESAYISQSkLDEPESNK K16(GlyGl) 39 4.07 -0.37 YHR117W VLNENLSkDEGR K8(GlyGl) 40 1.21 -0.37 YKL180W SVkFVQGLLQNAAANAEAK K3(GlyGl) 72 0.26 -0.37 YBR118W FQEIVkETSNFIK K6(GlyGl) 40 3.06 -0.38 YLR342W ILAEETAAYEGNENEAEkEDALK K18(GlyGl) 97 1.23 -0.38 YBR159W TGkYcAITGASDGIGK K3(GlyGl), C5(Carbamidomethyl) 43 0.26 -0.38 YDR127W DVLkPMGcK K4(GlyGl), C8(Carbamidomethyl) 36 0.12 -0.38 YNR016C EILIQGALPSVkER K12(GlyGl) 41 5.64 -0.39 YMR079W kLLEDAGFIER K1(GlyGl) 65 2.94 -0.39 YJL012C EDWTGEkSVK K7(GlyGl) 33 2.37 -0.39 YBR011C LEITkEETLNPIIQDTK K5(GlyGl) 39 0.44 -0.39 YLL024C LIGDAAkNQAAMNPANTVFDAK K7(GlyGl) 91 2.92 -0.4 YLR248W IDTLTEkISR K7(GlyGl) 34 1.58 -0.4 YNL121C TMEEkLQAITFAEAAK K5(GlyGl) 29 0.99 -0.4 YLR249W QINENDAEAMNkIFK K12(GlyGl) 50 5.64 -0.41 YDR385W ETVESESSQTALSkSPNK K14(GlyGl) 76 2.25 -0.41 YCR088W IDPSSDIANLkNESK K11(GlyGl) 64 5.25 -0.44 YGR192C ELDTAQkHIDAGAK K7(GlyGl) 29 1.07 -0.44 YAL053W DDTFGkNLNANTNTAR K6(GlyGl) 53 0.78 -0.44 YLR249W MPELIPVLSETMWDTkK K16(GlyGl) 76 5.48 -0.45 YKL056C IIYkDIFSNDELLSDAYDAK K4(GlyGl) 81 3.12 -0.45 YGL173C IVVNEkESQDLK K6(GlyGl) 38 2.91 -0.45 YDR385W AEQLYEGPADDANcIAIkNcDPK C14(Carbamidomethyl), K18(GlyGl), C20(Carbamidomethyl) 67 2.04 -0.45 YDL229W;YNL209W IINEPTAAAIAYGLGAGkSEK K18(GlyGl) 41 5.07 -0.46 YKL081W;YPL048W EIVDGkVLK K6(GlyGl) 44 4.47 -0.46 YFL004W KPLNLLkNAGPVNVEAK K7(GlyGl) 66 4.81 -0.48 YLL024C LIDVDGkPQIQVEFK K7(GlyGl) 42 3.56 -0.48 YOL109W NEATPEAEQVkK K11(GlyGl) 50 1.37 -0.48 YER056C SPVIGSSLENEkK K12(GlyGl) 56 0.87 -0.48 YER074W-A SNDETPVFGQDQNTTkSK K16(GlyGl) 65 0.56 -0.49 YAL003W IETLkQLNASLADK K5(GlyGl) 74 3.5 -0.5 YLR249W ATETVDNkDIER K8(GlyGl) 47 5.64 -0.51 YGL210W ENADDNVAVGLIGNkSDLAHLR K15(GlyGl) 29 3.78 -0.51 YLR249W cPAAkAYEELSNTDLEFK C1(Carbamidomethyl), K5(GlyGl) 71 1.52 -0.51 YOL109W AETAAQDVQQkLEETK K11(GlyGl) 57 1.65 -0.52 YHL001W VLIDGPkAGVPR K7(GlyGl) 36 0.33 -0.52 YBR082C IAkELSDLER K3(GlyGl) 35 5.49 -0.53 YDL229W;YNL209W LESYVASIEQTVTDPVLSSkLK K20(GlyGl) 96 4.93 -0.53 YKR093W QGDFPVIEEEkTQAVTLK K11(GlyGl) 74 2.13 -0.53 YOR198C NTENEQPASIFNkVDGK K13(GlyGl) 49 4.77 -0.54 YBL042C ITSkIDANVIEK K4(GlyGl) 30 -1.41 -0.54 YKL081W HPLEALGkSTFVLDDWK K8(GlyGl) 46 3.07 -0.55 YBR118W FDELLEkNDR K7(GlyGl) 45 1.8 -0.55 YEL034W LEDLSPSTHNMEVPVVkR K17(GlyGl) 37 4 -0.56   168 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YHR042W GkSITTDEATELIK K2(GlyGl) 56 5 -0.57 YPR133W-A FGLPQQEVSEEEkR K13(GlyGl) 30 4.36 -0.57 YGR037C EKPGIFNMkDR K9(GlyGl) 33 4.97 -0.58 YOL109W LEETkESLQNK K5(GlyGl) 47 1.18 -0.58 YPL106C EELEELVkPLLER K8(GlyGl) 43 4.9 -0.59 YDR508C NDLDDVSHYEMkEIQPK K12(GlyGl) 29 1.98 -0.59 YPR163C VDAAVEkLQDK K7(GlyGl) 44 5.43 -0.6 YOL086C VVGLSTLPEIYEkMEK K13(GlyGl) 60 4.17 -0.6 YBR118W VETGVIkPGMVVTFAPAGVTTEVK K7(GlyGl) 62 1.93 -0.6 YMR229C EDSTkQPSTSSLVR K5(GlyGl) 28 5.55 -0.61 YJR072C TkGEVNENSAPDLQR K2(GlyGl) 45 4.97 -0.61 YJR137C VEDTPDLPQkTGIR K10(GlyGl) 47 4.55 -0.64 YFR037C VGkEGEEVGEGDSIAK K3(GlyGl) 29 1.8 -0.64 YOL058W kAHVDLEGLTLDK K1(GlyGl) 32 0.12 -0.64 YKL060C ITkSLETFR K3(GlyGl) 35 5.64 -0.65 YNR006W NSASSEPIVPVVESkNEVK K15(GlyGl) 52 2.4 -0.66 YBR068C kETSPDSISIR K1(GlyGl) 35 2.84 -0.67 YGR192C kVVITAPSSTAPMFVMGVNEEK K1(GlyGl) 65 0.78 -0.68 YCR012W VLENTEIGDSIFDkAGAEIVPK K14(GlyGl) 71 1.94 -0.72 YGR192C TASGNIIPSSTGAAkAVGK K15(GlyGl) 73 0.42 -0.72 YDR155C kVESLGSPSGATK K1(GlyGl) 45 3.58 -0.74 YDR098C-B;YNL284C-B EVHTNQDPLDVSASkIQEYDK K15(GlyGl) 43 5.64 -0.75 YPL225W FPEYkDVER K5(GlyGl) 30 4.47 -0.75 YOL109W EQAEASIDNLkNEATPEAEQVK K11(GlyGl) 108 1.88 -0.75 YDR508C EkQIGSIEPENEVEYFEK K2(GlyGl) 86 1.4 -0.77 YDR046C LEEDNDLEDGTkSMK K12(GlyGl) 42 0.22 -0.79 YBR048W TPkTAIEGSYIDK K3(GlyGl) 44 2.52 -0.84 YHL015W EkVEEQEQQQQQIIK K2(GlyGl) 66 -0.93 -0.89 YOR327C LTSIEDkADNLAISAQGFK K7(GlyGl) 66 0.96 -0.93 YDR385W DTDAEGkPLER K7(GlyGl) 45 2.33 -0.94 YBR177C DNkEQVDFDEFANK K3(GlyGl) 42 3.24 -0.99 YBR302C SVDVLSFkQLESQK K8(GlyGl) 39 0.73 -0.99 YOR089C IPLkTAEEQNSASNER K4(GlyGl) 34 2.11 -1.1 YDL126C NAPAIIFIDEIDSIAPkR K17(GlyGl) 37 -2.45 -1.13 YAL030W LTSIEDkADNLAVSAQGFK K7(GlyGl) 56 1.44 -1.22 YOR375C VDIALPcATQNEVSGEEAkALVAQGVK C7(Carbamidomethyl), K19(GlyGl) 73 -1.21 -1.25 YBR025C DIEFAQkALEGAEK K7(GlyGl) 36 5.15 -1.27 YDR172W IkGVEEEDISPGFVLTSPK K2(GlyGl) 48 5.64 -1.35 YDR266C EDFNkFSSYNEDYSK K5(GlyGl) 39 5.39 -1.47 YML072C FDTSITkPGVLDDLGK K7(GlyGl) 43 3.67 -1.53 *ORF: yeast open reading frame; Peptide Sequence: peptides that contain di-gly (ubiquitylation) sites on lysine (lowercase); Modifications: modifications on the peptide in the left column with modified a.a. indicated; IonScore: Mascot ion score reported by Proteome Discoverer. For peptides that were identified more than once, the average ion scores are shown; Log: Log2 ratio; L(light): ubp2Δ HS, M(medium): WT HS, H(heavy): WT noHS.    169 Supplementary Table 3.6 DiGly peptides comparison between ubp3Δ and WT upon HS ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YOR332W ADQEYEIEkTNIVR K9(GlyGl) 59 0.57 5.64 YPL106C AEEWLYDEGFDSIkAK K14(GlyGl) 57 0.57 5.64 YGL105W AGDkVFFEGFGDEAPMK K4(GlyGl) 45 0.57 5.64 YGL245W APAAkLDDATEDVFNK K5(GlyGl) 70 0.57 5.64 YJL080C APIEIPLEkVcGSTEGENAEK K9(GlyGl), C11(Carbamidomethyl) 36 0.57 5.64 YNL301C APkGQNTLILR K3(GlyGl) 37 0.57 5.64 YOL136C ATLDcQDSVFFDNHkSSLLSTEVPR C5(Carbamidomethyl), K15(Label:13C(6)15N(2)+GlyGly) 31 0.57 5.64 YER165W AVEALNDSELNGEkLYVGR K14(GlyGl) 71 0.57 5.64 YBR025C cEDVFEYkDDSAIK C1(Carbamidomethyl), K8(GlyGl) 35 0.57 5.64 YLR044C DAkNPVILADAccSR K3(GlyGl), C12(Carbamidomethyl), C13(Carbamidomethyl) 52 0.57 5.64 YKL145W DFLkAVDK K4(GlyGl) 32 0.57 5.64 YBR025C DIEFAQkALEGAEK K7(GlyGl) 54 0.57 5.64 YBR143C DkSFAIDK K2(GlyGl) 40 0.57 5.64 YDL229W;YNL209W