Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Characterization of degradative protein quality control mechanisms using model substrates derived from… Comyn, Sophie 2016

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata


24-ubc_2017_february_comyn_sophie.pdf [ 3.4MB ]
JSON: 24-1.0340620.json
JSON-LD: 24-1.0340620-ld.json
RDF/XML (Pretty): 24-1.0340620-rdf.xml
RDF/JSON: 24-1.0340620-rdf.json
Turtle: 24-1.0340620-turtle.txt
N-Triples: 24-1.0340620-rdf-ntriples.txt
Original Record: 24-1.0340620-source.json
Full Text

Full Text

CHARACTERIZATION OF DEGRADATIVE PROTEINQUALITY CONTROL MECHANISMS USING MODELSUBSTRATES DERIVED FROM TEMPERATURESENSITIVE ALLELESbySophie ComynM.Sc., The University of Alberta, 2011B.Sc., The University of British Columbia, 2007A THESIS SUBMITTED IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFDoctor of PhilosophyinTHE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES(Genome Science and Technology)The University of British Columbia(Vancouver)December 2016c￿ Sophie Comyn, 2016AbstractThe purpose of protein homeostasis (proteostasis) is to maintain proteome integrity,thereby promoting viability at both the cellular and organism levels. Exposure toa range of acute stresses often produces misfolded proteins, which present a chal-lenge to maintaining proteostatic balance. The accumulation of misfolded proteinscan lead to the formation of potentially toxic protein aggregates, which are charac-teristic of a number of neurodegenerative diseases such as Alzheimer’s and Parkin-son’s. Therefore, a number of protein quality control pathways exist to promoteprotein folding by molecular chaperones or target terminally misfolded proteins fordegradation via the ubiquitin proteasome system or autophagy. Within the cytosolthe mechanisms responsible for targeting substrates for proteasomal degradationremain to be fully elucidated.In this thesis, we established and employed thermosensitive model substratesto screen for factors that promote proteasomal degradation of proteins misfoldedas the result of missense mutations in Saccharomyces cerevisiae. Using a genome-wide flow cytometry based screen we identified the prefoldin chaperone subunitGim3 as well as the E3 ubiquitin ligase Ubr1. An absence of Gim3 leads to theaccumulation of model substrates in cytosolic inclusions and their delayed degra-dation. We propose that Gim3 promotes degradation by maintaining substrate sol-ubility.In the course of screening for factors involved in degradative protein qual-ity control, we identified secondary mutations in the general stress response geneWHI2 among a number of E3 ligase deletion strains. We demonstrate that an ab-sence ofWHI2 is responsible for the observed impairment in the proteolytic degra-dation of Guk1-7. We propose a link between mutations inWHI2 to a deficiency iniithe Msn2/4 transcriptional response, thereby altering the cell’s capacity to degrademisfolded cytosolic proteins.Collectively, the data in this thesis generated with the Guk1-7 model substrateunderscores how changes in the elaborate protein quality control network can per-turb proteostasis. Given that proteostasis is altered in a number of diseases rangingfrom cancer to ageing, identifying the factors that mediate protein quality controland understanding the interplay between members of the proteostatic network areimportant not only for understanding the basic biological processes but also forpotential therapeutic applications.iiiPrefaceChapter 1 is partially based on a first author publication. Comyn SA, Chan GT,Mayor T. (2014). False start: cotranslational protein ubiquitination and cytosolicprotein quality control. J Proteomics 100:92-101. doi: 10.1016/j.jprot.2013.08.005.This is a peer reviewed review article. I co-wrote most of the manuscript withThibault Mayor, whereas Gerard Chan helped with some sections. I prepared thefigure.Chapter 2 is based on a first author publication. Comyn SA, Young BP, LoewenCJ, Mayor T. (2016). Prefoldin promotes proteasomal degradation of cytosolicproteins with missense mutations by maintaining substrate solubility. PLoS Genet-ics 12(7):e1006184. doi: 10.1371/journal.pgen.1006184. I performed all of theexperiments and Barry Young prepared the barcoded yeast deletion collection usedfor the screen and all subsequent validation experiments. I and Thibault Mayor co-wrote the manuscript with input from Drs. Barry Young and Christopher Loewen.The plasmid BPM866 (pFA6a-mCherry-KanMX6) and the yeast strain YTM1919(Hsp42-mCherry), which were used in this study, were made by Mang Zhu usinga codon optimized mCherry template prepared by Dr. Patrick Chan.Chapter 3 is based on a first author publication being prepared for submission.Comyn SA, Flibotte S, Spear ED, Michaelis S, Mayor T. Recurrent background mu-tations in WHI2 alter proteostasis and impair degradation of cytosolic misfoldedproteins in Saccharomyces cerevisiae. All the experiments were designed by my-self and Thibault Mayor. I performed most of the experiments and co-wrote themanuscript with Thibault Mayor. Illumina library preparation and whole genomesequencing was conducted by Ana Kuzmin at the NextGen Sequencing facility atthe UBC Biodiversity Research Centre. Dr. Ste´phane Flibotte performed the se-ivquencing analysis which led to the identification of theWHI2mutation. Eric Spearand Susan Michaelis performed experiments that are not presented in this thesisbut form part of this publication.vTable of ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iiPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ivTable of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viList of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xList of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiGlossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiiiAcknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Protein Misfolding and Protein Homeostasis . . . . . . . . . . . . 11.2 Protein Folding and Cytosolic Molecular Chaperones . . . . . . . 31.2.1 Nascent Protein Folding . . . . . . . . . . . . . . . . . . 31.2.2 Hsp70, Hsp40, and Hsp90 . . . . . . . . . . . . . . . . . 41.2.3 TRiC/CCT Chaperonin and Prefoldin . . . . . . . . . . . 51.3 Molecular Chaperones and Protein Degradation . . . . . . . . . . 81.4 Ubiquitin Proteasome System (UPS) . . . . . . . . . . . . . . . . 81.4.1 ER Associated Degradation (ERAD) . . . . . . . . . . . . 101.4.2 Nuclear Protein Quality Control . . . . . . . . . . . . . . 121.5 Cytosolic E3 Ubiquitin Ligases Involved in Protein Quality Control 131.5.1 CHIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13vi1.5.2 Ubr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141.5.3 Hul5 and Rsp5 . . . . . . . . . . . . . . . . . . . . . . . 151.5.4 Ltn1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151.6 Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.7 Spatial Protein Quality Control: CytoQ, IPOD, and INQ . . . . . 181.8 Stress Responses . . . . . . . . . . . . . . . . . . . . . . . . . . 191.8.1 Heat Shock and General Stress Response . . . . . . . . . 201.9 Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211.10 Model Substrates Used to Study Proteostasis . . . . . . . . . . . . 231.11 Research Objective . . . . . . . . . . . . . . . . . . . . . . . . . 261.11.1 Specific Aims . . . . . . . . . . . . . . . . . . . . . . . . 262 Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteinswith Missense Mutations by Maintaining Substrate Solubility . . . . 272.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . 292.2.1 Yeast Strains, Plasmids, and Media . . . . . . . . . . . . 292.2.2 Stability Effect of Guk1-7 Mutations . . . . . . . . . . . 342.2.3 Cellular Thermal Shift Assay (CETSA) . . . . . . . . . . 342.2.4 Solubility Assay . . . . . . . . . . . . . . . . . . . . . . 342.2.5 Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . 352.2.6 Degradation Assay . . . . . . . . . . . . . . . . . . . . . 352.2.7 Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . 362.2.8 GFP Pulldown . . . . . . . . . . . . . . . . . . . . . . . 362.2.9 Proteasome Function . . . . . . . . . . . . . . . . . . . . 362.2.10 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . 372.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372.3.1 Guk1-7 is Thermally Unstable . . . . . . . . . . . . . . . 372.3.2 Fluorescence-Based Assay to Assess Protein Stability . . 382.3.3 Guk1-7 Degradation is Proteasome Dependent . . . . . . 442.3.4 FACS-Based Screen for Protein Homeostasis Factors . . . 452.3.5 Ubr1 Stabilizes Guk1 Missense Mutant . . . . . . . . . . 482.3.6 Gim3 Impairs Guk1-7-GFP Degradation . . . . . . . . . . 51vii2.3.7 Gim3 Facilitates the Clearance of Insoluble Guk1 andMain-tains Guk1-7 Solubility . . . . . . . . . . . . . . . . . . . 542.3.8 Gim3 Has a General Effect Towards Thermally Destabi-lized Proteins . . . . . . . . . . . . . . . . . . . . . . . . 592.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622.5 Supplemental Data . . . . . . . . . . . . . . . . . . . . . . . . . 673 Recurrent Background Mutations in WHI2 Alter Proteostasis andImpair Degradation of CytosolicMisfolded Proteins in Saccharomycescerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743.2.1 Yeast Strains, Media, and Growth Conditions . . . . . . . 743.2.2 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . 773.2.3 Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . 773.2.4 Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . 803.2.5 WHI2 Plate Assay . . . . . . . . . . . . . . . . . . . . . 803.2.6 Turnover Assay . . . . . . . . . . . . . . . . . . . . . . . 813.2.7 Solubility Assay . . . . . . . . . . . . . . . . . . . . . . 813.2.8 Guk1-7-GFP Ubiquitination . . . . . . . . . . . . . . . . 813.2.9 Cellular Thermal Shift Assay (CETSA) . . . . . . . . . . 823.2.10 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . 823.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833.3.1 Multiple Strains From the Yeast Knockout Collection Dis-play Impaired Proteostasis . . . . . . . . . . . . . . . . . 833.3.2 A Secondary Mutation in WHI2 Co-Segregates with In-creased Guk1-7-GFP Stability . . . . . . . . . . . . . . . 853.3.3 Guk1-7-GFP Degradation is Impaired Owing to SecondaryMutations inWHI2 . . . . . . . . . . . . . . . . . . . . . 923.3.4 Reduced Proteostasic Capacity inWHI2Mutants is Linkedto Msn2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 953.3.5 Mutant WHI2 Impairs Guk1-7-GFP Degradation by Re-ducing Substrate Ubiquitination . . . . . . . . . . . . . . 96viii3.3.6 Essential E3 Ligase Rsp5 and Molecular Chaperones Ydj1and Ssa1 are Required for Guk1-7-GFP Degradation . . . 993.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1064.1 Chapter Summaries . . . . . . . . . . . . . . . . . . . . . . . . . 1064.2 General Discussion . . . . . . . . . . . . . . . . . . . . . . . . . 1074.2.1 Using Temperature Sensitive Alleles asModel Protein Qual-ity Control Substrates . . . . . . . . . . . . . . . . . . . . 1074.2.2 Flow Cytometry: An Ideal Method for Identifying and Char-acterizing Protein Quality Control Factors . . . . . . . . . 1094.2.3 Triage Decisions: Simply a Matter of Kinetic Partitioning? 1104.2.4 The Importance Of, and Difficulty In, Maintaining Pro-teostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . 1114.3 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . 1134.3.1 Flow Cytometry Screens for E3 Ligases Targeting HumanDisease Alleles . . . . . . . . . . . . . . . . . . . . . . . 1134.3.2 Characterizing the Role of the E3 Ligase Ubr1 in Cyto-plasmic Protein Quality Control . . . . . . . . . . . . . . 113Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116ixList of TablesTable 2.1 Yeast strains used in Chapter 2 . . . . . . . . . . . . . . . . . 30Table 2.2 Plasmids used in Chapter 2 . . . . . . . . . . . . . . . . . . . 33Table 2.3 Summary of FACS screen validation . . . . . . . . . . . . . . 48Table 3.1 Yeast strains used in Chapter 3 . . . . . . . . . . . . . . . . . 74Table 3.2 E3 ligase collection used for screening . . . . . . . . . . . . . 78Table 3.3 Plasmids used in Chapter 3 . . . . . . . . . . . . . . . . . . . 79xList of FiguresFigure 1.1 Proteostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Figure 1.2 Hsp70 reaction cycle . . . . . . . . . . . . . . . . . . . . . . 6Figure 1.3 E3 ubiquitin ligases . . . . . . . . . . . . . . . . . . . . . . . 10Figure 1.4 Protein quality control . . . . . . . . . . . . . . . . . . . . . 11Figure 1.5 The general stress response . . . . . . . . . . . . . . . . . . . 22Figure 2.1 Guk1-7 is thermally unstable . . . . . . . . . . . . . . . . . . 40Figure 2.2 Misfolded Guk1-7 is degraded at the non-permissive temperature 42Figure 2.3 Guk1-7 degradation is proteasome dependent . . . . . . . . . 44Figure 2.4 FACS-based screen . . . . . . . . . . . . . . . . . . . . . . . 47Figure 2.5 Ubr1 promotes Guk1-7-GFP degradation . . . . . . . . . . . 50Figure 2.6 Absence of Gim3 reduces Guk1-7 turnover . . . . . . . . . . 53Figure 2.7 Gim3 facilitates clearance of insoluble Guk1-7 . . . . . . . . 57Figure 2.8 Guk1-7-GFP puncta colocalize with Q-body markers . . . . . 58Figure 2.9 Gim3 helps maintain Guk1-7 solubility . . . . . . . . . . . . 59Figure 2.10 Thermosensitive alleles are stabilized by prefoldin subunits . . 61Figure 2.11 Model for stabilization of temperature sensitive alleles by Gim3 65Figure 2.12 Guk1-7-GFP flow cytometry . . . . . . . . . . . . . . . . . . 68Figure 2.13 Ubr1 does not act with San1 in the degradation of Guk1-7-GFP 69Figure 2.14 Guk1-7-GFP Gim3 interaction and viability assay . . . . . . . 