DMkTWPFK K3(GlyGl) 30 0.57 5.64 YKL060C DSkSPIILQTSNGGAAYFAGK K3(GlyGl) 72 0.57 5.64 YJR070C DSLDDLNkAAK K8(GlyGl) 47 0.57 5.64 YGR264C DSSSFkNIGAVK K6(GlyGl) 43 0.57 5.64 YNL281W DYLNGkLTSLSTVSSDGK K6(GlyGl) 58 0.57 5.64 YKL060C EDLYTkPEQVYNVYK K6(GlyGl) 36 0.57 5.64 YMR229C EDSTkQPSTSSLVR K5(Label:2H(4)+GlyGly) 29 0.57 5.64 YHR039C-A EFEQkNAGGVGELEK K5(GlyGl) 46 0.57 5.64 YPR062W EGGVPIGGcLINNkDGSVLGR C9(Carbamidomethyl), K14(GlyGl) 54 0.57 5.64 YDL229W;YNL209W EIAEAkIGK K6(GlyGl) 34 0.57 5.64 YNR016C EILIQGALPSVkER K12(Label:2H(4)+GlyGly) 38 0.57 5.64 YNL121C EkFGDIDTATATPTELSTQPAK K2(GlyGl) 50 0.57 5.64 YGR037C EKPGIFNMkDR K9(GlyGl) 31 0.57 5.64 YEL037C EkVPLDLEPSNTILETK K2(GlyGl) 30 0.57 5.64 YMR309C ENEDSMAkFR K8(GlyGl) 29 0.57 5.64 YPL106C ENEMLAQDkLVAETEDR K9(Label:2H(4)+GlyGly) 62 0.57 5.64 YPR163C ENkVDAAVEK K3(GlyGl) 41 0.57 5.64 YIL121W ETADkLALTR K5(Label:13C(6)15N(2)+GlyGly) 74 0.57 5.64 YDR365W-B EVHTNQDPLDVSASkTEEcEK K15(GlyGl), C19(Carbamidomethyl) 65 0.57 5.64 YNL121C FDDcETLFSEAkR C4(Carbamidomethyl), K12(Label:2H(4)+GlyGly) 41 0.57 5.64 YDL229W;YNL209W FDDESVQkDMK K8(GlyGl) 43 0.57 5.64 YLR354C FDLNEDAMATEkLSEGIR K12(Label:13C(6)15N(2)+GlyGly) 64 0.57 5.64 YDL229W;YNL209W FEDLNAALFkSTLEPVEQVLK K10(GlyGl) 78 0.57 5.64 YKL152C FGEEkFNTYR K5(GlyGl) 29 0.57 5.64 YDL126C FGLSPSkGVLFYGPPGTGK K7(GlyGl) 32 0.57 5.64 YER165W FGPIVSASLEkDADGK K11(GlyGl) 43 0.57 5.64 YBR061C GGTFVAkIFR K7(Label:2H(4)+GlyGly) 31 0.57 5.64 YPL106C GkLEEEYAPFASDAEK K2(GlyGl) 36 0.57 5.64 YHR042W GkSITTDEATELIK K2(GlyGl) 35 0.57 5.64   170 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YPL239W GTEIEQVTkEATEALR K9(GlyGl) 58 0.57 5.64 YER143W GVGTGkIIGR K6(GlyGl) 35 0.57 5.64 YPR035W HAEHIkLYGSDNDMR K6(Label:13C(6)15N(2)+GlyGly) 32 0.57 5.64 YKL081W HPLEALGkSTFVLDDWK K8(GlyGl) 51 0.57 5.64 YGL008C HYGDQTFSSSTVkR K13(GlyGl) 46 0.57 5.64 YCR088W IDLQkVIAEEK K5(GlyGl) 49 0.57 5.64 YOR361C IGQEMEkSMNFK K7(GlyGl) 65 0.57 5.64 YNL178W INELTLLVQkR K10(GlyGl) 45 0.57 5.64 YJL012C IPkGTTFDTQIR K3(Label:2H(4)+GlyGly) 49 0.57 5.64 YHR108W ISESDLAVLkPSNQLK K10(GlyGl) 37 0.57 5.64 YDL229W;YNL209W ISkSQIDEVVLVGGSTR K3(GlyGl) 38 0.57 5.64 YFL022C ITDFNVTkGPEFSTDLTK K8(GlyGl) 68 0.57 5.64 YJL080C IVkDAENSVTK K3(GlyGl) 59 0.57 5.64 YER165W kAIEQLNYTPIK K1(GlyGl) 35 0.57 5.64 YOL139C kFEENVSVDDTTATPK K1(GlyGl) 28 0.57 5.64 YER072W kIALPTR K1(Label:2H(4)+GlyGly) 28 0.57 5.64 YGR175C kIGDLDFSDR K1(GlyGl) 49 0.57 5.64 YKL081W KKDEAkPADDAAPAK K6(GlyGl) 45 0.57 5.64 YJR139C kLGYTEPDPR K1(GlyGl) 29 0.57 5.64 YJR010C-A kLVFPIDFPSQR K1(GlyGl) 32 0.57 5.64 YJL080C kSGDIVILGPR K1(GlyGl) 38 0.57 5.64 YDL126C kTPLEPGLELTAIAK K1(GlyGl) 37 0.57 5.64 YGL008C kVTAVVESPEGER K1(GlyGl) 30 0.57 5.64 YAL005C;YLL024C LDkSQVDEIVLVGGSTR K3(GlyGl) 58 0.57 5.64 YEL034W LEDLSPSTHNMEVPVVkR K17(GlyGl) 39 0.57 5.64 YNL121C LFEEQLDkNNEDEK K8(GlyGl) 55 0.57 5.64 YFL045C LGkELASQSFINWLGEEK K3(GlyGl) 50 0.57 5.64 YLL024C LIGDAAkNQAAMNPANTVFDAK K7(GlyGl) 92 0.57 5.64 YGL195W LLDTLSDESkSGDR K10(Label:2H(4)+GlyGly) 37 0.57 5.64 YLR249W LLPGLkSNFATIADPEAR K6(Label:2H(4)+GlyGly) 35 0.57 5.64 YDL229W;YNL209W LLSDFFDGkQLEK K9(GlyGl) 30 0.57 5.64 YER124C LNDDQEDIVFTkR K12(Label:13C(6)15N(2)+GlyGly) 45 0.57 5.64 YPL106C LNELIEkENEMLAQDK K7(GlyGl) 40 0.57 5.64 YBR118W LPLQDVYkIGGIGTVPVGR K8(Label:2H(4)+GlyGly) 39 0.57 5.64 YKL056C LQETNPEEVPkFEK K11(GlyGl) 52 0.57 5.64 YAL005C;YLL024C LSkEDIEK K3(GlyGl) 32 0.57 5.64 YLR342W LVGDESEkAAGDASR K8(Label:13C(6)15N(2)+GlyGly) 60 0.57 5.64 YDR155C LYNDIVPkTAENFR K8(GlyGl) 31 0.57 5.64 YPR041W MPPIQAkVEGR K7(GlyGl) 31 0.57 5.64 YDL229W;YNL209W MVNQAEEFkAADEAFAK K9(GlyGl) 69 0.57 5.64 YML028W NGTVLPcNWTPGAATIkPTVEDSK C7(Carbamidomethyl), K17(GlyGl) 67 0.57 5.64 YDR266C NLPTLkSPSASYDPFATTVK K6(GlyGl) 58 0.57 5.64 YLR249W NLTEEVWAVkDGR K10(GlyGl) 49 0.57 5.64 YPR041W NPETEIIITkDNDLVR K10(GlyGl) 52 0.57 5.64 YNL121C NPTVENFIEATNLLEkASK K16(GlyGl) 70 0.57 5.64 YER165W NQQIVAGkPLYVAIAQR K8(GlyGl) 86 0.57 5.64 YIL053W PLTTkPLSLK K5(GlyGl) 36 0.57 5.64   171 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YJL034W;YAL005C;YLL024C QATkDAGTIAGLNVLR K4(GlyGl) 39 0.57 5.64 YLR249W QINENDAEAMNkIFK K12(GlyGl) 49 0.57 5.64 YLR179C QPkGADSSTFTK K3(GlyGl) 62 0.57 5.64 YPL106C QSISEAFGkPLSTTLNQDEAIAK K9(GlyGl) 83 0.57 5.64 YDL229W;YNL209W RFDDESVQkDMK K9(GlyGl) 30 0.57 5.64 YGR180C SATPSkEINFDDDF K6(GlyGl) 44 0.57 5.64 YNL287W SETTLDTTPEAESVPEkR K17(Label:2H(4)+GlyGly) 42 0.57 5.64 YAL003W SIVTLDVkPWDDETNLEEMVANVK K8(GlyGl) 61 0.57 5.64 YDR155C SIYGGkFPDENFK K6(GlyGl) 47 0.57 5.64 YPL106C SkQEASQMAAMAEK K2(GlyGl) 71 0.57 5.64 YIL078W SkQQSLYLDPEPTFIEER K2(Label:2H(4)+GlyGly) 51 0.57 5.64 YNL112W SNYNQPQELIkPNWDEELPK K11(GlyGl) 46 0.57 5.64 YDR266C SPSASYDPFATTVkK K14(GlyGl) 57 0.57 5.64 YDL229W;YNL209W STLEPVEQVLkDAK K11(GlyGl) 58 0.57 5.64 YGR295C SVDVLSVkQLESQK K8(GlyGl) 40 0.57 5.64 YHR108W TASEIQkEQEIAQAAK K7(GlyGl) 75 0.57 5.64 YGL008C TDTSSSSSSSSASSVSAHQPTQEkPAK K24(GlyGl) 57 0.57 5.64 YEL037C TkLAQSIScEESQIK K2(GlyGl), C9(Carbamidomethyl) 82 0.57 5.64 YCR088W TPSPAPAAkISSR K9(GlyGl) 56 0.57 5.64 YLR264W TPVTLAkVIK K7(GlyGl) 26 0.57 5.64 YOR063W VGkGDDEANGATSFDR K3(Label:2H(4)+GlyGly) 29 0.57 5.64 YGR085C VLEQLSGQTPVQSkAR K14(GlyGl) 57 0.57 5.64 YDL229W;YNL209W VNckENTLLGEFDLK C3(Carbamidomethyl), K4(GlyGl) 66 0.57 5.64 YGL097W VPTFSkYNIVQLAPGK K6(GlyGl) 51 0.57 5.64 YDL229W;YNL209W VQkLLSDFFDGK K3(GlyGl) 46 0.57 5.64 YPL106C VTEPVTkALAQAK K7(GlyGl) 62 0.57 5.64 YDL126C VVNQLLTEMDGMNAkK K15(GlyGl) 57 0.57 5.64 YIL053W VVVFEDAPAGIAAGkAAGcK K15(GlyGl), C19(Carbamidomethyl) 59 0.57 5.64 YER166W WNEkYDIAAASLANR K4(Label:2H(4)+GlyGly) 41 0.57 5.64 YDR429C YGSEkGSPAGPSAVTAR K5(GlyGl) 58 0.57 5.64 YHL015W YIDLEAPVQIVkR K12(GlyGl) 45 0.57 5.64 YGL105W YPVSFTkEQSAQAAQWESVLK K7(GlyGl) 76 0.57 5.64 YFL062W SVDVLSFkQLESQK K8(GlyGl) 41 -0.49 5.32 YGL123W NTEEkGWVPVTK K5(GlyGl) 49 -2.41 5.28 YDR497C VHELkYEPTQEIIEDI K5(Label:13C(6)15N(2)+GlyGly) 48 4.16 4.35 YLR075W kGSLENNIR K1(Label:13C(6)15N(2)+GlyGly) 53 -5.4 4.18 YLR167W LIFAGkQLEDGR K6(GlyGl) 58 1.25 4.02 YDL126C ETVEYPVLHPDQYTkFGLSPSK K15(GlyGl) 40 2.51 3.69 YDL229W;YNL209W QATkDAGAISGLNVLR K4(GlyGl) 60 3.35 3.59 YNL178W ALPDAVTIIEPkEEEPILAPsVK K12(GlyGl), S21(Phospho) 57 -0.19 3.43 YBR290W kYLNQSQNQA K1(GlyGl) 73 -1.17 3.37 YDL171C LDNkLIDEAEVTLDR K4(Label:2H(4)+GlyGly) 51 0.46 3.34 YDR038C DGVkITPLTDcDVETIR K4(Label:13C(6)15N(2)+GlyGly), C11(Carbamidomethyl) 77 -1.61 2.98 YJR070C TVAEEFATkPEEAK K9(GlyGl) 54 4.94 2.95 YBR082C IAkELSDLER K3(GlyGl) 33 4.84 2.65   172 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YGR295C;YFL062W LFNSEkSWSPVGLEDAK K6(GlyGl) 42 -2.18 2.59 YGR082W ELEAASkFYK K7(GlyGl) 53 0.94 2.58 YDR155C FPDENFkK K7(GlyGl) 38 4.33 2.44 YGL008C kGLDAIDK K1(GlyGl) 54 1.85 2.42 YAL005C;YLL024C EDIEkMVAEAEK K5(GlyGl) 61 2.95 2.37 YLR249W FPEPGYLEGVkTK K11(GlyGl) 63 4.37 2.31 YNL144C NQSSNDSkWAPATQLVSR K8(Label:13C(6)15N(2)+GlyGly) 59 0.82 2.28 YFL062W NEkSVDVLSFK K3(GlyGl) 65 -0.43 2.11 YDL229W;YNL209W LSSEEIEkMVNQAEEFK K8(GlyGl) 62 3.63 2.09 YGR085C GPkAEEILER K3(GlyGl) 30 -0.37 2.05 YLR043C VVGANPAAIkQAIAANA K10(GlyGl) 56 4.92 1.96 YDR098C-B;YDR365W-B EVDPNISESNILPSkK K15(GlyGl) 32 1.52 1.85 YLR297W LQSNDEEkQALAEK K8(GlyGl) 47 -1.42 1.83 YKL006C-A LEGLANkLATFR K7(GlyGl) 53 0.98 1.82 YAL030W;YOR327C DNINkVAER K5(GlyGl) 42 0.33 1.7 YDR044W LPEDPkTGLPVTDGVK K6(GlyGl) 36 -2.61 1.66 YDR098C-B;YDR365W-B DTWNTkNMR K6(GlyGl) 26 0.36 1.64 YOL119C kNSDQVDLDVNK K1(GlyGl) 37 -0.03 1.62 YDR155C kVESLGSPSGATK K1(GlyGl) 73 4.71 1.59 YDL064W VLLQAkQYSK K6(GlyGl) 45 0.5 1.57 YLR414C SRPTVIYANAPIEEkPLI K15(GlyGl) 59 -0.99 1.56 YER133W GSkPGQQVDLEENEIR K3(GlyGl) 48 5.64 1.5 YAL003W IETLkQLNASLADK K5(GlyGl) 73 3.87 1.5 YCR005C ELVkNIESK K4(GlyGl) 39 -3.39 1.5 YGL008C GYLVAMTGDGVNDAPSLkK K18(GlyGl) 71 0.15 1.46 YDR508C kSSYITVDGIK K1(GlyGl) 51 0.35 1.45 YBR106W DYDLkEIDSAIK K5(GlyGl) 35 1.09 1.42 YDR098C-B;YDR365W-B SLEDNETEIkVSR K10(GlyGl) 41 1.33 1.39 YKL182W EFDETIFNLPkNK K11(GlyGl) 60 1.19 1.39 YPR170W-B kPSIELR K1(GlyGl) 37 0.73 1.39 YBR118W FDELLEkNDR K7(GlyGl) 34 1.76 1.35 YDL126C AEQEPEVDPVPYITkEHFAEAmK K15(GlyGl), M22(Oxidation) 45 3.39 1.34 YOL040C GVDLEkLLEMSTEDFVK K6(GlyGl) 56 -2.33 1.33 YML038C NELkGFQDFEQLGSK K4(GlyGl) 48 -0.95 1.3 YGL008C kADTGIAVEGATDAAR K1(GlyGl) 52 0.42 1.26 YDR508C NDLDDVSHYEMkEIQPK K12(GlyGl) 34 1.78 1.22 YPL143W IEGVATPQDAQFYLGkR K16(GlyGl) 50 0.45 1.21 YLR167W TLSDYNIQkESTLHLVLR K9(GlyGl) 72 0.31 1.21 YJR105W LLNENEkAGVK K7(GlyGl) 37 4.83 1.17 YPL106C QEASQMAAMAEkLAAQR K12(GlyGl) 66 3.89 1.17 YFL039C EITALAPSSMkVK K11(GlyGl) 41 3.86 1.15 YOL119C EGPSSGYNPNFNAADAILkK K19(GlyGl) 88 0.65 1.13 YDR177W LGVkSLDPNDNNTANR K4(GlyGl) 63 2.97 1.08 YOL119C NSDQVDLDVNkLTNVTSR K11(GlyGl) 78 1.62 1.08 YER056C LEEGNNVYEIQDLEkR K15(GlyGl) 67 -0.21 1.05 YJR105W ENDAILVDAkSGDAK K10(GlyGl) 68 4.46 1.04   173 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YGR060W IPSAkEQLYcLK K5(GlyGl), C10(Carbamidomethyl) 67 -1.37 1.04 YHL015W VkQLENVSSNIVK K2(GlyGl) 32 0.69 1.02 YBL042C ESEMGDATkITSK K9(GlyGl) 62 1.2 0.99 YOL109W NEATPEAEQVkK K11(GlyGl) 43 1.16 0.99 YLR197W LkEVQEQINDFGAFTK K2(GlyGl) 45 2.65 0.98 YLR413W IIEEHESPIDAEkNFAR K13(GlyGl) 48 1.89 0.98 YKL056C AkLQETNPEEVPK K2(GlyGl) 42 4.42 0.96 YBL042C ITSkIDANVIEK K4(GlyGl) 36 0.18 0.96 YLR259C VGGASEVEVGEkK K12(GlyGl) 50 1.04 0.95 YER056C SPVIGSSLENEkK K12(GlyGl) 58 0.77 0.89 YDL171C VVDLEDAVPDSkQLEK K12(GlyGl) 30 4.23 0.87 YFL010C GPPPGVNNEkSSR K10(GlyGl) 51 0.28 0.85 YER009W DIVEkLVSLPFQK K5(GlyGl) 36 0.09 0.85 YCL025C QAQELEkNESSDNIGANTGHK K7(GlyGl) 41 -0.93 0.84 YER056C MLEEGNNVYEIQDLEkR K16(GlyGl) 64 0.91 0.82 YAL053W DDTFGkNLNANTNTAR K6(GlyGl) 47 0.45 0.81 YLR100W VQEVINQIkDFYNK K9(GlyGl) 52 2.87 0.8 YLR109W DTTHIkFASDPGcAFTK K6(GlyGl), C13(Carbamidomethyl) 48 0.86 0.77 YLR044C VATTGEWDkLTQDK K9(GlyGl) 41 5.64 0.73 YOL127W VIEQPITSETAMkK K13(GlyGl) 53 -0.24 0.73 YDL229W;YNL209W VEkAVITVPAYFNDAQR K3(GlyGl) 32 5.43 0.7 YDR098C-B EVHTNQDPLDVSASkIQEYDK K15(GlyGl) 38 5.4 0.69 YOL109W AETAAQDVQQkLEETK K11(GlyGl) 52 1.78 0.66 YEL037C TkVTEPPIAPESATTPGR K2(GlyGl) 47 3.43 0.63 YDL136W EQLASQLVDLkK K11(GlyGl) 35 0.13 0.63 YOR369C LVEGLANDPENkVPLIK K12(GlyGl) 58 2.22 0.62 YHL001W VLIDGPkAGVPR K7(GlyGl) 56 0.16 0.62 YBR179C DLSPETYkR K8(GlyGl) 30 -0.68 0.62 YKL060C ITkSLETFR K3(GlyGl) 36 5.13 0.61 YDL229W;YNL209W IINEPTAAAIAYGLGAGkSEK K18(GlyGl) 45 5.23 0.59 YER145C EVLHVkADSL K6(GlyGl) 54 1.44 0.59 YOL109W LEETkESLQNK K5(GlyGl) 50 0.97 0.59 YOR369C VPLIkVADAK K5(GlyGl) 29 1.31 0.58 YGL147C DGAkFIEVR K4(GlyGl) 43 0.39 0.58 YDR155C VESLGSPSGATkAR K12(GlyGl) 56 5.64 0.57 YAL005C;YLL024C LIDVDGkPQIQVEFK K7(GlyGl) 31 3.47 0.56 YKL180W ETAQAINGWELTkAQK K13(GlyGl) 58 0.29 0.56 YCR012W VLENTEIGDSIFDkAGAEIVPK K14(GlyGl) 68 2.43 0.52 YOR327C LTSIEDkADNLAISAQGFK K7(GlyGl) 42 0.31 0.51 YLR044C kLIDLTQFPAFVTPMGK K1(GlyGl) 38 3.24 0.5 YML097C FNELFSPIGEPTQEEALkSEQSNK K18(GlyGl) 41 1.86 0.49 YGR135W TLSkTTDSSALTYDR K4(GlyGl) 69 0.66 0.49 YDR155C KVESLGSPSGATkAR K13(GlyGl) 37 -1.76 0.49 YLR167W TLTGkTITLEVESSDTIDNVK K5(GlyGl) 104 0.41 0.47 YNR006W NSASSEPIVPVVESkNEVK K15(GlyGl) 54 3.07 0.46 YBR106W NDMTTMkYVEPGNAMSGEGEK K7(GlyGl) 65 0.23 0.46 YLR167W QIFVkTLTGK K5(GlyGl) 63 0.12 0.44 YPL265W NSSSLDSDHDAYYSkQNPDNFPVK K15(GlyGl) 75 2.15 0.42   174 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YDL229W;YNL209W LIGDAAkNQAALNPR K7(GlyGl) 62 5.06 