71Figure 3.1 Flow cytometry based screen for E3 ligases targeting Guk1-7-GFPfor degradation . . . . . . . . . . . . . . . . . . . . . . . . . 85Figure 3.2 Guk1-7-GFP degradation in E3 ligase deletion strains . . . . . 87xiFigure 3.3 Guk1-7-GFP stability is not a direct effect of E3 ligase deletion 88Figure 3.4 Mutations in WHI2 segregate with the Guk1-7-GFP stabilityphenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90Figure 3.5 das1∆ tetrad analysis and WHI2 addback . . . . . . . . . . . 91Figure 3.6 Absence ofWHI2 leads to Guk1-7-GFP stability . . . . . . . 94Figure 3.7 Msn2 is linked to reduced proteostatic capacity inWHI2 mutants 97Figure 3.8 whi2∆ promotes Guk1-7-GFP stability through reduced ubiq-uitination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98Figure 3.9 A role for essential E3 ligases and molecular chaperones inGuk1-7-GFP degradation . . . . . . . . . . . . . . . . . . . . 101xiiGlossary∆∆G Free energy changeABCE1 ATP binding cassette subfamily E member 1ADP Adenosine diphosphateANOVA Analysis of varianceARS Autonomously replicating sequenceAsi1 Amino acid sensor independentAtg8 Autophagy relatedATP Adenosine triphosphateBmh2 Brain modulosignalin homologuebp Base pairBra7 Fluorocytosine resistanceBtn2 Batten diseaseCCT Chaperonin containing TCP-1Cdc48 Cell division cycleCEN Yeast centromereCETSA Cellular thermal shift assayCHX CycloheximideCHIP C-terminus of Hsc70-interacting proteinCFTR Cystic fibrosis transmembrane conductance regulatorCPY CarboxypeptidaseCytoQ Cytosolic quality control compartmentDas1 Dst1-delta6-azaurail sensitivityDBD DNA binding domainDeg1 Depressed growth rateDIC Differential interference contrastDNA Deoxynucleic acidxiiiDoa10 Suppressor of mRNA stability mutantDsRed Discosoma sp. red fluorescent proteinE1 Ubiquitin activating enzymeE2 Ubiquitin conjugating enzymeE3 Ubiquitin ligase enzymeEDTA Ethylenediamine tetraacetic acidEGFP Enhanced GFPER Endoplasmic reticulumERAD ER associated degradationERISQ Excess ribosomal protein quality controlEV Empty vectorFACS Fluorescence activated cell sortingFap1 FKBP12-associated proteinFDA Food and drug administrationFes1 Factor exchange for Ssa1pFITC Fluorescein isothiocyanateg GravityGDP Guanosine diphosphateGFP Green fluorescent proteinGim Gene involved in microtubule biogenesisGlo4 GlyoxalaseGMP Guanosine diphosphateGPD Triose-phosphate dehydrogenaseGPS Global protein stability analysisGuk1 Guanylate kinaseGus1 Glutamyl-tRNA synthetaseHbs1 Hsp70 subfamily B suppressorHCl Hydrogen chlorideHECT Homologous to the E6AP carboxyl terminus domainHEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHGMD Human gene mutation databaseHOP Hsp90 organizing proteinHrd1 HMG-coA reductase degradationxivHrt3 High level expression reduces Ty3 transpositionHSD Honest significant differenceHsp Heat shock proteinHul5 HECT ubiquitin ligaseINQ Intranuclear quality control compartmentIPOD Immobile protein depositIRES Internal ribosome entry siteJUNQ Juxtanuclear quality control compartmentkDa Kilo daltonLtn1 RING domain mutant killed by Rtf1 deletionµg MicrogramµL Microlitreµm MicrometermL MillilitremM MillimolarM MolarMr Molecular weightmin MinuteMAT Mating typeMPP11 DnaJ Hsp family member C2Msn Multicopy suppressor of SNF1 mutationMup3 Methionine uptaken NumberNAC Nascent chain associated complexNaCl Sodium chlorideNEDD4 Neural precursor cell expressed, developmentally downregulated 4NEF Nucleotide exchange factorNEMF Nuclear export mediator factorNES Nuclear export sequenceNLS Nuclear localization sequenceNMP Nucleoside monophosphate kinaseNP-40 Nonidet P-40Npl4 Nuclear protein localizationxvOD Optical densityORF Open reading frameP P-valuep62 Nucleoporin 62PAH Phenylalanine hydroxylasePBS Phosphate buffered salinePCR Polymerase chain reactionPDB Protein data bankPep4 Carboxy peptidase Y-deficientPgk1 3-phosphoglycerate kinasePKA Protein kinase APMSF Phenylmethane sulfonyl fluoridePolyQ PolyglutaminePrb1 Proteinase BPro3 Proline requiringPrc1 Proteinase CRAC Ribosome associated complexRBR RING-between-RINGRim15 Regulator of IME2RING Really interesting new geneRNA Ribonucleic acidRpt6 Regulatory particle triple-A proteinRQC Ribosome quality control complexRqc1 Ribosome quality control 1Rsp5 Reverses Spt-phenotypeS SolubleSan1 Sir antagonistSD Standard deviationSDG Saccharomyces genome databaseSDS Sodium dodecyl sulfateSDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresisSec61 SecretorySILAC Stable isotope labelling with amino acids in cell culturexviSir4 Silent information regulatorSis1 Slt4 suppressorSNV Single nucleotide variantSsa Stress sensitive subfamily ASsb Stress sensitive subfamily BSse1 Stress seventy subfamily ESti1 Stress inducibleSTRE Stress response elementst TimeT Total cell lysateTAD Transcriptional activating domainTae2 Translation-associated element 2TAP Tandem affinity purificationTcp Tailless complex polypeptideTom1 Trigger of mitosisTOR Target of rapamycinTPR Tetratricopeptide repeat domainTRiC TCP-1 ring complexTx-100 Triton X-100Ub UbiquitinUbc Ubiquitin conjugatingUbe2W Ubiquitin conjugating enzyme E2 WUbp Ubiquitin specific proteaseUbr1 Ubiquitin protein ligase E3 component N-recognin 1Ufd Ubiquitin fusion degradationUgp1 UDP-glucose pyrophosphorylaseUPS Ubiquitin proteasome systemUTR Untranslated regionVHL von Hippel LandauWhi2 WhiskeyX TimesYdj1 Yeast dnaJYPD Yeast extract peptone dextrosexviiAcknowledgementsI would like to thank everyone who has contributed to the work contained in thisthesis and who has helped me throughout the course of my degree. I would like toexpress my appreciation to my supervisor, Thibault Mayor, and to my supervisorycommittee members Christopher Loewen, Vivien Measday, and Michel Robergefor their advice, encouragement, guidance, and mentorship. Thank you to membersof the Mayor lab for valuable feedback and camaraderie. To Elizabeth, Allym,Aruna, and Carla, thank you for making me welcome in your lab and for yourkindness and generosity. Thank you to Megan Kofoed for all of her assistance withthe numerous yeast collections. Thank you to members of the Hansen lab, bothpast and present, for their friendship and making the office a fun place to be overthe past six years. I am especially grateful to Cheryl and Tan for their friendshipand insightful discussions. Finally, I would like to express my gratitude to myparents for their support and patience, without which none of this would have beenpossible.xviiiChapter 1Introduction1.1 Protein Misfolding and Protein HomeostasisProtein homeostasis, or proteostasis, preserves proteome integrity and thereby pro-motes viability at both the cellular and organism levels. To do so, proteostasis ismaintained by a network of interconnected pathways that influence the fate of pro-teins by directing their translation, folding, localization, and degradation [1]. Mis-folded proteins are one of many factors that challenge a cell’s ability to maintainproteostatic balance.Protein misfolding can result from a number of processes such as mutation; er-rors during transcription, RNA processing and translation; trapping of a folding in-termediate; failure to incorporate into multimeric complexes; or post-translationaldamage [2, 3]. The risk to the cell of misfolded or partially folded proteins may beattributed, at least in part, to the exposure of hydrophobic amino acid residues thatin the native state would be sequestered to the core of the protein, or at protein-protein interaction interfaces, but once exposed can engage in unspecific interac-tions with other polypeptides. These exposed hydrophobic regions of misfoldedproteins also have an inherent propensity to aggregate, forming associations notnative to the cell [4]. For example, artificial beta-sheet proteins expressed in humanHEK293T cells were found to coaggregate with proteins that have many functionalinteraction partners suggesting that the aggregates competitively bind to functionalprotein-protein interaction interfaces [5]. Moreover, the relative cytotoxicity of the1aggregates correlated with the number of interaction partners ascribed to the coag-gregating proteins. More recently, Kim et al. identified and analysed aberrant pro-tein interactions involving soluble oligomers and insoluble inclusions of the mutanthuntingtin protein [6]. Expressing a fragment of huntingtin, containing the Hunt-ington’s disease causing polyglutamine (PolyQ) repeat expansion, they found thatinsoluble inclusions predominantly interacted with members of the protein qualitycontrol machinery representing ∼85 proteins. Soluble oligomers interacted withupwards of 800 different proteins representing diverse cellular functions such astranscription, translation, and RNA-binding. Within the cytosol, macromolecularcrowding creates an environment that increases the tendency of folding interme-diates and misfolded proteins to aggregate, as aggregation is highly concentrationdependent [7]. The native conformation of a protein, however, must balance struc-tural stability with conformational flexibility that is associated with protein func-tion [8]. As such, a tightly regulated network of molecular chaperones contendwith a constant flux of protein intermediates, misfolded proteins, and aggregateformation [9].Proteostasis depends on balancing the folding capacity of chaperone networkswith the quantity of proteins in non-native conformations (Figure 1.1). When thelevel of misfolded proteins rises, the folding capacity of the cell can be temporarilyaugmented to meet the increased demand through the activation of signaling path-ways modulated by the transcription factors heat shock factor 1 (Hsf1) andMsn2/4.Stressors that precipitate protein misfolding disrupt equilibrium and if the cellularresponse is overwhelmed and insufficient to meet the increase in need, then it canlead to the accumulation and aggregation of misfolded proteins [10]. Although theexact mechanism that results in the formation of cellular aggregates has yet to befully elucidated, their presence is associated with a number of neurodegenerativeconditions such as Huntington’s, Parkinson’s, and Alzheimer’s diseases, as well asageing [11, 12]. Maintaining proteome integrity, therefore, requires an integratedprotein quality control network that monitors the proteome, mediates protein re-folding by molecular chaperones, and removes terminally misfolded proteins viathe ubiquitin proteasome system or autophagy [1].2ProteostasisStresses ResponsesPerturbations Adaptive ResponsesAgeingEnvironmental StressesGenetic MutationsMisfolded ProteinsProteasome DeficiencyUnassembled SubunitsAutophagyGeneral Stress ResponseHeat Shock ResponseMolecular ChaperonesSpatial PQCTranslation ControlUbiquitin Proteasome SystemFigure 1.1: Proteostasis. Proteostasis depends upon balancing the perturba-tions that disrupt protein folding with the network of pathways that direct thelevels, conformational state, and distribution of the proteome.1.2 Protein Folding and Cytosolic Molecular ChaperonesMolecular chaperones promote protein homeostasis by preventing protein aggrega-tion, assisting protein folding, and targeting terminally misfolded clients for degra-dation. Broadly, chaperones can be defined as any protein that recognizes andinteracts with proteins found in a non-native state for the purpose of stabilizing andpromoting folding into an active conformation without forming part of the finalstructure [10]. There are a number of distinct conserved chaperone families, oneof which is the heat shock protein (Hsp) family whose members are classified bytheir molecular weights (e.g. Hsp40, Hsp70, Hsp90, and the small Hsps). Withinthe context of protein quality control in the eukaryotic cytosol, the main chaperonemachineries involved are the: ribosome associated chaperones, Hsp70/40, Hsp90,TRiC/CCT chaperonin, and prefoldin.1.2.1 Nascent Protein FoldingA number of chaperones bind to, or associate with, the ribosome to both promoteprotein folding and prevent misfolding or aggregation of the nascent polypeptideas it emerges from the ribosome. As translation occurs at a slower rate than proteinfolding, nascent polypeptide chains emerge from the ribosome in a partially foldedaggregation prone state. Also, because the size of the ribosome exit tunnel is suchthat folding beyond the formation of alpha helical structures is prohibited, only lim-ited folding can proceed until a domain (generally 50 to 300 amino acids in length)3exits the ribosome [13]. Therefore, molecular chaperones interact co-translationalywith nascent polypeptide chains to prevent aggregation and premature non-nativefolding from occurring before the polypeptide has been fully translated. Moreover,while the ribosome exit sites are positioned in the polysome in such a way as tominimize aggregation of the nascent polypeptides, ribosome associated chaperonesare needed to further prevent aggregation of the numerous identical proteins beingtranslated from the polysome [14, 15]. The ribosome associated complex (RAC)and nascent chain associated complex (NAC) are the first chaperone complexesto interact with the nascent polypeptide as it exits the ribosome. In mammals,RAC is formed by the association of Hsp70L1 and the J-domain containing pro-tein MPP11. In yeast, RAC consists of the Hsp70 Ssz1 and the ribosome bindingHsp40 zuotin. Zuotin in turn acts in concert with the Hsp70s Ssb1 and Ssb2 that arealso associated to the ribosome [16–18]. Deleting the genes encoding the dimericNAC complex subunits and Ssb