0.4 YER043C EcINIkPQVDR C2(Carbamidomethyl), K6(GlyGl) 29 1.63 0.4 YGL045W VSVPSEDkQELEQK K8(GlyGl) 33 -2.68 0.4 YAL005C;YLL024C DNNLLGkFELSGIPPAPR K7(GlyGl) 47 4.47 0.39 YKL056C IIYkDIFSNDELLSDAYDAK K4(GlyGl) 80 3.65 0.38 YHL015W EkVEEQEQQQQQIIK K2(GlyGl) 97 -0.49 0.38 YGR192C VVDLVEHVAkA K10(GlyGl) 35 2.16 0.37 YAL038W EVLGEQGkDVK K8(GlyGl) 39 0.5 0.36 YDL126C AAAPTVVFLDELDSIAkAR K17(GlyGl) 83 3.67 0.35 YML063W NVGkTLVNK K4(GlyGl) 38 1.84 0.35 YBL057C cPDkFTMDELYAK C1(Carbamidomethyl), K4(GlyGl) 59 -0.36 0.31 YBR118W MDSVkWDESR K5(GlyGl) 28 4.65 0.3 YKL081W;YPL048W EIVDGkVLK K6(GlyGl) 43 4.43 0.27 YHR203C IDLASGkITDFIK K7(GlyGl) 64 0.49 0.27 YML097C SEQSNkEEDVSSLIK K6(GlyGl) 52 1.17 0.26 YKL182W cIAAcTGVPDDkWEQTYK C1(Carbamidomethyl), C5(Carbamidomethyl), K12(GlyGl) 59 -0.19 0.26 YDL229W;YNL209W STGkSSNITISNAVGR K4(GlyGl) 91 5.64 0.25 YGL206C DGTNSVAIVDLAkGNEVTR K13(Label:2H(4)+GlyGly) 81 1.37 0.25 YLL024C NTISEAGDkLEQADKDAVTK K9(GlyGl) 33 0.91 0.25 YML097C EEDVSSLIkK K9(GlyGl) 55 -0.45 0.25 YOL086C ANGTTVLVGMPAGAkccSDVFNQVVK K15(GlyGl), C16(Carbamidomethyl), C17(Carbamidomethyl) 79 5.14 0.24 YDL229W;YNL209W kTGLDISDDAR K1(GlyGl) 54 3.11 0.24 YKL180W SVkFVQGLLQNAAANAEAK K3(GlyGl) 77 0.07 0.23 YOR204W VGSTSENITQkVLYVENQDK K11(GlyGl) 74 5.51 0.21 YOL086C VLGIDGGEGkEELFR K10(GlyGl) 32 5 0.2 YOL004W DFkSQAIDTPGVIER K3(GlyGl) 52 4.72 0.2 YPL265W QNPDNFPVkEQEIYNIDLEENNVSSR K9(GlyGl) 72 2.56 0.2 YEL037C LIYSGkVLQDSK K6(GlyGl) 41 0.35 0.2 YHL015W QLENVSSNIVkNAEQHNLVK K11(GlyGl) 45 1.72 0.19 YGR136W TGDkIQVLEK K4(GlyGl) 63 1.33 0.19 YDR266C EDFNkFSSYNEDYSK K5(GlyGl) 67 4.33 0.18 YPR163C VDAAVEkLQDK K7(GlyGl) 53 4.17 0.17 YMR230W DFNQAkHEEIDTK K6(GlyGl) 28 0.41 0.17 YDR385W TGTLTTSETAHNMkVMK K14(GlyGl) 51 4.09 0.12 YLR175W EDFVIkPEAAGASTDTSEWPLLLK K6(GlyGl) 91 2.49 0.11 YNL178W TGPkALPDAVTIIEPK K4(GlyGl) 43 2.41 0.11 YAL005C;YLL024C NTISEAGDkLEQADK K9(GlyGl) 86 1.81 0.11 YML097C SEQSNKEEDVSSLIkK K15(GlyGl) 39 -0.31 0.11 YOL086C LPLVGGHEGAGVVVGMGENVkGWK K21(GlyGl) 71 5.07 0.1 YDL229W;YNL209W LESYVASIEQTVTDPVLSSkLK K20(GlyGl) 104 4.44 0.1 YGR192C TASGNIIPSSTGAAkAVGK K15(GlyGl) 70 0.73 0.1 YOL086C SPIkVVGLSTLPEIYEK K4(GlyGl) 43 4.26 0.09 YKR093W QGDFPVIEEEkTQAVTLK K11(GlyGl) 76 3.94 0.09 YCR031C DESSPYAAMLAAQDVAAkcK K18(GlyGl), C19(Carbamidomethyl) 57 0.79 0.08   175 ORF Sequence Modifications Ion Score  Log(M/H) Log(L/M) YGL245W ANFEIDLPDAkMGEVVTR K11(GlyGl) 60 3.27 0.04 YOR046C IDNEkEDTSEVSTK K5(GlyGl) 85 3.92 0.03 YOL086C MEkGQIVGR K3(GlyGl) 31 5.24 0.02 YIL053W TYDAIAkFAPDFADEEYVNK K7(GlyGl) 71 3.71 0.01 YOL086C VVGLSTLPEIYEkMEK K13(GlyGl) 37 3.29 0.01 YBR068C kETSPDSISIR K1(GlyGl) 40 3.66 0 YDR172W IkGVEEEDISPGFVLTSPK K2(GlyGl) 43 5.64 -0.02 YBR118W FLkSGDAALVK K3(GlyGl) 35 4.82 -0.05 YHR174W IGLDcASSEFFkDGK C5(Carbamidomethyl), K12(GlyGl) 59 2.31 -0.05 YPL231W SLGVkSLGGGAALK K5(GlyGl) 45 3.54 -0.07 YDR508C QIGSIEPENEVEYFEkTVEK K16(GlyGl) 48 0.5 -0.07 YHR174W kAADALLLK K1(GlyGl) 53 0.81 -0.11 YGR192C kVVITAPSSTAPMFVMGVNEEK K1(GlyGl) 57 0.51 -0.12 YHR203C ITDEEASYkLGK K9(GlyGl) 62 5.47 -0.17 YIL053W EkWAVATSGTR K2(GlyGl) 43 3.82 -0.18 YMR079W kLLEDAGFIER K1(GlyGl) 62 3.69 -0.22 YOR104W NDAkNDTFYDEVK K4(GlyGl) 39 0.8 -0.22 YJL080C EIISkAGGEEIR K5(GlyGl) 41 1.91 -0.23 YDR508C EkQIGSIEPENEVEYFEK K2(GlyGl) 58 1.26 -0.23 YBR048W TPkTAIEGSYIDK K3(GlyGl) 49 3.38 -0.24 YBR159W TGkYcAITGASDGIGK K3(GlyGl), C5(Carbamidomethyl) 38 -0.12 -0.24 YPL106C TDLPEGEEkPR K9(GlyGl) 35 3.28 -0.33 YLR249W AAATAAMTkATETVDNK K9(GlyGl) 60 5.44 -0.35 YHR174W YDLDFkNPESDK K6(GlyGl) 49 3.08 -0.44 YNL121C FGDIDTATATPTELSTQPAkER K20(GlyGl) 61 3.9 -0.49 YBL047C NDPIVDASLSkGPIVNR K11(GlyGl) 59 1.56 -0.51 YDR385W DTDAEGkPLER K7(GlyGl) 45 2.54 -0.53 YLR249W MPELIPVLSETMWDTkK K16(GlyGl) 69 5.4 -0.57 YLR249W DSLGALSkALK K8(GlyGl) 56 3.43 -0.58 YJL080C VcGSTEGENAEkTK C2(Carbamidomethyl), K12(GlyGl) 44 2.12 -0.58 YDR385W DLEGkALLK K5(GlyGl) 46 3.34 -0.64 YLR249W ATETVDNkDIER K8(GlyGl) 44 5.64 -0.65 YOR198C NILPDLSSNALETkPAR K14(GlyGl) 38 5.18 -0.66 YLR249W cPAAkAYEELSNTDLEFK C1(Carbamidomethyl), K5(GlyGl) 78 0.87 -0.68 YLR249W LVEDPQVIAPFLGkLLPGLK K14(GlyGl) 67 3.87 -0.78 YBR118W VETGVIkPGMVVTFAPAGVTTEVK K7(GlyGl) 61 4.16 -0.84 YGL009C EFEYkDQDQSSPK K5(GlyGl) 47 3.84 -0.88 YIL053W YGEHSIEVPGAVkLcNALNALPK K13(GlyGl), C15(Carbamidomethyl) 28 4.6 -1 YDR064W kGLTPSQIGVLLR K1(GlyGl) 29 1.59 -1.14 YJR123W TIAETLAEELINAAkGSSTSYAIK K15(GlyGl) 86 4.18 -2.73 *ORF: yeast open reading frame; Peptide Sequence: peptides that contain di-gly (ubiquitylation) sites on lysine (lowercase); Modifications: modifications on the peptide in the left column with modified a.a. indicated; IonScore: Mascot ion score reported by Proteome Discoverer. For peptides that were identified more than once, the average ion scores are shown; Log: Log2 ratio; L(light): ubp3Δ HS, M(medium): WT HS, H(heavy): WT noHS.   176 Chapter 4: Conclusions  4.1 Chapter summary In Chapter 2, using quantitative mass spectrometric analysis we established that transient and acute heat-stress mainly caused the ubiquitylation of misfolded proteins in cytosol. HS stress provided us a complementary approach to further study the degradation pathways that may be involved in PQC. We found that Hul5 is a major ubiquitin ligase that participated in the cytosolic HS ubiquitylation response. This HS-responding activity of Hul5 required its ligase activity, as the mutation of Hul5 ligase active site led to both an impaired ubiquitylation response and reduced cell fitness after HS. In addition, we showed that Hul5 interacted with both Ubc4 and Ubc5, the two major cytosolic E2 conjugating enzymes required for the ubiquitylation and degradation of heat-induced misfolded proteins[209, 210]. We found that upon HS, Hul5 targeted for degradation, not only newly-synthesized but also long-lived misfolded proteins. Hul5 was also required for targeting misfolded proteins induced by inactivation of major yeast Hsp70 cytosolic folding machinery (Ssa1-4). Furthermore, cytosolic localization was required for Hul5 to perform its PQC function. Lastly, we found that Hul5 provided constant PQC protection by ubiquitylating the low-solubility cytosolic misfold proteins under unstressed and HS conditions. Altogether, we present a novel Hul5-dependent PQC degradation pathway that has housekeeping function of eliminating cytosolic misfolded proteins.   