proteins in Saccharomyces cerevisiae resulted indecreased viability under conditions of protein folding stress [19]. Moreover, theabundance of ribosomal particles was altered in these mutants suggesting that ribo-some biogenesis is linked to the protein folding capacity of the ribosome associatedchaperones. Partially folded proteins are transferred by Hsp70 chaperones fromthe ribosome associated complex to further downstream folding pathways such asHsp70, prefoldin, and chaperonin.1.2.2 Hsp70, Hsp40, and Hsp90The Hsp70 family of molecular chaperones promotes protein folding through reit-erative cycles of ATP-dependent client capture and release (Figure 1.2). Hsp70s donot function independently, but as part of an Hsp70 core machinery consisting of anHsp70, an Hsp40 (J domain protein), and a nucleotide exchange factor (NEF) thatcoordinate their activities to increase the efficiency of Hsp70 client folding [20, 21].Hsp70 consists of an N-terminal ATPase domain and a C-terminal substrate bind-ing domain that binds to short 5-7 amino acid stretches of hydrophobic residueson client proteins. Dimerization is necessary for Hsp70 chaperone activity and ef-ficient Hsp40 interaction [22]. Initial client binding occurs while Hsp70 is in theATP-bound state and the Hsp70-client interaction is stabilized through ATP hy-4drolysis. The intrinsic ATPase activity of Hsp70 is relatively low however, andis enhanced by association with an Hsp40. As the number of Hsp40 proteins inthe cell greatly outnumbers that of the Hsp70s, it is thought that the repertoire ofHsp40 proteins help target Hsp70 to its clients, or bind them directly to enhancethe specificity of the system [23]. How each Hsp40 protein recognizes its cohort ofsubstrates remains to be fully understood. Once Hsp70 is bound to a client protein,Hsp40, via its conserved J domain, stimulates ATP hydrolysis and the subsequentactivity of a NEF promotes ADP dissociation and client release. Upon releasefrom Hsp70 the bound hydrophobic region is free to refold, however, it may re-associate with Hsp70 if folding is not complete. There are four non-ribosomalbinding Hsp70s (Ssa1-4) in the cytosol of S. cerevisiae and their ATP hydrolysisis enhanced by Ydj1 and Sis1 Hsp40 co-chaperones [24]. In addition to Fes1, aconfirmed NEF in the yeast cytosol, the Hsp110 Sse1 has also been reported toact as a NEF for the Hsp70s Ssa1 and Ssb1 [24]. The Hsp70 family can act bothco- and post-translationaly and are hubs to direct substrates to further downstreamchaperone networks such as Hsp90 and chaperonin [25].Hsp90 acts downstream of Hsp70 and Hsp40 to assist in the folding of nascenttranscription factors, protein kinases, and steroid hormone receptors [26]. Sub-strate transfer from the Hsp70/Hsp40 system to Hsp90 is mediated by the Hsp90organizing protein (HOP, or Sti1 in yeast) [27]. The tetratricopeptide repeat do-main (TPR) of HOP interacts with the MEEVD sequence on the C-terminal ofHsp90 bridging the two chaperone machineries and facilitating substrate transfer.Once bound to a substrate, Hsp90 ATPase activity is stimulated through an inter-action with Aha1. Chemical inhibition of Hsp90 function leads to the proteasomaldegradation of many Hsp90 substrates potentially as the result of increased inter-action with Hsp70 and Hsp70 associated factors [28].1.2.3 TRiC/CCT Chaperonin and PrefoldinChaperonin, also known as the TCP-1 ring complex (TRiC) or; the chaperonincontaining TCP-1 (CCT), functions in folding newly translated proteins and pre-venting protein aggregation in the cytosol [29, 30]. Essential in all three domains oflife, it has been estimated that upwards of 10% of the eukaryotic proteome transits5Hsp70ATPOpenOpenClosedPartially foldedproteinADPATPHsp40Hsp70Hsp40Non-native proteinNon-native proteinHsp40PiNEFATP123Figure 1.2: Hsp70 reaction cycle. 1) Hsp40 binds to misfolded substrates anddelivers them to an ATP bound Hsp70. 2) Substrates bind to Hsp70 via hy-drophobic patches (blue) and ATP hydrolysis to ADP, accelerated by Hsp40,switches Hsp70 to the closed substrate binding conformation. 3) Hsp40 dis-sociates from Hsp70 and a nucleotide exchange factor (NEF) exchanges ATPfor ADP causing Hsp70 to open and release the misfolded substrate enablingit to fold. Substrates can re-engage with Hsp40 to continue Hsp70 cycling orfold into their native conformation.through CCT, including cytoskeleton proteins and cell cycle regulators [31]. Chap-eronin is a large cylindrical 1 MDa protein complex formed through the stackingof two identical rings, each comprising eight subunits (CCT1-8) [30]. The apicaldomains of the ring subunits act as a lid to enclose the partially unfolded sub-strate in the central cavity mitigating the effects of macromolecular crowding onprotein folding. In eukaryotes, the chaperonin lid does not close entirely therebyaccommodating extended polypeptide chains and single domains of multidomainsubstrates [32]. CCT engages substrates while in the ATP-bound state with ATP6hydrolysis inducing conformational changes that lead to the closure of the chap-eronin lid and substrate encapsulation. Each ring is divided into two hemispheresbased on ATP binding affinity that leads to a cycle of asymmetric conformationalchanges [33]. CCT subunits 3, 6, 7, and 8 constitute one of the hemispheres andhave low affinity for ATP under physiological conditions and were found to be dis-pensable for chaperonin activity [33]. While all eight subunits have a conservedATP binding domain, their sequences are only ∼40% identical. The apical do-mains of each subunit, therefore, are thought to recognize different substrate mo-tifs, which is underscored by each subunit having its own specific patterns of polarand hydrophobic residues [34]. The pattern of amino acid residues in the each api-cal domain is thought to allow chaperonin to bind and fold a range of structurallydiverse proteins. Although initially identified through its requirement for fold-ing the cytoskeletal proteins actin and tubulin, the eukaryotic chaperonin has beenshown to act in the folding of a number of other substrates [35]. The human chaper-onin interactome is enriched for proteins predicted to be aggregation prone, whichcontain multiple domains and have complex topologies [36]. The importance ofCCT in human health is underscored by a mutation in CCT5 being identified asthe cause of autosomal recessive mutilating sensory neuropathology with spasticparaplegia [37]. CCT is also required for the replication of human pathogens suchas HIV and hepatitis C as well as folding a number of cancer associated proteins,such as p53 and the von Hippel Lindau tumor suppressor [38–40].Prefoldin is a hetero-oligomeric protein complex composed of six subunitsranging in size from 14–23 kDa [41]. Conserved in archaea and eukaryotes, butabsent in prokaryotes, the prefoldin hexamer forms a “jellyfish-like” structure withN- and C-terminal coiled-coil regions of each subunit forming “tentacles” that em-anate from a central region [42]. Misfolded substrates are transferred from pre-foldin to the TRiC/CCT chaperonin in an ATP-independent manner through di-rect binding of the two chaperone complexes [41]. In addition to its role in aidingnascent proteins, such as actin and tubulin, to attain their functional conforma-tions, the prefoldin chaperone complex has been shown to prevent huntingtin andalpha-synuclein aggregate formation [43, 44].71.3 Molecular Chaperones and Protein DegradationWhile molecular chaperones promote protein homeostasis by preventing proteinaggregation and promoting protein folding, they can also mediate the targetingof terminally misfolded clients for degradation; either indirectly, by maintainingmisfolded proteins in a non-aggregated degradation competent state, or directlythrough facilitating the recognition and/or transfer of substrates to degradativequality control pathways [45–49]. In an elegant study using the von Hippel Lin-dau (VHL) tumor suppressor protein as a misfolded model substrate, Frydman andcolleagues showed that a different set of chaperone proteins and co-factors mediatefolding and degradation, respectively [50]. Hsp90 and the co-chaperone Sti1 wererequired for degradation but not the folding of VHL, while the converse was truefor the chaperonin TRiC/CCT. The Ssa1/2 cytosolic Hsp70s, and the nucleotideexchange factor Sse1 were also required for degradation of VHL, as well as othercytosolic misfolded proteins [50, 51]. Although the yeast Hsp70 cofactor Ydj1 wasnot required for VHL degradation, it has been shown to mediate the degradation ofER proteins with exposed misfolded cytosolic domains and the degradation of cy-tosolic proteins after heat shock [52, 53]. Most recently Fes1, another yeast Hsp70NEF, was shown to promote proteasomal degradation of additional misfolded pro-teins [54]. In higher eukaryotes, the Bag6 Hsp70 cofactor that can bind to theproteasome via its ubiquitin-like domain is required for the efficient degradationof defective nascent polypeptides [55]. Although the degradation of distinct sub-strates has been demonstrated to require different chaperones and co-chaperones,in most cases it remains unclear how two competing systems (i.e. folding anddegradation machineries) triage misfolded proteins in the cell.1.4 Ubiquitin Proteasome System (UPS)In eukaryotes, the ubiquitin proteasome system plays a critical role in protein qual-ity control by selectively targeting intracellular proteins for degradation throughthe covalent attachment of polyubiquitin chains. Ubiquitin is a highly conserved8.5 kDa protein that is primarily conjugated onto lysine residues of target sub-strates through the activity of an enzymatic cascade involving ubiquitin activating(E1), ubiquitin conjugating (E2), and ubiquitin ligase (E3) enzymes [56]. Substrate8specificity and recruitment is mediated by an E3 ubiquitin ligase, either alone or incombination with an E2 conjugating enzyme. The inherent complexity of the ubiq-uitin system is reflected in the sheer number of putative E3 ligases (90 and 600)encoded in the genome of yeast and human, respectively [57]. Moreover, the im-portance of E3 ligases in substrate targeting is emphasized by the fact that the num-ber of putative E3 ligases greatly outnumbers that of E2 conjugating enzymes byapproximately 15:1 [58]. E3 ubiquitin ligases belong to one of three families char-acterized by their namesake domains (Figure 1.3). The really interesting new gene(RING) family are the most abundant E3 ligases with ∼600 members in humansand can be found as monomers, dimers, or as part of multisubunit complexes [59].The RING domain can be located anywhere on the protein and consists of a con-served consensus sequence of cysteine and/or histidine residues, which coordinatewith two zinc atoms to stabilize the domain structure [58]. RING E3 ligases act asscaffolds to orient the substrate with an E2-ubiquitin conjugate for efficient ubiqui-tin transfer. Approximately thirty proteins belong to the homologous to the E6APcarboxyl terminus domain (HECT) family with all known HECT domains locatedat the C-terminus of the protein. HECT E3 ligases play a direct role in substrateubiquitination through a stepwise process. First, the HECT E3 ligase, via an N-lobe E2 binding domain, receives an E2-ubiquitin conjugate to then form an E3-ubiquitin conjugate. Ubiquitin is then conjugated to a substrate from the E3 activesite via a C-lobe catalytic cysteine [58]. The final family, the RING-between-RINGor RBR E3 ligases, are the least abundant containing only 13 members in humans.RBR ligases contain an N-terminal RING domain (RING1), like the RING ligases,followed by in between RING and RING2 domains, which are unique to RBR pro-teins and do not contain the cysteine RING consensus sequence. In addition, likeHECT ligases, RBR ligases form a thioester bond with ubiquitin. Mutations inParkin, one of the most studied members of this family, are associated with earlyonset Parkinson’s disease [60].Ubiquitin has seven lysine residues, all of which can be conjugated to ubiquitinmolecules to form polyubiquitin chains. The K48 chain linkage is considered tobe the predominant signal recognized by the cell for proteasomal degradation. The26S proteasome is a large 2.5 MDa protein complex responsible for the selectiverecognition and degradation of ubiquitin conjugated proteins. It is composed of the9RING HECT RBRE2 conjugating enzyme RING domainSubstrate UbiquitinN-lobeC-lobeIBRRING1 RING2Figure 1.3: Schematic of the three E3 ubiquitin ligase families and E3 catal-ysed ubiquitin transfer.central barrel-like 20S core particle, which contains the proteolytic peptidases, andtwo 19S regulatory particles. The regulatory particle is responsible for substraterecognition, ubiquitin chain removal, and protein unfolding and translocation intothe catalytic core particle [61, 62]. In addition, the system is under constant flux asubiquitination can be reversed by deubiquitinating enzymes.There are multiple pathways that can target misfolded proteins for proteaso-mal degradation. Different types of protein damage are more prevalent in differentcellular compartments owing to the nature of the subcellular environment neces-sitating compartment-specific quality control pathways, with systems having beendescribed for the ER, nucleus, and cytoplasm [63].1.4.1 ER Associated Degradation (ERAD)Approximately one third of all eukaryotic proteins are membrane or secreted pro-teins that must pass through the ER [64]. Protein folding is monitored by ERquality control machinery and non-native or unassembled subunits are targeted fordegradation by the ER associated degradation (ERAD) pathway. If misfolded pro-teins are left to accumulate in the ER, a stress response is triggered in an attemptto rebalance the protein quality control system and to clear misfolded proteins.ERAD was initially discovered through studies of the cystic fibrosis transmem-brane conductance regulator (CFTR). While some components of the ERAD path-10way, such as the E3 ligases, are better defined in yeast, the identification of humanhomologs to several of the yeast genes involved would suggest that ERAD mayplay a role in ER proteostasis in higher eukaryotes as well. ER proteins targetedfor degradation must be retranslocated from the ER in an ATP-dependent mannerwhere they are degraded by the ubiquitin proteasome system [64]. The multispanER membrane RING E3 ligases Doa10 and Hrd1 ubiquitinate substrates on the cy-tosolic side of the ER following substrate retranslocation by a complex containingthe AAA-ATPase Cdc48, Ufd1, and Npl4 (p97, UFD1, and NPL4 in mammals)(Figure 1.4) [65]. The mechanism determining whether a substrate is targeted byDoa10 or Hrd1 is thought to be based upon the location of the degradation sig-nal. ER lumen and membrane substrates are generally recognized by Hrd1 andcytosolic substrates by Doa10 [66, 67].NucleusSan1, Asi1, Tom1Endoplasmic reticulumHrd1, Doa10CytoplasmUbr1, Hul5, Rsp5, Ltn1INQCytoQVacuoleIPODFigure 1.4: Schematic representation of the spatial distribution of proteinquality control compartments in yeast.111.4.2 Nuclear Protein Quality ControlDespite the fact that the majority of proteasomes are located within the nucleusunder non-stress conditions, our understanding of nuclear protein quality controlis relatively limited compared to that of the ER and cytoplasm [68]. In yeast, theprimary model organism used for nuclear protein quality control studies, the RINGE3 ligase San1, in conjunction with the E2 ubiquitin conjugating enzyme Ubc1,is responsible for the ubiquitination of misfolded nuclear proteins, thereby tar-geting them for degradation by the ubiquitin proteasome system in the nucleus(Figure 1.4) [69–71]. San1 recognizes exposed hydrophobic residues on misfoldedproteins that are normally buried in the native conformation [69]. These hydropho-bic stretches interact with substrate recognition sites on San1 that are interspersedbetween N- and C-terminal intrinsically disordered domains [72]. It is thought thatthese disordered regions, which lack secondary structure, provide flexibility suchthat San1 can bind to a large number of substrates with different conformations.The AAA-ATPase Cdc48/p97 has also been shown to be required for the degra-dation of some highly insoluble San1 substrates [73]. While the role of molecu-lar chaperones in nuclear protein quality control remains unclear, the chaperonesSis1 and Sse1 are required for the nuclear targeting of misfolded cytoplasmic pro-teins [74–76]. Moreover, inhibition of Hsp42 leads to the accumulation of cyto-plasmic proteins in the nucleus [77]. This would suggest a mechanism wherebysome proteins are normally retained in the cytosol and when this fails it leads tothe accumulation and aggregation of proteins within the nucleus. Why misfoldedcytoplasmic proteins are imported into the nucleus remains unknown, however, anumber of possible explanations have been proposed. The first, is that proteinsunder 40 kDa in size passively diffuse through nuclear pores into the nucleus. Thesecond, is that nuclear import is an active response in cases where cytoplasmicprotein quality control becomes overwhelmed. The third, is that it could be ad-vantageous to separate nuclear degradation from cytosolic protein folding. Morework will be required to determine whether one, or all, of these explanations iscorrect [78, 79]. It remains unclear as to which E3 ligase is required for nuclearprotein quality control in mammals.In addition to San1, two other nuclear E3 ligases have recently been character-12ized. Asi1 is a RING E3 ligase located in the inner nuclear membrane. The Asicomplex, consisting of the proteins Asi1-3, acts in parallel with Hrd1 and Doa10of the ERAD pathway to degrade soluble and integral membrane proteins [80]. Itis not yet known if Asi1 has a more general role in nuclear protein quality control,or how its substrates are recognized. Recently, the HECT E3 ligase Tom1 wasidentified in a screen, along with the E2 conjugating enzymes Ubc4 and Ubc5, tobe responsible for targeting overexpressed and unassembled ribosomal proteins fordegradation [81]. Tom1 specifically targets residues that would normally be hiddenin mature ribosome assemblies. Cells lacking TOM1 contained aggregated riboso-mal proteins. This new pathway named excess ribosomal protein quality control(ERISQ) is conserved as the human Tom1 homolog Huwe1 demonstrated a similarfunction in human cells.1.5 Cytosolic E3 Ubiquitin Ligases Involved in ProteinQuality ControlSeveral ubiquitin ligases in higher and lower eukaryotes are proposed to targetmisfolded cytosolic proteins for degradation, a number of which are described indetail below. Many of these E3 ligases act in conjunction with molecular chaper-one partners to target their misfolded clients for degradation. Their substrates arediverse ranging from nascent polypeptides that fail to attain their native confor-mation, stalled translation products, N-terminally destabilized polypeptides, andproteins which have become misfolded. How do E3 ligases recognize their sub-strates? Does each pathway target a specific subset of misfolded proteins? Doseveral ubiquitin ligases target the same proteins, potentially recognizing differentdomains or conformations? The search for the answers to these questions drivescurrent work in the cytoplasmic protein quality control field.1.5.1 CHIPC-terminus of Hsc70-interacting protein (CHIP) E3 ligase was shown over tenyears ago to be part of a major pathway targeting cytosolic misfolded proteinsfor degradation. A chaperone dependent ligase, CHIP interacts with both Hsp70and Hsp90 via its TPR domain, as well as with misfolded proteins that are then13ubiquitinated and targeted for degradation [49, 82–84]. Several co-factors werefound to influence CHIP activity. For instance, the Bag2 Hsp70 co-chaperone wasfound to interact and inhibit CHIP activity, favoring folding over degradation ofthe substrate [85, 86]. In contrast, the related Bag1 Hsp70 co-chaperone that con-tains a proteasomal interacting ubiquitin-like domain promotes CHIP activity andmay facilitate substrate delivery to the proteasome [87, 88]. In addition, CHIPauto-ubiquitination on lysine 2, mediated by the E2 conjugating enzyme Ube2W,enhances the E3 ligase activity of CHIP [89]. Preventing this self-ubiquitinationresults in a reduction of CHIP’s ability to ubiquitinate a variety of substrates. Thedeubiquitinating enzyme Ataxin 3 has been shown to regulate the ability of CHIPto ubiquitinate itself, as well as regulate the polyubiquitin chain lengths of CHIPsubstrates [90]. CHIP in turn, has been observed to ubiquitinate the polyglutamineexpanded form of Ataxin 3, targeting it for degradation [91]. It is still unclear whatcriterion determines the targeting of such proteins for degradation. Other ubiq-uitin ligases, like Parkin (for which mutations are linked to Parkinson’s disease)and Dorfin have been implicated in the targeting of cytosolic misfolded proteins,although more recent work indicates that Parkin may instead target defective mi-tochondria for macroautophagy [92–96]. Intriguingly, these ubiquitin ligases aremostly absent in lower eukaryotes like S. cerevisiae.1.5.2 Ubr1Ubr1 was first identified and characterized as being the E3 ligase of the N-end rule,a pathway whereby the half-life of a protein correlates with the identity of the N-terminal amino acid residue that is recognized by the ubiquitin ligase [97]. Ubr1recognizes N-end rule substrates through two domains: the UBR box (binds TypeI (Arg, Lys, or His) basic N-terminal amino acids) and the ClpS domain (bindsType II (Phe, Leu, Trp, Tyr, Ile) bulky hydrophobic residues) [98]. A number ofreports however, now lend support to Ubr1 playing a role in protein degradationindependent of the N-end rule [99–101]. Subsequently, it was shown that bothSan1 and Ubr1 are key E3 ligases in the cytosolic quality control machinery (Fig-ure 1.4) [74]. The cytosolic Ubr1, alone or together with the nuclear ubiquitinligase San1, was found to target a large variety of cytosolic misfolded proteins14including artificial model substrates, thermosensitive mutant alleles and unfoldedkinases [74, 101–104]. In some cases, Ubr1 ubiquitinates cytosolic substrates withthe assistance of Sse1 and Ssa1 chaperones, while the nuclear localized San1 ubiq-uitinates substrates that are delivered to it from the cytosol with the help of theSis1 Hsp40 [48, 75]. Ubr1 is highly conserved with one mammalian homologueknown to play a role in protein quality control [105]. Mutations in human Ubr1 areresponsible for the autosomal recessive Johanson-Blizzard syndrome characterizedby developmental abnormalities and pancreatic insufficiency [106].1.5.3 Hul5 and Rsp5Exposure to heat shock stress induces a conserved cytoprotective heat shock re-sponse that, in addition to transcriptional induction and repression, results in in-creased protein ubiquitination and degradation of primarily cytosolic proteins [107–109]. The HECT E3 ligases Hul5 and Rsp5 are both required for the increasedubiquitination of cytosolic proteins observed following heat shock (Figure 1.4) [52,109]. Hul5 is a proteasome associated protein, with chain elongation activity in op-position to the proteasome bound deubiquitinating enzyme Ubp6 [110, 111]. WhileHul5 is mainly nuclear in unstressed cells, it relocalizes to the cytoplasm upon heatshock and its cytosolic localization is required for the targeting of cytosolic mis-folded proteins for proteasomal degradation [109]. As an E4 ligase, Hul5 promotesthe elongation of polyubiquitin chains initiated by other E3 ligases, thereby in-creasing substrate processivity at the proteasome [110]. Rsp5 is essential for yeastviability and has a role in a number of cellular processes such as endocytosis, RNAexport, and lipid biosynthesis [112–114]. Following heat shock, Rsp5 interactswith the Hsp40 chaperone Ydj1 to promote substrate ubiquitination [52]. Rsp5’srole in ubiquitinating proteins following heat shock is conserved as the homologNEDD4 is also required for heat shock induced ubiquitination in higher eukary-otes [52].1.5.4 Ltn1Nascent polypeptides on stalled ribosomes have been shown to be ubiquitinatedand targeted for proteasomal degradation by the E3 ligase Ltn1 (Figure 1.4) [115].15Ltn1 targets non-stop proteins (derived from non-stop mRNA lacking a terminationcodon) and proteins containing polylysine stretches for ubiquitination and subse-quent degradation in yeast, but was shown not to play a role in general cytoso-lic quality control when tested against the VHL quality control substrate [115].Stalled 80S ribosomes are dissociated by the ribosome recycling factors Hbs1-Pelota-ABCE1 into 40S small subunits and 60S nascent chain tRNA complexesthat facilitate the recognition of the nascent polypeptide by Ltn1 [116]. ExposedtRNA is recognized by Rqc2 (NEMF in mammals) prohibiting 40S reassociationand promoting Ltn1 recruitment [117]. Ltn1 associates with the 60S ribosomeand functions as part of a ribosome quality control complex (RQC) comprisingCdc48, the translation-associated element 2 (Tae2), and the protein ribosome qual-ity control 1 (Rqc1) [118, 119]. Ltn1 binds to ribosomal proteins in a way suchthat its RING domain is oriented towards the exit tunnel [120]. Following ubiq-uitination by Ltn1, and as a prerequisite for proteasomal degradation, the tRNA-linked polypeptide is dissociated from the 60S ribosome through the activity of theCdc48-Ufd1-Npl4 complex [121]. Recently, Ltn1 has also been shown to mediatethe degradation of translationally stalled ER proteins [122]. This function requirescytosolic exposure of the nascent polypeptide at the ribosome-Sec61 transloca-tion channel junction [117]. Targeting proteins during cotranslational translocationprevents complete translocation into the ER, thereby eliminating the need to re-translocate the protein back into the cytosol and bypassing the ERAD network.Ltn1’s structure, determined by single-particle electron microscopy, is similar tothe cullin subunit of the cullin-RING ubiquitin ligases, but has significant confor-mational variability that could be integral for its function [123]. The importance ofLtn1 is underscored by the results of an N-ethyl-N-nitrosourea mutagenesis screenthat identified homozygous lister mouse mutants that are viable, but display pro-gressive early onset neurodegeneration [124].1.6 AutophagyAutophagy, the process whereby cytoplasmic components are degraded by thelysosome, is important for recycling amino acids during nutrient starvation and forthe clearance of aggregated proteins and damaged organelles, such as mitochondria16and ribosomes. Three types of autophagy have been described, each categorized bythe mechanisms required to deliver substrates to the lysosome [125]. Cellular com-ponents destined for degradation via macroautophagy are encapsulated through theformation of double membraned autophagosomes that fuse with the lysosome (orvacuole in fungi) delivering their contents to be degraded by enzymes. In microau-tophagy, the lysosomal