177 In Chapter 3, we uncovered another major PQC pathway that depended on the ubiquitin ligase Rsp5 (a yeast homolog of the mammalian Nedd4 ligase) to ubiquitylate HS-induced misfolded proteins. We found that Rsp5 was required for the ubiquitylation and degradation of heat-induced cytosolic misfolded proteins. This function of Rsp5 required its E3 catalytic activity and WW-domains that mediate the interaction with PY-motif-containing substrates and adaptor proteins. We found that Hsp40 co-chaperone Ydj1 was an adaptor protein that assisted Rsp5 with recognizing its cytosolic PQC targets. In addition, Rsp5 was also capable to recognize directly proteins containing PY-motifs. These PY motifs are presumably exposed on heat-induced misfolded substrates, and otherwise buried in the native-protein structures. We also showed that two deubiquitinases Ubp2 and Ubp3 removed K63-linkage built by Rsp5 upon HS stress, which was required for the proteasomal degradation of the heat-induced misfolded proteins. We therefore propose that Rsp5 ubiquitylates heat-induced cytosolic misfolded proteins recruited by 1) the co-chaperone Ydj1, and/or 2) their exposed PY-motifs for proteasomal degradation; and the ubiquitylation of these HS-induced Rsp5 substrates is modulated by the deubiquitinases Ubp2 and Ubp3.           178 4.2 Common discussion 4.2.1 The HS stress We found that HS stress triggered a rapid increase of polyubiquitylation of mainly cytosolic proteins in yeast cells. This increase in ubiquitylation occurred within minutes suggesting that the UPS components involved in the response are 1) most likely present in the cell prior to the stress and 2) do not require regulation at the transcriptional level (at least in the time frame used). One question raised by our findings is why cytosolic proteins, and not other proteins, are first ubiquitylated during HS. For instance, misfolded ER proteins can be efficiently targeted for proteasomal degradation by the ERAD pathway, but ER proteins were not prominently represented among the proteins ubiquitylated upon the short HS we applied. One possibility is that the ubiquitylation of ER proteins, which have to be first retrotranslocated to the cytoplasm, is a slower process. It was also shown that HS induced increased sumoylation (another ubiquitin-like post-translational modification) of mainly nuclear proteins in plant cells, as well as in mammalian cells[211, 212]. Therefore, sumoylation may prevent the ubiquitylation of misfolded proteins in the nucleus by occupying the exposed lysine residues. Curiously, Rsp5 was showed to be also involved in the PQC of plasma membrane proteins, which were targeted for degradation to the lysosome with the help of ART substrate-adaptor proteins[74]. Deletion of the 10 different yeast ART genes did not lead to a reduction of the increased ubiquitylation observed upon short HS, indicating that their targets may not be readily ubiquitylated. Degradation of misfolded plasma membrane proteins upon HS was measured in a longer time frame (>1 hr). It is   179 not yet clear how Rsp5 may prioritize its misfolded substrates upon stress. Possibly, Rsp5 may first ubiquitylate cytosolic misfolded proteins before being specifically recruited to the plasma membrane.    4.2.2 The ligase activity of Hul5 and Rsp5 upon HS stress As HS presumably challenges the ability of most proteins to maintain their native conformation, one question is how Hul5 and Rsp5 are able to remain active under heat-stress conditions. One possibility is that the HECT ligase domain itself is more resistant to thermal fluctuations to ensure that both ligases are maintained active upon HS. In the case of Rsp5, HS might increase the ubiquitin ligase activity. For instance the C2-domain (at the N-terminus) of the Rsp5 mammalian homologs Nedd4 and Nedd4-like ligases can inhibit its ligase activity both in vitro and in vivo[213]. As the C2-domain is also conserved on Rsp5, a similar auto-inhibitory regulatory mechanism might also apply to Rsp5. Under HS conditions, the higher temperature might disrupt the auto-inhibition mediated by the C2-domain leading to the enhancement of Rsp5 activity. Furthermore, our data showed that upon HS there was an increased interaction between Rsp5 and the Hsp40 co-chaperone Ydj1, which may act as an Rsp5 adaptor to recruit misfolded proteins upon HS. One cannot exclude that Ydj1 may also help to stabilize the Rsp5 structure via its co-chaperone activity. Once Ydj1 is bound to Rsp5, there must be a mechanism to prevent the transfer of the substrates to Hsp70; otherwise, the misfolded proteins would not be efficiently refolded or eliminated.    180 On the other hand, less is known about Hul5 function under thermal stress. Notably, no other structural element or domain has been identified besides its HECT domain. It is therefore not clear how Hul5 may specifically maintain its activity under stress conditions.  Interestingly, the majority of HECT ligases have at least one long disordered stretch (≥ 30 residues) in their N-terminal region (HECT domains are located in the C-terminal region)[214]. In fact most HECT ligases are relatively large proteins in which many long disordered segments inter-mixed with ordered domains[215]. The presence of disordered segments was also found on the yeast San1 RING ligase that targets nuclear misfolded proteins[76]. The disordered regions of HECT ligases like Rsp5 and Hul5 might increase their flexibility to facilitate the recognition of a broad-range of misfolded proteins that may adopt variable and diverse configurations.   4.2.3 The ubiquitin linkages catalyzed by Hul5 and Rsp5 Our data suggest that both the Hul5 and Rsp5 pathways direct cytosolic misfolded proteins for degradation. It is generally thought that proteins targeted for proteasomal degradation are preferably conjugated to K48, K29, and K11 linked-poly-ubiquitin chains[24, 216]. In contrast, both Hul5 and Rsp5 were shown to predominantly assemble K63-linked chains in vitro[108, 217]. Therefore, it is intriguing that both Hul5 and Rps5 target substrates for proteasomal degradation, while these two ubiquitin ligases catalyze a non-favorable ubiquitin linkage type. One possibility is that the proteasome can also process K63-linked substrates, as long as they are delivered to the proteasome loci. A   181 recent study showed that purified proteasomes bind and degrade K48- and K63-linked substrates, while in the presence of a cell extract only K48-linked conjugates bind to the proteasome[218]. It was suggested that potent K63-interacting proteins (e.g., ESCRT0) could bind to proteins conjugated with K63 chains, thereby preventing their proteasomal degradation[218]. Therefore, if a protein is conjugated with a K63-chain nearby a proteasome subunit, it may be efficiently recognized and degraded by the proteasome. This possibility is supported by the fact that Hul5 is associated with proteasomes and was proposed to promote proteasome processivity by elongating ubiquitin chains on the substrates[108, 135].   Another possibility is that Rsp5 and Hul5 might be able to catalyze K48 linkage in vivo after binding to a specific interacting partner or under certain conditions (e.g., HS). Our data suggested that HS predominantly leads to an increase of K48-linked ubiquitin chains. We also found that both Ubp2 and Ubp3 specifically promoted lower levels of K63 ubiquitin linkages in the cell upon HS. Importantly, the absence of either Ubp2 or Ubp3 prevented the proteasomal degradation of heat-induced misfolded proteins. As mentioned previously, the two deubiquitinases may edit Rsp5 substrates to remove the K63-linked ubiquitin. In this case, yet another ubiquitin ligase may then build on K48 chains. Alternatively, the two deubiquitinases when associated to Rsp5 could also modulate the ubiquitin ligase activity, independently of their catalytic activity. For instance, the deubiquitinases could bind to the previously attached ubiquitin (i.e., on the elongated chain) and place the K48 of the distal ubiquitin near the Rsp5 catalytic cysteine, which would then select this lysine for conjugation to the next ubiquitin. In this   182 case, Rsp5 would directly assemble K48-linked ubiquitin chains on its substrates to promote their proteasomal degradation.   4.2.4 The degradation “timer” in the cytosol A critical challenge for the cell is to distinguish between transiently and terminally misfolded proteins, and to only eliminate the latter. This is a harrowing task due to the large variability of protein structures and of their folding rates. In addition, as cytosolic proteins are synthesized in the cytoplasm, both “naturally unfolded” (i.e., during translation) and folded species cohabit in the same compartment. For ERAD, the fates of misfolded proteins are mainly determined by association with chaperones (e.g., BiP) and their state of the appended glycan chain (glycosylation) before being re-translocated to the cytosol for ubiquitylation and proteasomal degradation[219]. The uncoupling of both processes (between folding in the ER and degradation in the cytoplasm) may provide an additional step to better control the triage of misfolded proteins. In the cytosol, to prevent the pre-mature degradation of proteins that are not terminally misfolded, we propose that both Hul5 and Rsp5 pathways may rely on a “timer” system that controls the rate of ubiquitylation of misfolded substrates.  We found several elements that may regulate or participate in the Hul5 pathway to prevent premature targeting of transiently misfolded proteins. Firstly, Hul5 is mainly localized in the nucleus in physiological conditions. We showed that Hul5 needs to re-localize to the cytoplasm prior to catalyzing the ubiquitylation of the heat-induced misfolded proteins. This step may ensure that there is less Hul5 in the cytosol in   183 unstressed conditions to potentially prevent the premature targeting of transiently misfolded proteins. Secondly, the ubiquitylation of Hul5 substrates may be a two-step process. When we validated Hul5 candidate substrates identified by mass spectrometry, we found that several of them remain mono-ubiquitylated in the absence of Hul5 (e.g., Lsm7). These results indicate that another E3 is adding the first ubiquitin moiety before Hul5. In this case, Hul5 may work primarily as an ubiquitin chain assembly factor (also called E4, as previously defined)[220]. Indeed, it has been previously suggested that Hul5 may be an E4 enzyme that antagonizes the deubiquitinase Ubp6, also associated with the proteasome[108]. Note that Rsp5 could likely