membrane is remodeled to capture cellular componentsbringing them directly into the lysosome in a fashion reminiscent of phagocytosis.Finally, chaperone-mediated autophagy requires the selective import of unfoldedproteins into the lysosome through a combination of chaperone mediated substratetargeting and a set of dedicated receptors and translocation machinery. While au-tophagy was originally viewed as a non-selective process, whereby the lysosomeindiscriminately engulfed portions of the cytosol, many studies now demonstratethat macroautophagy can selectively target protein aggregates and organelles forlysosomal degradation. Moreover, the ubiquitin proteasome system and autophagyare interconnected, as a compensatory increase in autophagy is observed whenproteasome activity is impaired or inhibited [126]. Under starvation conditions,ribosomes and proteasomes undergo selective lysosomal degradation in a processcalled ribophagy and proteaphagy, respectively [127–129]. The E3 ubiquitin ligaseLtn1 protects 60S ribosomal subunits from starvation-induced selective ribophagyin a process antagonised by the deubiquitinating enzyme Ubp3 [130]. Similarly, se-lective mitochondrial degradation, or mitophagy, is important for maintaining mi-tochondrial integrity and for limiting the production of potentially harmful reactiveoxygen species [131]. Parkin, an E3 ligase of the outer mitochondrial membrane,has been associated with mitophagy suggesting that some outer mitochondrialmembrane proteins require ubiquitination in order to promote selective macroau-tophagy [132]. In cases where damaged proteins presumably can no longer beprocessed by the proteasome, protein aggregates accumulate adjacent to the vac-uole, presumably to be cleared by autophagy [125]. Such aggregates colocalizewith Atg8, the homolog to the mammalian autophagosome LC3 protein, whichacts as a receptor for ubiquitin binding proteins. For instance, p62 and Nbr1 pro-mote the turnover of polyubiquitinated protein aggregates by selectively binding toK63 ubiquitin chains, which are recognized through a ubiquitin binding domain,while also binding to LC3 to shuttle the substrates to autophagosomes [133]. While17it is clear that a level of reciprocity exists between the autophagy and UPS path-ways, a greater appreciation of the protein quality control elements will be neededbefore we can truly understand how substrates are triaged between these two com-partments.1.7 Spatial Protein Quality Control: CytoQ, IPOD, andINQProtein aggregation has traditionally been viewed as a last resort when proteinquality control is exhausted. More recently however, the perception of spatial se-questration of misfolded proteins has changed, and it is now believed to representan early event in protein quality control and to occur even under physiologicalconditions. In S. cerevisiae, there are three spatially distinct protein quality con-trol compartments that sequester misfolded or aggregated proteins into inclusionswithin the cell. These are: the cytosolic quality control compartment (CytoQ), theimmobile protein deposit (IPOD), and the intranuclear quality control compart-ment (INQ). These compartments are not unique to yeast as similar cytoplasmicinclusions have been described in mammalian cells [134, 135].CytoQ inclusions (also referred to as stress foci or Q-bodies) are found through-out the cytosol and require Hsp42 for their formation following heat stress [136,137]. Hsp42, along with Hsp26 constitute the cytosolic members of the smallheat shock family of molecular chaperones and, like all small heat shock proteins,have a conserved alpha-crystallin C-terminal domain [138, 139]. Functional understress and non-stress conditions, Hsp42 binds to misfolded proteins to prevent pro-tein aggregation. In addition to being a monomer, Hsp42 can also form barrel likeoligomeric structures from hexameric rings of dimers if present at high concentra-tions. Hsp42 is exclusive to CytoQ and is used as a marker for this compartment.A single IPOD inclusion is found adjacent to the vacuole and formation is in-dependent of stress [135]. This deposit does not co-localize with proteasomes andcontains insoluble non-ubiquitinated proteins as well as amyloid proteins [135]. Todate, most attention has been paid to the INQ quality control compartment. Orig-inally thought to associate with the nucleus while remaining in the cytosol, thejuxtanuclear quality control compartment (JUNQ) has recently been discovered to18reside within the nucleus in close proximity to the nucleolus and has, as a con-sequence, been renamed the intranuclear quality control compartment (INQ) [77,135]. Cytosolic proteins require active transport through the nuclear pore complexto reach INQ and substrate targeting and aggregation is mediated by Sis1 in thecytosol and Btn2 within the nucleus [77, 140]. Sis1 alone however, is not suffi-cient to target proteins for nuclear import suggesting that other factors remain tobe discovered [140]. Sis1 levels are relatively high under both physiological andstress conditions, while Btn2 is barely detectable and must be rapidly induced uponheat shock. Even under stress conditions Btn2 is rapidly degraded and inhibitingits degradation stabilizes INQ deposits underscoring its importance in nuclear in-clusion formation [77]. Hsp104 is an AAA-ATPase that associates with aggregatesto assist with their disassembly [141]. Hsp104 is used as a general aggregationmarker and is conserved in fungi and plants but no metazoan homolog has yetbeen identified. While not essential for viability, Hsp104 is required for inducedthermotolerance in yeast [142]. Ubiquitination was once thought to be the sortingsignal dictating protein sorting to the INQ compartment [135]. INQ’s associationwith Hsp104 however, suggests instead that sequestration of misfolded proteins oc-curs prior to, or independently from, the decision to refold or degrade a misfoldedsubstrate.1.8 Stress ResponsesExposure to a range of intrinsic or extrinsic stressors can precipitate protein mis-folding overwhelming the proteostasis capacity of the cell. Depending on the na-ture of the stress, the cell can elicit a number of cellular responses to ensure survivaland restore proteostasis. Common to most of these is the induction of molecularchaperones and other factors required to mitigate the stress as well as a decreasein the transcription, translation, and splicing of all other factors not essential to thestress response [143]. While a number of pathways have been described, the mostwidely studied are the heat shock and general stress responses.191.8.1 Heat Shock and General Stress ResponseExposure to elevated temperatures results in a highly conserved physiological heatshock response, which is characterized by induced expression of genes includingmembers of the heat shock molecular chaperone family. While the heat shockresponse is cytoprotective, many of the genes induced are not required for surviv-ing the initial stress, but are instead necessary for surviving subsequent stresses,thereby forming acquired stress resistence [144]. Genes are induced through bind-ing of the heat shock transcription factor Hsf1 to heat shock elements in promoterregions [141]. Vertebrates and plants have four Hsf proteins, with the Hsf1 iso-form primarily responsible for the heat shock response [145]. In contrast, inverte-brates and yeast have a single Hsf1 protein. Low level Hsf1 activity is essentialfor yeast viability and is required for basal expression of Hsp70 and Hsp90 chap-erones [146]. In higher eukaryotes under non-stress conditions, Hsf1 is maintainedin an inactive monomeric form in the cytoplasm through an interaction with Hsp90proteins. Exposure to stress releases Hsf1 resulting in its trimerization, which isrequired for DNA binding and gene induction [141].A broad range of environmental stresses such as heat, nutrient starvation, os-motic shock, and oxidation precipitate a transcriptional response in eukaryotes.This general stress response, resulting in the induction of approximately 200 genes,is mediated by the zinc-finger transcription factors Msn2 and Msn4 which bindto stress response elements (STRE) in the promoter regions of target genes (Fig-ure 1.5) [147]. Originally, the heat shock response was considered to be a subsetof the general stress response, but a recent report suggests that, in yeast at least,the heat shock response is largely Hsf1 independent and, instead, the heat shocktranscriptional response is predominantly driven by Msn2/4 activity [146]. Msn2and Msn4 are partially redundant transcription factors that share 41% sequenceidentity at the amino acid level. Neither gene is essential in yeast and they are notconserved from yeast to metazoans [145]. While Msn4 expression is induced bystress, Msn2 is constitutively expressed and is thought to play the dominant rolein stress response as overexpression of MSN4 can only partially suppress the phe-notype of the msn2∆ mutant [148]. Msn2 contains an N-terminal transcriptionalactivating domain (TAD), a nuclear export sequence (NES), a nuclear localization20sequence (NLS), and a C-terminal zinc finger DNA binding domain (DBD) [149].Structurally, Msn2 is predicted to be intrinsically disordered with the exceptionof two structured regions in the TAD domain. The sequences of these structuredmotifs are highly conserved in yeast and mutations result in decreased Msn2 ac-tivity and nuclear localization [149]. Msn2 activity is thought to be regulated bytwo nutrient sensing pathways: protein kinase A (PKA) and target of rapamycin(TOR) [141]. Under non-stress conditions, cAMP dependent PKA phosphorylationnegatively regulates Msn2 by phosphorylating the nuclear localization sequence,thereby retaining Msn2 in the cytoplasm [150]. Nuclear exclusion in the absenceof stress is also thought to be mediated by an interaction between Msn2 and the 14-3-3 protein homolog Bmh2, which is enhanced by TOR activity [150]. A secondPKA consensus site on Msn2 regulates nuclear export, which requires the Msn5exportin receptor that controls the nuclear localization of many transcription fac-tors (Figure 1.5) [150, 151]. Msn2 is primarily found in the nucleus under certainconditions such as when TOR activity is inhibited, in msn5∆ cells, or when PKAlevels decrease [150–152]. Interestingly, Msn2/4 display oscillatory nucleocyto-plasmic shuttling under intermediate stress conditions that is regulated by PKAlevels in the case of Msn2, but not for Msn4 [153]. How this oscillatory shuttlingrelates to transcriptional activity remains unknown. Msn2/4 bind to a five base pair(CCCCT or AGGGG) consensus binding site resulting in a transcriptional responsethat is both transient and scales with the magnitude of the stress [147]. This is inpart the product of a linear relationship between induced gene expression and theconcentration of nuclear Msn2, which is produced by low Msn2 binding affinityand a limited number of Msn2 molecules relative to the number of STRE bindingsites in the genome [154–156]. The combination of environmental sensing path-ways regulating Msn2 localization and activity and the linear relationship betweenMsn2 concentration and target gene expression means that Msn2/4 can mediate acommensurate homeostatic response to a range of extrinsic stresses.1.9 DiseasesProtein homeostasis networks maintain proteome integrity and are essential forcell viability. Perturbations that disrupt the equilibrium of this system can lead21NucleusCytoplasmSTREMsn2/4Msn2/4P Bmh2TORPKAMsn5Figure 1.5: The general stress response. Under a range of stress conditionsthe transcription factors Msn2 and Msn4 bind to stress response elements(STREs) in the promoter regions of target genes. Nuclear localization andimport is regulated by the PKA and TOR pathways. Nuclear export is medi-ated by PKA activity and the exportin a class of diseases known as proteopathies, which range from lysosomal stor-age diseases to cystic fibrosis and neurodegenerative disorders [157]. Protein mis-folding, which can lead to protein aggregation, is characteristic of a number ofproteopathies. Moreover, it is thought that an age related decline in the cell’s ca-pacity to respond to the presence of misfolded proteins underlies the late onset ofneurodegenerative diseases such as Alzheimer’s and Parkinson’s [12]. The effectof missense mutations on protein stability is of particular interest in the contextof disease as missense mutations represent more than half of all mutations in theHuman Gene Mutation Database (HGMD) [158]. Sahni and colleagues tested ap-proximately 3000 human disease associated missense alleles and found about onethird of the mutations altered protein stability and resulted in an increased engage-ment with components of the protein homeostasis network [159].22There is currently great interest in the potential for developing therapeutics thattarget proteostatic imbalance and components of the ubiquitin proteasome system.One such example is Bortezomib, a proteasome inhibitor used to treat relapsedmultiple myeloma [160]. The selectivity of proteasome inhibition to kill tumorcells as opposed to normal healthy cells is thought to be attributed to tumor cellsbeing more sensitive to proteasome inhibition due to higher concentrations of ab-normal proteins [161]. Recently, selective proteasome inhibition by a compoundtargeting the kinetoplastid proteasome was shown to clear mice of the parasitesresponsible for leishmaniasis, sleeping sickness, and Chagas disease, which leadto 50,000 deaths