be the upstream E3 for a subset of Hul5 substrates (e.g., Pin3, see also point below), but not for all of them (e.g., Lsm7 in Fig. S3.3f). This two-step ubiquitylation (i.e., first by an E3 then by Hul5) may delay the targeting of the substrates to the proteasome, and transiently misfolded proteins may refold to escape degradation. The “timer” process may ensure that the Hul5-pathway only targets proteins that are misfolded or aggregated for an extended period. In the Rsp5 pathway, we proposed that the deubiquitinases Ubp2 and Ubp3 presumably remove K63 ubiquitin chains catalyzed by Rsp5 under heat-stress conditions. This step may ensure that the K48-linked chains of Rsp5 substrates are assembled at a slower rate (either by Rsp5 directly or another ubiquitin ligase). Similar to Hul5 substrate processing, the involvement of Ubp2 and Ubp3 could provide a time-limiting step in the pathway to allow sufficient time for refolding.         184 4.2.5 The relationship between the Hul5 and Rsp5 pathways   Pin3/Lsb2 was identified an Rsp5 substrate by others[130, 221]. Pin3 is one of the prion-like proteins in yeast. It contains short stretches of glutamine residues and binds to actin patches.  Overexpression of Pin3 promoted the conversion of the translation termination factor Sup35 into its prion form [PSI+]; while deletion of PIN3 destabilized [PSI+][130]. In a previous study, Pin3 was also found to be targeted by Rsp5 via a PY motif on Pin3. In this case, ubiquitylation of Pin3 by Rsp5 reduced its ability to induce [PSI+] formation[130]. We found that in the absence of Hul5 activity, the poly-ubiquitylation of Pin3 was nearly abolished upon HS, but not the mono-ubiquitylation (Fig. 2.7c) indicating that Rsp5 might be the upstream E3 of Hul5 for this particular substrate.  Besides Pin3, the ubiquitylation of another Rsp5 substrate Pdc1 (the major isozyme of the three pyruvate decarboxylase in yeast) was also partially mediated by Hul5 (Fig. 3.3f). The data on Pin3 and Pdc1 suggest that these two ubiquitin ligases might share a common pool of substrates. However, we also believe that Hul5 and Rsp5 constitute two independent PQC pathways.  We found that absence of both ligases led to the complete abolition of the HS-induced increased ubiquitylation, while the absence of one ligase only caused a partial defect. Notably, based on the proteomic data, ubiquitylation of several proteins was not affected by the absence of either Hul5 or Rsp5. Indeed, the ubiquitylation of the Hul5 substrate Lsm7 was not affected by the absence of Rsp5, and ubiquitylation of two other Rsp5 substrates, Cdc19 and Sup45, was not affected by the deletion of HUL5. One possibility is that cells employ multiple pathways to target some of the most “toxic-prone” proteins such as intrinsic   185 aggregation-prone proteins (e.g., Pin3) and enzymes with important cellular functions (e.g., Pdc1) in order to maintain fitness and viability under stress conditions.  4.3 Future directions The work presented in this thesis raises a number of important questions and novel hypotheses, which should be pursued in the future.  For the Hul5 PQC pathway, one major question is how misfolded proteins are recognized. It is not clear if substrate recognition by Hul5 requires additional adaptor proteins or whether Hul5 is able to interact directly with the misfolded proteins. Hul5 is a relatively large protein that consists of 910 amino acids. Its HECT domain at the C-terminus of the protein is approximately 450 amino acids, which leaves a large remaining portion of the protein without any known functional domain or motif. The N-terminal region of Hul5 could well contain one or several motifs that bind to substrates or additional factors. Alternatively, Hul5 may not directly recognize misfolded proteins but instead only target misfolded mono-ubiquitylated proteins recruited to the proteasome. Since we found that retaining Hul5 in the nucleus, abolished its ability to ubiquitylate and target cytosolic misfolded proteins, additional factors may instead be required to promote the relocalization of Hul5 to the cytosol. One future approach for studying the Hul5 targeting mechanism, would be to assess the degradation/ubiquitylation of Hul5 substrates identified by mass spectrometry (e.g., Lsm7 and Slh1) in cells that express Hul5 N-terminal truncations. This would help to determine the region of Hul5 that might be involved in substrate recognition or the   186 recruitment of additional factors.  The region of interest could then be used as a bait to identify interacting factors using mass spectrometry analysis to then probe further its function in the Hul5 PQC pathway.  As discussed previously, Hul5 most likely works together with another ligase that would add the first ubiquitin moiety on a targeted cytosolic misfolded protein. Therefore, another attractive follow-up study would be to search for the potential Hul5 E3 partner(s). Here, the Hul5 substrates identified in this work can be again used to screen for the participating E3 ligases. More specifically, absence of the upstream E3 would fully abolish substrate ubiquitylation. One possibility is that multiple E3s each target a different subset of misfolded proteins, but not efficiently, so that Hul5 is then required to target the broader range of proteins (only proteins conjugated to ubiquitin chains of four or more moieties would be targeted for proteasomal degradation).  