annually and affect more than 20 million people globally [162].Therapies targeting molecular chaperones are also being developed for the treat-ment of diseases ranging from cancer to neurodegeneration. For instance, a recom-binant human HSP70 therapy was shown to reduce a number of disease associatedneurological symptoms in mouse models of lysosomal storage diseases [163]. Inaddition, the drug Lumacaftor, which acts as a chaperone, was recently approved bythe food and drug administration (FDA) to treat patients with the F508∆ mutationin CFTR [164]. Together, these examples highlight the exciting potential targetingprotein homeostasis networks have for drug development and clinical applications.1.10 Model Substrates Used to Study ProteostasisProtein quality control pathways have been identified and characterized using awide range of model substrates. These substrates are essential components of ge-netic screens that have been used to probe protein quality control and will con-tinue to be vital if we are to understand what aspects within misfolded proteinsare necessary for recognition by molecular chaperones and E3 ubiquitin ligases totarget them for degradation. Model substrates used in the study of ER, nuclear, andcytoplasmic protein quality control include: VHL, CPY*, Ura3, Ubc9, and GFPfusions.VHL is an E3 ligase that acts as a tumor suppressor with mutations leadingto a disease of the same name. VHL folding and stability is coupled to its as-sembly into a complex containing elongin B and C. An absence of the elonginpartners, or mutations that disrupt binding, results in VHL being degraded by the23proteasome [165]. Folding defective mutants of VHL were used to examine howdifferent molecular chaperones contribute to the triage decision of whether to foldor degrade misfolded proteins. McClellan and colleagues demonstrated that somechaperones, such as the TRiC chaperonin, were only required for folding, whereasHsp90 was necessary for VHL degradation, and Hsp70 had a role in both foldingand degradation [50].The vacuolar carboxypeptidase (CPY) encoded by the gene PRC1, has beeninstrumental in the study of ER and cytoplasmic protein quality control. Mutantprc1-1 (or CPY*) is retained in the ER and targeted for degradation while thewild type protein is located in the vacuole. Genetic screens looking for mutantsthat are defective in CPY* degradation isolated key factors of the ERAD pathway,including the E3 ligase Hrd1 and E2 conjuating enzymes Ubc6 and Ubc7 [166].∆ssCPY*, a truncated version of the mutant CPY* protein that has had its signalsequence removed restricting its localization to the cytosol, has been used in thediscovery of cytosolic protein quality control pathways [53, 74, 103]. Primarily,∆ssCPY* substrates have been used to delineate the role of Ubr1 and San1 in thedegradation of misfolded cytoplasmic proteins [74, 103].Fused to a model substrate or short peptide, Ura3 is used as a reporter proteinin genetic screens to identify protein quality control components. It has been usedto screen for mutations in the Type I and II substrate binding sites of Ubr1 andmore recently used to generate a new panel of model substrates through fusionwith a degron library [98]. Screening this panel of substrates revealed a globalrequirement for the molecular chaperones Ssa1, 2 and Ydj1 as well as a novel rolefor Ltn1 in a mechanism distinct from ribosomal quality control [167]. The E3ligase Doa10 was also identified as the primary ligase required for these substrates.Ubc9 is essential for yeast viability and is required for cyclin degradation [168].A temperature sensitive allele of Ubc9 was identified and found to undergo con-ditional proteasomal degradation [169]. More recently, Ubc9 has been used as agreen fluorescent protein (GFP) fusion protein in the study of the INQ and CytoQpathways [135, 136]. As is the case with Ubc9, the majority of the work presentedin this thesis relies upon fusing novel model substrates to GFP to study cytosolicprotein quality control. GFP is a 27 kDa protein originally isolated from the jelly-fish Aequorea victoria that emits green light at a wavelength of 509 nm and can be24used to tag proteins at their N- or C-termini [170]. It forms a cylindrical beta barrelstructure consisting of eleven beta strands with a central alpha helix that is cova-lently bonded to the chromophore. The GFP chromophore is formed through thecyclisation and oxidation of three amino acids (Ser65, Tyr66, and Gly67), whichoccurs within two to four hours of synthesis [171, 172]. The S65T GFP mutantis more amenable to biological applications as it has a faster maturation time, ismore resistant to photobleaching, and its single excitation peak at 490 nm meansthat it can be used with fluorescein isothiocyanate (FITC) filter sets [173]. Theadvent of whole proteome GFP tagging collections has meant that it was possibleto perform high throughput studies using flow cytometry to identify factors thatinfluence protein stability or abundance and shifted the focus of flow cytometryscreens away from single substrates or a small collection of deletion strains. Twomethodologies highlight these advances: global protein stability profiling and tan-dem fluorescent protein timers. Global protein stability (GPS) analysis is a methodfor analysing protein turnover at the proteome level in mammalian cells [174]. Twofluorescent proteins, an internal control DsRed and an EGFP fusion with a proteinof interest, are translated from a single mRNA transcript containing an internalribosome entry site (IRES). The EGFP/DsRed ratio of a cell represents the sta-bility of the protein of interest as both fluorescent proteins are produced from thesame mRNA. Changes to the stability of the GFP fusion protein will therefore bereflected by a change in the EGFP/DsRed ratio. EGFP/DsRed constructs were cre-ated for the entire human ORFeome containing approximately 8000 human proteinencoding open reading frames (ORFs) and pooled transformed cells are fluores-cence activated cell sorted (FACS) into bins based on the GFP/DsRed ratio andthen microarray analysis is performed to identify the tagged ORF. This approachwas used to successfully identify substrates of the SCF ubiquitin ligase in mam-malian cells [175]. The tandem fluorescent protein timer method uses a similar dualfluorescent protein approach, however, in this case, the two proteins are fused andmature with different kinetics. The fluorescence ratio of the two proteins providesa measure of protein age and has been used to identify regulators of the N-end rulepathway in yeast [176].251.11 Research ObjectiveProtein homeostasis encompasses the network of pathways that influence the fateof proteins from synthesis to degradation for the purpose of maintaining proteomeintegrity, thereby promoting viability at both the cellular and organism levels. Mis-folded proteins challenge the cell’s capacity to maintain the proteostatic balanceand may divert resources away from essential cellular processes or result in the pro-duction of potentially toxic protein aggregates. Consequently, cells have adoptednumerous protein quality control pathways to prevent aberrant protein aggregation,promote protein folding, and to target terminally misfolded proteins for degrada-tion. Previous work from the Mayor lab identified a panel of temperature sensitivealleles of essential genes encoding for cytosolic proteins in S. cerevisiae that aredegraded in a proteasome-dependent manner once shifted to an elevated tempera-ture of 37◦C. The protein quality control pathways responsible for the degradationof a number of these alleles, which contain potentially destabilizing missense mu-tations, are unknown. Recently, there has been renewed interest in the role thatmissense mutations play in genetic disease as they can induce protein instabilitywhich leads to premature and/or increased rates of protein degradation and, asa consequence, loss of function phenotypes. My hypothesis is that a number ofquality control pathways, both known and as yet undiscovered, are present withinthe cytoplasm to aid the cell in the recognition, refolding and/or degradation ofproteins destabilized by missense mutations. This thesis is focused on identifyingand characterizing cytosolic protein quality control factors that induce proteasome-mediated degradation of thermally unstable model substrates.1.11.1 Specific Aims1. Develop a flow cytometry based assay to monitor the stability of a GFP-tagged substrate.2. Use genetic screens to identify protein quality control factors that promoteproteasomal degradation of a model substrate.3. Perform in depth characterization of the factors identified in Aim2.This work was performed using the model organism Saccharomyces cerevisiaewith a combination of cell biology, biochemical, and genetic approaches.26Chapter 2Prefoldin Promotes ProteasomalDegradation of Cytosolic Proteinswith Missense Mutations byMaintaining Substrate Solubility2.1 IntroductionThe protein homeostasis network encompasses systems required by the cell to gen-erate and maintain the correct levels, conformational state, and distribution of itsproteome [1]. Misfolded proteins threaten this balance by triggering loss of func-tion phenotypes, diverting resources away from producing essential protein prod-ucts, or precipitating the production of potentially toxic protein aggregates [4]. Thepresence of protein aggregates is characteristic of a number of neurodegenerativediseases such as Parkinson’s and Alzheimer’s disease, and a decrease in the proteinhomeostasis capacity of the cell is thought to underlie the later stages of cellularageing [11, 12, 177]. It is, therefore, not surprising that the cell has evolved a num-ber of protein quality control pathways aimed at preventing protein aggregation,promoting protein folding, and targeting terminally misfolded proteins for degra-dation [178–180]. These pathways triage misfolded proteins, which will face three27main possible fates: to be refolded back to their functional native conformation;to be targeted for degradation; or to be sequestered into spatially distinct qualitycontrol compartments.Proteins are selectively targeted to the eukaryotic ubiquitin proteasome sys-tem by the covalent attachment of polyubiquitin chains catalyzed by a cascadeof E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin ligase)enzymes [62, 181]. Substrate recruitment and specificity is determined by the E3ubiquitin ligases, either alone or in concert with an E2 conjugating enzyme or othersubstrate adaptors. A number of subcellular compartment-specific quality controlpathways have been identified, each associated with a particular E3 ligase or set ofligases [63, 70, 178]. In yeast, the San1 ligase is responsible for ubiquitinating nu-clear misfolded proteins [70]. Experiments have shown that San1 binds misfoldedproteins through recognition sequences located in disordered regions of its N- andC-terminal domains [72]. In contrast to the nucleus, a number of ligases have beenidentified to target cytosolic proteins for degradation in yeast. While initially char-acterized for its role as the recognin of the N-end rule pathway, Ubr1 has also beenshown to target misfolded cytoplasmic proteins for degradation [74, 76, 102–104].It does so either alone, or in conjunction with other E3 ligases such as Ubr2 in thecase of newly synthesized kinases, or with the nuclear San1 where both are requiredfor the complete degradation of the engineered ∆ssCPY*-GFP substrate [74, 104].Hul5, a nuclear protein that relocalizes to the cytoplasm upon heat shock, and Rsp5have been identified as the two ligases responsible for the marked increase in cy-toplasmic protein ubiquitination following heat shock stress [52, 182]. Finally, theribosome associated ligase Ltn1 targets non-stop polypeptides stalled during trans-lation for degradation [115].Recently, the importance of spatial organization in protein quality control hasgained recognition. Under normal physiological conditions, misfolded proteins canbe concentrated into dynamic Q-bodies where they can be refolded by chaperonesor degraded [136]. However, if the protein quality control systems become over-whelmed, misfolded proteins can be sequestered into discrete cellular inclusions.The INQ compartment acts to concentrate detergent soluble misfolded proteinscapable of being refolded, or degraded, and contains 26S proteasomes and chap-erones such as the disaggregase Hsp104 [77, 135]. The IPOD by contrast contains28insoluble non-ubiquitinated proteins; does not co-localize with proteasomes; andis the site of amyloidogenic protein sequestration, perhaps to prevent their toxicinteraction with quality control machinery [135]. The IPOD is also postulated tobe the site of yeast prion maturation [183].In this study we performed a screen to identify factors involved in degradativeprotein quality control of a model substrate that misfolds as the result of destabiliz-ing missense mutations. We show that our model substrate is thermally unstable,undergoes proteasome mediated degradation, and forms Q-body like inclusions.We then identified and characterized the prefoldin chaperone subunit Gim3 as afactor important for maintaining our substrate protein’s solubility, and thereby fa-cilitating its degradation.2.2 Materials and Methods2.2.1 Yeast Strains, Plasmids, and MediaAll yeast deletion strains used in this study are derived from BY4741 or BY4742wild type (WT) strains and are listed in Table 2.1. The temperature sensitive alleleswere generously provided by Dr. P. Hieter. The Cup1-Deg1-GFP plasmid wasa gift from T. Sommer [184]. The Hsp104-mCherry and Hsp42-mCherry strainswere constructed by homologous recombination of a PCR product amplified froma plasmid containing a yeast codon optimized mCherry ORF (BPM 866). Guk1and Guk1-7 GFP-tagged fusion plasmids (BPM 453, BPM 458) were constructedby inserting ORFs amplified from genomic DNA, with primers containing BamHIand XbaI restriction enzyme recognition sequences, into PGPD-GFP(S65T) (BPM241). Ugp1-3 (BPM 457), Pro3-1 (BPM 507), and Gus1-3 (BPM 500) GFP taggedplasmids were produced in the same fashion using: BamHI and NotI; BamHI andNotI; and NotI and XbaI, respectively. The histidine tagged fusions were producedby cloning PCR amplified inserts into PGPD (BPM 171) using BamHI and SalI(BPM 659, BPM 717). All plasmids used in this study are listed in Table 2.2. Cellswere grown in synthetic drop out media following standard procedures.29Table 2.1: Yeast strains used in Chapter 2Strain ID Alias Genotype SourceYTM 408 BY4741 ura3∆0, leu2∆0, his3∆1, met15∆0OpenBiosystemsCollectionYTM 703 ubr1∆san1∆ his3∆1, leu2∆0, met15∆0, ura3∆0,san1∆::His3MX6, ubr1∆::KanMXKhosrow-Khavaret al. 2012YTM 736 Guk1-7-13mychis3∆1, leu2∆0, LYS2,met15∆0, ura3∆0, guk1-7-13myc::KanMX6::URA3, CAN1Khosrow-Khavaret al. 2012YTM 749 Gus1-3ura3∆0, leu2∆0, his3∆1, LYS,MET, can1∆::Leu2-MFA1pr::His3,Gus1-3::UraP. HieterYTM 755 Pro3-1ura3∆0, leu2∆0, his3∆1, LYS,MET, can1∆::Leu2-MFA1pr::His3,Pro3-1::UraP. HieterYTM 758 Guk1-7ura3∆0, leu2∆0, his3∆1, LYS,MET, can1∆::Leu2-MFA1pr::His3,Guk1-7::UraP. HieterYTM 766 Ugp1-3ura3∆0, leu2∆0, his3∆1, LYS,MET, can1∆::Leu2-MFA1pr::His3,Ugp1-3::UraP. HieterYTM 938 san1∆ his3∆1, leu2∆0, met15∆0, ura3∆0,san1∆::KanMXOpenBiosystemsCollectionYTM 981 ubr1∆ his3∆1, leu2∆0, met15∆0, ura3∆0,ubr1∆::KanMXOpenBiosystemsCollectionYTM 1183 tda2∆ his3∆1, leu2∆0, met15∆0, ura3∆0,tda2∆::KanMXOpenBiosystemsCollectionYTM 1184 yak1∆ his3∆1, leu2∆0, met15∆0, ura3∆0,yak1∆::KanMXOpenBiosystemsCollectionContinued on next page30Strain ID Alias Genotype SourceYTM 1185 rim15∆ his3∆1, leu2∆0, met15∆0, ura3∆0,rim15∆::KanMXOpenBiosystemsCollectionYTM 1186 gim3∆ his3∆1, leu2∆0, met15∆0, ura3∆0,gim3∆::KanMXOpenBiosystemsCollectionYTM 1187 YOR364W∆ his3∆1, leu2∆0, met15∆0, ura3∆0,YOR364W∆::KanMXOpenBiosystemsCollectionYTM 1290 vhr1∆ his3∆1, leu2∆0, met15∆0, ura3∆0,vhr1∆::KanMXOpenBiosystemsCollectionYTM 1293 sli15∆ his3∆1, leu2∆0, met15∆0, ura3∆0,sli15∆::KanMXOpenBiosystemsCollectionYTM 1294 fau1∆ his3∆1, leu2∆0, met15∆0, ura3∆0,fau1∆::KanMXOpenBiosystemsCollectionYTM 1301 gim5∆ his3∆1, leu2∆0, met15∆0, ura3∆0,gim5∆::KanMXOpenBiosystemsCollectionYTM 1302 gim6∆ his3∆1, leu2∆0, met15∆0, ura3∆0,gim6∆::KanMXOpenBiosystemsCollectionYTM 1304 gim1∆ his3∆1, leu2∆0, met15∆0, ura3∆0,gim1∆::KanMXOpenBiosystemsCollectionYTM 1305 gim4∆ his3∆1, leu2∆0, met15∆0, ura3∆0,gim4∆::KanMXOpenBiosystemsCollectionYTM 1306 gim2∆ his3∆1, leu2∆0, met15∆0, ura3∆0,gim2∆::KanMXOpenBiosystemsCollectionYTM 1356 rpt6-20 his3∆1, leu2∆0, met15∆0, ura3∆0,RPT6::rpt6-20::KanMXBoone tscollectionContinued on next page31Strain ID Alias Genotype SourceYTM 1357 pep4∆prb1∆his3∆1, leu2∆0, met15∆0,ura3∆0, lys2∆0, PRB1::KanMX6,PEP4::His3MX6Fang et al.2015YTM 1489 Gim3-TAP his3∆1, leu2∆0, met15∆0, ura3∆0,gim3::TAP::His3MXOpenBiosystemsCollectionYTM 1677 tcp1-1 his3∆1, leu2∆0, met15∆0, ura3∆0,TCP1::tcp1-1-KanMAX6 P. HieterYTM 1678 tcp4-1 his3∆1, leu2∆0, met15∆0, ura3∆0,CCT4::cct4-1-KanMAX6 P. HieterYTM 1692 bra7∆ his3∆1, leu2∆0, met15∆0, ura3∆0,bra7∆::KanMXOpenBiosystemsCollectionYTM 1693 asp1∆ his3∆1, leu2∆0, met15∆0, ura3∆0,asp1∆::KanMXOpenBiosystemsCollectionYTM 1694 mal11∆ his3∆1, leu2∆0, met15∆0, ura3∆0,mal11∆::KanMXOpenBiosystemsCollectionYTM 1695 mup3∆ his3∆1, leu2∆0, met15∆0, ura3∆0,mup3∆::KanMXOpenBiosystemsCollectionYTM 1696 pol4∆ his3∆1, leu2∆0, met15∆0, ura3∆0,pol4∆::KanMXOpenBiosystemsCollectionYTM 1697 pph22∆ his3∆1, leu2∆0, met15∆0, ura3∆0,pph22∆::KanMXOpenBiosystemsCollectionYTM 1855 ubr1∆gim3∆ his3∆1, leu2∆0, met15∆0, ura3∆0,ubr1∆::KanMX, gim3∆::His3MX6 This thesisYTM 1901 Hsp104-mCherry his3∆1, leu2∆0, met15∆0, ura3∆0,Hsp104-mCherry::His3MX6 This thesisYTM 1902 Guk1-7-13mycgim3∆, his3∆1, leu2∆0, LYS, MET15,Guk1-7-13myc::KanMX6::Ura3,CAN1, gim3∆::His3MX6This thesisYTM 1919 Hsp42-mCherry his3∆1, leu2∆0, lys2∆0, ura3∆0,Hsp42-mCherry::KanMX6 This thesis32Table 2.2: Plasmids used in Chapter 2PlasmidID NameAuxo-trophicMarkerPlasmidType SourceBPM 42 pRS316 Ura CEN/ARS RJD CollectionBPM 45 pRS313 His CEN/ARS RJD CollectionBPM 171 PGPD His CEN/ARS F. Khosrow-KhavarBPM 241 PGPD-GFP(S65T) His CEN/ARS F. Khosrow-KhavarBPM 368 PUbr1-Ubr1 Leu CEN/ARS R. HamptonBPM 369 PUbr1-Ubr1(C1220S) Leu CEN/ARS R. HamptonBPM 453 PGPD-Guk1-GFP His CEN/ARS This thesisBPM 457 PGPD-Ugp1-3-GFP His CEN/ARS This thesisBPM 458 PGPD-Guk1-7-GFP His CEN/ARS This thesisBPM 500 PGPD-Gus1-3-GFP His CEN/ARS This thesisBPM 507 PGPD-Pro3-1-GFP His CEN/ARS This thesisBPM 509 PGPD-Guk1(E127K)-GFP His CEN/ARS This thesisBPM 510 PGPD-Guk1(T95A)-GFP His CEN/ARS This thesisBPM 511 PGPD-Guk1(F59H)-GFP His CEN/ARS This thesisBPM 513 PGPD-Guk1(A84T)-GFP His CEN/ARS This thesisBPM 551 PGPD-Gim3 Ura CEN/ARS This thesisBPM 572 PUbr1-Ubr1 Ura CEN/ARS This thesisBPM 659 PGPD-Guk1::His6 His CEN/ARS This thesisBPM 708 PCup1-Deg1-cNLS-GFP Ura CEN/ARS T. SommerBPM 717 PGPD-Guk1-7::His6 His CEN/ARS This thesisBPM 779 PGPD-GFP Ura CEN/ARS This thesisBPM 780 PGPD-Guk1-GFP Ura CEN/ARS This thesisBPM 781 PGPD-Guk1-7-GFP Ura CEN/ARS This thesisBPM 866 pFA6a-mCherry-KanMX6 KanMX CEN/ARS This thesisBPM 894 PGuk1-Guk1-GFP His CEN/ARS This thesisBPM 895 PGuk1-Guk1-7-GFP His CEN/ARS This thesis332.2.2 Stability Effect of Guk1-7 MutationsThe predicted thermodynamic stability changes of mutations in Guk1-7 were com-puted using FoldX (version 3.0). The protein structure of Guk1 was downloadedfrom the Protein Data Bank (PDB accession 1EX7) and was optimized using therepair function of FoldX. Structures corresponding to each of the single point mu-tations and all four point mutants combined were generated. The predicted effect ofmutations on protein structural stability was expressed as the predicted free energychange (∆∆G) and was obtained by subtracting the energy values of the mutantstructures from that of the wild type.2.2.3 Cellular Thermal Shift Assay (CETSA)A 50 mL yeast culture grown at 25◦C was collected at log phase and harvestedby centrifugation. Cells were then lysed with glass beads in 200 µL of nativelysis buffer (20 mM HEPES, pH 7.5, 0.5% NP-40, 200 mM NaCl, 1X proteaseinhibitor mix (Roche), 1 mM 1,10 phenanthroline, 1 mM EDTA). The solublefraction was collected by centrifugation (16,000 g, 10 min, 4◦C) and protein con-centration was determined by the DC Protein Assay (BioRad). Samples were nor-malized to 2 µg/µL and 50 µL aliquots were distributed into PCR strip tubes andrun on a PCRmachine with the following program: 25◦C, 3:00; Gradient 30–50◦C,10:00; 25◦C, 1:00. The soluble fraction was once again collected by centrifugation(16,000 g, 10 min, 4◦C). Equal volumes were resolved by SDS-PAGE. Membraneswere immunoblotted with mouse anti-HIS6 (Ablab, 1:2,500) and secondary anti-bodies (Mandel Scientific, 1:10,000) and then quantified using an Odyssey InfraredImaging System.2.2.4 Solubility AssayYeast cells were grown to log phase at 25◦C and then incubated for 20 min at ei-ther 25◦C or 37◦C. Cells were lysed with glass beads in native lysis buffer (20mM HEPES, pH 7.5, 0.5% NP-40, 200 mM NaCl, 1X protease inhibitor mix, 1mM 1,10 phenanthroline, 1 mM EDTA) and then precleared by centrifugation at2,000 g for 5 min at 4◦C. Sample protein concentrations were measured by the DCProtein Assay (BioRad) and normalized. Samples were further fractionated into34soluble and pellet fractions by centrifugation at 16,000 g for 10 min at 4◦C. Thepellet fractions were then washed twice with lysis buffer. Equal volumes of to-tal cell lysate, soluble, and pellet fractions were resolved by SDS-PAGE. Sampleswere analyzed by mouse anti-GFP (Roche, 1:2,500) and rabbit anti-Pgk1 antibod-ies (Acris Antibodies, 1:10,000) as a loading control.2.2.5 MicroscopyCells were grown in synthetic dropout media lacking histidine to log phase (OD600= 0.8–1.0) at 25◦C and then collected at the indicated time points following incu-bation at 25◦C or 37◦C with our without 100 µg/mL cycloheximide (CHX), asnoted. Samples were fixed in 3.7% formaldehyde for 15 minutes at room tempera-ture and then rinsed in 0.1 M potassium phosphate containing 1 M sorbitol beforebeing permeabilized with 0.1% Triton X-100 for ten minutes. Nuclei staining wasperformed by incubating permeabilized cells in Hoechst 33342 (25 µg/mL) for 10minutes before mounting cells on slides in mounting media (2% N-Propylgallate,80% glycerol, 0.02% sodium azide in 1X PBS). Cells were imaged with a ZeissAxio observer inverted microscope equipped with a 63x oil-immersion objectiveand a digital camera. Images were analyzed with Zeiss Axiovision software.2.2.6 Degradation AssayCells were grown to log phase in synthetic drop out media at 25◦C and cyclohex-imide was added to a final concentration of 100 µg/mL. Cells were then incubatedat either 25◦C or 37◦C, and at the indicated time points cells were collected by cen-trifugation. The cells were then resuspended in modified Laemmli buffer (50 mMTris-HCl, pH 6.8, 2% SDS, 10% glycerol), and lysed with glass beads. Protein con-centration was assessed by the DC Protein Assay (BioRad). Equal amounts of pro-tein were resolved by SDS-PAGE following the addition of 10X 2-mercaptoethanol(20%) and dye to each sample. Immunoblots were performed with a mouse anti-GFP primary antibody (Millipore, 1:2,500) and a rabbit anti-Pgk1 (1:10,000, AcrisAntibodies) as a loading control. Infrared secondary antibodies were used (MandelScientific, 1:10,000) and membranes were scanned and analyzed with an OdysseyInfrared imaging system (LI-COR).352.2.7 Flow CytometryYeast cells were grown in synthetic drop out media to log phase before the additionof 100 µg/mL cycloheximide and incubated at 25◦C or 37◦C as indicated. Sampleswere run on a BD FACSCalibur instrument (BD Biosciences) with a 488 laser andGPF was detected with a 530/30 filter. 50,000 events were collected. Analysis wasperformed with FlowJo (FlowJo Data Analysis Software, LLC). For multi-hourCHX chase experiments median GFP fluorescence values were normalized to thatof the first time point. FACS sorting was performed with a BD Influx instrumentby the UBC Flow facility.2.2.8 GFP PulldownGim3-TAP yeast cells transformed with a control empty vector (BPM 42), PGPD-GFP (BPM 779), PGPD-Guk1-GFP (BPM 780), or PGPD-Guk1-7-GFP (BPM781) were grown to log phase and then lysed with glass beads and native lysisbuffer (20 mM HEPES, pH 7.5, 0.5% NP-40, 200 mM NaCl, 1X protease inhibitormix, 1 mM 1,10 phenanthroline, 1 mM EDTA, 10 mM iodoacetamide). To pull-down GFP-tagged proteins, lysates were incubated for 2 hours at 4◦C with 20 µLGFP-Trap coupled agarose beads (Chromotek). Beads were washed three times inlysis buffer before samples were eluted with 3X SDS sample buffer. Nitrocellulosemembranes were probed with mouse anti-GFP (Roche, 1:2,500), rabbit anti-Pgk1(Acris Antibodies, 1:10,000), rabbit anti-TAP (Fisher, 1:2,500), and mouse anti-ubiquitin (Millipore, 1:2,500) primary antibodies.2.2.9 Proteasome FunctionYeast cultures were grown to saturation in synthetic drop out media overnight at30◦C and then diluted to OD600 = 0.2 and left to grow for 3 hours at 30◦C. 100 µMcopper sulphate was added to the culture and incubated at 30◦C for 4 hours. Aninitial sample was removed and then cycloheximide was added to the culture toa final concentration of 100 µg/mL. Samples were collected at the indicated timepoints. Cells were lysed with glass beads and lysis buffer (1% Tx-100, 0.1% SDS,150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.5, 1 mM PMSF, 1X proteaseinhibitor mix). Protein concentrations were assessed using the DC Protein Assay36(BioRad) and equal amounts were resolved by SDS-PAGE.2.2.10 Statistical AnalysisUnpaired two tailed Student’s t-tests were used to assess significance of differencesbetween wild type and gim3∆ or ubr1∆ strains. One-way ANOVA with post-hocTukey HSD (honest significant difference) was used to assess significance of dif-ferences between multiple deletion strains.2.3 Results2.3.1 Guk1-7 is Thermally UnstableOur lab previously identified a panel of temperature sensitive alleles of essentialgenes encoding for cytosolic proteins in Saccharomyces cerevisiae [101]. A largefraction of mutant proteins underwent proteasome-mediated degradation when in-cubated at the restrictive temperature of 37◦C, whereas the wild type proteins werestable. While approximately one third of the unstable alleles were found to besubstrates of the E3 ubiquitin ligase Ubr1 [101], the protein quality control path-ways responsible for the proteasomal degradation of the remaining mutant pro-teins are unknown. To screen for other proteins involved in proteasome mediateddegradation of thermosensitive mutant proteins, we sought to establish an assaybased on fluorescence intensity to facilitate the quantification of a model qual-ity control substrate fused to GFP. For this study, we selected the Guk1-7 allelethat contains four missense mutations generated by random PCR-based mutagen-esis [101, 185, 186]. Guk1 is a member of the nucleoside monophosphate kinase(NMP) family and converts GMP to GDP [187]. Similar to other members of theNMP family, Guk1’s structure contains a