The HS-induced increased ubiquitylation was uncovered in mammalian cells over 25 years ago, but no E2 or E3 enzyme has been identified in this response. We showed that both Hul5 and Rsp5 were the two main ubiquitin ligases responsible for this HS response in yeast. Both ligases have human homologs and it would be important to determine whether these homologs have the same function. The human Ube3B and Ube3C ubiquitin ligases are two homologs that share the highest sequence similarity with Hul5. An obvious future study direction would be to assess whether Ube3B or Ube3C is the functional homolog of Hul5 for its role in PQC, especially given that Ube3C has been recently found to promote degradation of some of the model misfolded proteins[145]. If Ube3C is also contributing to the increased ubiquitylation upon HS in mammalian cells, the next step would be to identify the Ube3C substrates. Otherwise, a   187 genome-wide RNA interference strategy may be appropriate to identify mammalian functional homologs of Hul5. This approach could also be extended to assess other components of the UPS (e.g., E2s) that are involved.  On the other hand, it may be easier to assess whether the Rsp5 mammalian homologs that belong to the NEDD4 ligase family share the same PQC function in higher eukaryotes. Unlike other HECT ligases, NEDD4 family members have a relatively well-defined organization of their functional domains. Despite their similarity, NEDD4 family members can each have very distinct physiological functions[222]. Therefore, while NEDD4 has a higher probability to be the functional homolog of Rsp5, other members in the family should also be assessed. Our data suggested that Rsp5 recognizes misfolded substrates directly or indirectly via substrate adaptor proteins. In both cases the interaction was mediated by WW-PY interactions. The next question to address is whether Rsp5 recognizes its PQC clients via one or several WW-domain(s) of Rsp5, and whether the same WW domain(s) mediate the binding of Ydj1. Interestingly, it was previously shown that each of the three Rsp5-WW domains had a binding preference for distinct substrates or adaptor proteins[168, 223-226]. Mutation of individual or a combination of WW domains could be used to assess Rsp5 activity in PQC. More specifically, ubiquitylation of different Rsp5 PQC substrates could be compared. For instance, Sup45 could be employed to assess which WW domain is important for Ydj1 association, while Pdc1 and Cdc19 ubiquitylation could be assessed in an ydj1∆ background. According to our data, the structurally buried PY motif might act as an intrinsic “degron” to trigger Rsp5 recognition of protein misfolding. It is a very attractive model,   188 as this direct recognition mechanism would enable the UPS to efficiently and specifically target misfolded PY-containing proteins. However, more experiments are needed to further support this hypothesis. For example, mutations that destabilize protein structure and expose the PY motifs in Rsp5 substrates could be used to assess whether the exposure of PY-motif is sufficient to trigger Rsp5 recognition, even in the absence of stress. A PY-motif swapping experiment could also be used to further determine whether the PY-motif is involved in misfolded protein recognition by Rsp5. The idea would be to add a PY motif in a protein, and to investigate whether this is sufficient to trigger Rsp5-dependent ubiquitylation upon misfolding. In this experiment, a careful selection of PY-recipient proteins is needed as addition of a PY-motif may induce misfolding by itself. Good candidate recipient proteins are PY-proteins that have paralogs (produced by specific gene or genome duplication) that do not contain any PY motif. These experiments would help to strengthen our model for Rsp5’s role in PQC.   Another question raised from our data is whether the function of Ubp2 and Ubp3 in promoting proteasomal degradation of heat-induced misfolded proteins is linked to their activity of removing Rsp5-catalyzed K63 linkages on these substrates. To answer this question, we can assess the proteasomal degradation of heat-induced misfolded proteins (35S-labeled newly-synthesized proteins) in ubp2Δ or ubp3Δ cells that express solely the K63R ubiquitin variant (only lysine 63 is mutated to arginine). If the expression of ubiquitin-K63R bypasses the defect on proteasomal degradation upon HS due to the lack of Ubp2/ Ubp3, Ubp2 and 3 are most likely involved in proteasomal degradation of heat-induced misfolded proteins by promoting the conjugation of non-  189 K63 linkages on these targets. A future study then can be done to determine whether these non-K63 linkages are catalyzed by Rsp5 or other ligase partner(s) of Rsp5.  Furthermore, as our data suggest that there is certain overlap between the Hul5 and Rsp5 pathways. Quantitative proteomic studies using different enrichment approaches for ubiquitylated proteins could be used to further determine the shared substrates of Hul5 and Rsp5 upon HS, which may help us to gain insight of the reasons and functions of the cross talk between the two pathways. My hypothesis was that there are additional PQC pathways that target cytosolic misfolded proteins and that HS can be used to identify novel PQC components. Thus far, I have identified two, novel PQC degradation pathways that required ubiquitin ligases Hul5 and Rsp5 to target cytosolic misfolded proteins for the proteasomal degradation. The discovery of these pathways will contribute to the advancement of our general knowledge on the PQC system and provides foundation for the discovery of novel therapeutic targets.        190 Bibliography 1.     Dobson CM. (2003) Protein folding and misfolding. Nature 6968(426): 884-90  2.     Tyedmers J, Mogk A, Bukau B. 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