core, a lid, and a dynamic NMP-bindingdomain [188]. Mutants of Guk1 are defective in mannose chain elongation, havehigher cell wall porosity, and are hypersensitive to larger molecular weight an-tibiotics [189]. We first predicted the structural stability effects of the missensemutations found in Guk1-7 using FoldX (Figure 2.1A) [190]. The predicted freeenergy changes (∆∆G) between the single point mutants and the wild type proteinwere modest, whereas the combined effect of all the mutations found in Guk1-737was much larger (∼7 kcal/mol). While this value is higher than that predicted formissense mutations in transmembrane domains of disease-associated proteins suchas cystic fibrosis transmembrane conductance regulator (CFTR) and rhodopsin(1.5 and 1.9 kcal/mol, respectively), it is in line with those predicted for muta-tions in phenylalanine hydroxylase (PAH) associated with mild or severe forms ofphenylketonuria (5.7 and 14.2 kcal/mol, respectively) [191, 192]. We then com-pared the thermodynamic stability of ectopically expressed wild type Guk1 withGuk1-7 in cellular lysates by a cellular thermal shift assay (CETSA) [193]. Inagreement with its predicted lower stability, Guk1-7 was less stable than the wildtype Guk1 at incubation temperatures above 38◦C (Figure 2.1B). We further exam-ined the solubility of Guk1 and Guk1-7 proteins in cells incubated at the normalgrowth temperature of 25◦C and following a short 20 minute incubation at 37◦C.Guk1 was found predominantly in the soluble form while Guk1-7 was enriched inthe NP-40 insoluble fraction at 25◦C and 37◦C (Figure 2.1C). Together these datasuggest that Guk1-7 is much less stable than the wild type protein and misfoldsforming NP-40 insoluble aggregates.2.3.2 Fluorescence-Based Assay to Assess Protein StabilityTo determine whether the ectopically expressed mutant protein was also degradedwhen fused to GFP, we first examined fluorescence levels by microscopy. Guk1-7-GFP fluorescence was on average 58% lower than that of Guk1-GFP at 25◦C(n = 101, 108), and was nearly undetectable with an average 87% loss of fluores-cence following a two hour incubation at 37◦C in the presence of the translationinhibitor cycloheximide (n = 168) (Figure 2.2A, Figure 2.12A). By contrast, thefluorescence of the wild type Guk1-GFP only slightly decreased by 29% between37◦C and 25◦C (n = 120). To verify that the loss of fluorescence was due to pro-teolysis and not misfolding of GFP, we examined levels of Guk1 and Guk1-7 byWestern blot in a cycloheximide chase assay. While Guk1-GFP levels remainedrelatively unchanged, the level of Guk1-7-GFP decreased by 30% after a four hourincubation at 25◦C, and decreased by 70% after the same period at 37◦C (Fig-ure 2.2B). We then verified whether adding a GFP tag alters Guk1 or Guk1-7 sol-ubility. Three times as much Guk1-7 as Guk1 was found in the NP-40 insoluble38pellet fraction at 25◦C, and this rose to nine times more upon the short incubationat 37◦C (Figure 2.2C). Although Guk1-7-GFP is less insoluble than Guk1-7-His6,presumably due to stabilization conferred by the GFP moiety, the GFP tagged mu-tant was both less soluble and more degraded than the wild type protein, and couldtherefore be employed as a model substrate.39!"!"#$"%&'(%")*+,-."%/"0'&1")23)404'4&567 68 97 98 87787:77:87;77 <(=:<(=:>?! @ .;8+,6?+,;8+,6?+,<(=:<(=:>?#<)/A&01")*52BB)23)35(2%"B/"0/"-C8DEF)6GH8)=/&5I#25)*;:J-KH9!F)7G98)=/&5I#25)*L8J-!D8KF):G7D)=/&5I#25)*L8J-M:;?NF):GOH)=/&5I#25)*L8J-C8DEP)KH9!P)!D8KP)M:;?N)*<(=:>?-F)?G:)=/&5I#25)*O:J-C8DEKH9!M:;?N!D8K<(=:>?<(=:<(=:>?!"#$G)*+,- 67 6:G; 66GH 6?GO 9;G8 9OG8 9HGH 87 Q%)*=R&-6?6?Q%)*=R&-6?6?6?6?Figure 2.1: Guk1-7 is thermally unstable. (A) Ribbon structure of Guk1(PDB 1EX7). Positions of the four missense mutations and predicted ∆∆Gvalues are indicated. Loss of fluorescence measured by flow cytometry after atwo hour incubation at 37◦C with cycloheximide is indicated in brackets. (B)Cellular thermal shift assay of Guk1 and Guk1-7 fused to a six histidine tag inlysates derived from cells grown at 25◦C. One representative anti-His WesternBlot is shown. The graph represents the means and standard deviations ofGuk1 levels from three independent experiments. (C) Guk1 and Guk1-7 fusedto a six histidine tag were expressed in cells grown at 25◦C or shifted to 37◦Cfor 20 min. Total cell lysate (T), soluble (S), and pellet fractions (P) wereimmunoblotted with anti-His antibodies.40!"!"#$%&!'()* +,)*- ' . - ' ./01234 5 !'()*+,)*'()*+,)*%1#$%1#$6,+7+898-7:;<7,898$7,;$$7=898'7';<$7.898,7.;!>??>@A28B#CDE(-(-(-(-%&! F0>G/3@CH*'()*+,)*'()*+,)*%1#$6%&!%1#$6,6%&!A>2">IJ?K8@LM>8N8*FO'()* +,)*- ' . - ' .%1#$6,6%&!8N8*FO %1#$6%&!8N8*FOA28B#CDE(-(-!>2G>P@D">80Q8JPJ@JD?%1#$6%&!8'()*%1#$6%&!8+,)*%1#$6,6%&!8'()*%1#$6,6%&!8+,)*#4JR>8B/0123E- ' .$--,((-'(-Figure 2.2: Misfolded Guk1-7 is degraded at the non-permissive temperature.41Figure 2.2: (Previous page) Misfolded Guk1-7 is degraded at the non-permissive temperature. (A) Wild type cells expressing ectopic Guk1-GFPor Guk1-7-GFP were grown at 25◦C and then incubated in the presence of thetranslation inhibitor cycloheximide (CHX) at 25◦C or 37◦C for 2 hours priorto fixation and imaging. Scale bar represents 5 µm. (B) Cycloheximide chaseassay. Wild type cells expressing ectopic Guk1-GFP or Guk1-7-GFP wereincubated with CHX for 4 hours at 25◦C or 37◦C and samples were collectedat the indicated time points. Guk1-GFP and Guk1-7-GFP was immunoblottedwith anti-GFP antibodies and a representative blot is shown. GFP levels werenormalized to Pgk1 levels and shown in the graph below with results repre-senting the means and standard deviations of three independent experiments.(C) Guk1-GFP and Guk1-7-GFP were ectopically expressed in wild type cellsgrown at 25◦C or shifted to 37◦C for 20 min. Total cell lysate (T), soluble (S),and pellet fractions (P) were immunoblotted with anti-GFP antibodies. Theratio of the pellet fraction to total cell lysate is noted and represents the meanand standard deviation of three independent experiments.In order to use Guk1-7-GFP as a model substrate to screen for factors impor-tant in maintaining cytosolic protein homeostasis, we established a flow cytometryassay to monitor protein stability. Cultures were incubated at 25◦C or 37◦C inthe presence of cycloheximide for two hours and then the GFP fluorescence inten-sity from single cells was measured by flow cytometry. The relative difference inmedian intensity values between 37◦C and 25◦C was used as a measure of pro-tein stability. In a wild type strain at 25◦C Guk1-7-GFP fluorescence intensity islower than that of the wild type allele, suggesting that the model substrate is in-herently unstable even at lower temperatures. After shifting the cells to 37◦C inthe presence of CHX for two hours, GFP intensity levels remained nearly constantfor Guk1-GFP (5% loss) but decreased for Guk1-7-GFP (60% loss; Figure 2.3A).These data are consistent with our previous fluorescence microscopy and CHX-chase observations (Figure 2.2A, 2.2B). The data obtained from flow cytometrymeasurements was comparable to that acquired using traditional Western blottingtechniques (Figure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igure 2.3: Guk1-7 degradation is proteasome dependent.43Figure 2.3: (Previous page) Guk1-7 degradation is proteasome dependent.(A) Flow cytometry profiles of wild type cells expressing Guk1-GFP orGuk1-7-GFP were incubated at 25◦C or 37◦C for two hours in the pres-ence of CHX. Fluorescence in cells with the control empty vector (EV) arealso shown. Lines demark median GFP fluorescence values and i denotesthe difference in median intensity values used to measure protein stability.(B) Comparison of quantitation of Guk1-7 levels in a CHX chase assay byWestern blot or flow cytometry. (C) Wild type and rpt6-20 cells expressingGuk1-7-GFP were incubated with CHX at 25◦C or 37◦C and samples wereanalysed by flow cytometry at the indicated time points. The results rep-resent the means and standard deviations of three independent experiments.(D) Guk1-7-GFP expressing wild type or pep4∆prb1∆ cells were incubatedat 25◦C or 37◦C in the presence of CHX and samples were analyzed by flowcytometry at the indicated time points. The results represent the means andstandard deviations of three independent experiments. (E) rpt6-20 cell ex-pressing Guk1-GFP or Guk1-7-GFP were grown at 25◦C and then shiftedto 37◦C for 1 hour prior to their fixation and imaging. Scale bar represents5 µm. (F) Guk1-GFP and Guk1-7-GFP expressing cells were incubated at25◦C and cell lysates were immunoprecipitated using GFP-Trap beads andthen immunoblotted with anti-ubiquitin, anti-GFP, and anti-Pgk1 antibodies.The Guk1 and Guk1-7 constructs used in this report are ectopically expressedfrom the constitutive GPD promoter. To ensure that the overexpression from aplasmid does not influence the stability of our model substrate, we expressed bothGuk1-GFP and Guk1-7-GFP from their endogenous locus and promoters, and per-formed a cycloheximide assay. Consistent with our previous CHX chase observa-tions, Guk1-GFP levels remained relatively constant and Guk1-7-GFP levels de-creased by approximately 30% after four hours at 25◦C and by 60% when incu-bated at 37◦C (Figure 2.12B). Together, this data suggested that the flow cytometryassay was suitable for monitoring protein levels and for screening purposes.2.3.3 Guk1-7 Degradation is Proteasome DependentWe next verified that degradation of the ectopically expressed model GFP-fusionsubstrate was proteasome-dependent. When we assayed levels of Guk1-7 at 37◦Cin the temperature sensitive proteasome mutant rpt6-20 [52], degradation of the44mutant protein was largely stopped (Figure 2.3C). Conversely, no difference inGuk1-7-GFP stability was seen between the wild type strain or a double mutant ofthe two main lysosomal proteases (Figure 2.3D). These results suggest that loss ofGuk1-7-GFP fluorescence is primarily caused by proteasomal degradation. Fluo-rescence microscopy revealed that Guk1-GFP was evenly distributed with no in-clusions in rpt6-20 cells at both 25◦C and 37◦C, as was the case for Guk1-7-GFPat 25◦C (n = 126, 104, 118, respectively) (Figure 2.3E). Guk1-7-GFP inclusionswere detected in 69% of the cells incubated at 37◦C (n = 120), of which 94%of cells contained a single inclusion and 5% contained two. These data indicatethat non-degraded Guk1-7-GFP was prone to aggregation at the non-permissivetemperature. Finally, we asked whether the difference in protein stability betweenGuk1-GFP and Guk1-7-GFP was also reflected by their respective ubiquitinationlevels. We found that Guk1-7-GFP, but not Guk1-GFP, was ubiquitinated at 25◦C(Figure 2.3F). In this case, we collected cell lysates from cultures incubated at thelower growth temperature, as we encountered issues with our model substrate be-ing mostly lost to the insoluble pellet fraction when cultures were grown at highertemperatures. Together these experiments suggest that misfolded Guk1-7 is tar-geted for degradation by the ubiquitin proteasome system.2.3.4 FACS-Based Screen for Protein Homeostasis FactorsTo identify novel factors involved in targeting proteins destabilized by missensemutations for degradation, we performed a genome-wide screen based on flow cy-tometry using the Guk1-7-GFP allele. A schematic of the screen is depicted in Fig-ure 2.4A. First, we pooled and bulk transformed the yeast non-essential knockoutcollection with a low copy number plasmid containing Guk1-7-GFP (Figure 2.4Ai). Growth prior to and after transformation was limited, to avoid under repre-sentation of slow growing strains. Pooled transformants were grown in selectivemedia at 25◦C and then subjected to an initial FACS presort to obtain a narrowfluorescence range, which reduces cell-to-cell variability of GFP fusion expression(Figure 2.4A ii; compare grey and green profiles for before and after presort, re-spectively). Presorted cells were then incubated at 37◦C in the presence of CHXfor two hours (Figure 2.4A iii) and then sorted again, selecting for cells with GFP45fluorescence in the top 10% range (Figure 2.4A iv). Samples were collected overa short fifteen minute period to minimize shifting of the population during thehandling time. Cells were recovered in selective liquid media and the screen wasrepeated two more times for a total of three rounds of enrichment (Figure 2.4Av). Following the final FACS sorting, cells were collected on solid selective mediaplates (Figure 2.4A vi).We selected 170 colonies, which had been isolated using the FACS screen de-scribed above, for validation using the flow cytometry assay. In approximately twothirds of the colonies tested, Guk1-7-GFP was more stable than in the wild typecells (Figure 2.4B). The yeast knockout collection was created by replacing eachyeast open reading frame with a KanMX module (conferring resistance to the an-tibiotic geneticin) and a unique 20 base pair nucleotide sequence, referred to asa molecular barcode. Universal priming sites located upstream and downstreamof the barcodes are used for PCR amplification of the barcode region. Sequenc-ing or microarray of the resulting amplicon can be used to reveal the identity of thecorresponding yeast deletion strain. We therefore selected fifty colonies at random,spanning the range of Guk1-7-GFP stabilities, and their corresponding gene knock-outs were identified by Sanger sequencing of the unique strain-specific barcodes.From these fifty colonies, we identified fifteen different gene deletions (Table 2.3).Next, to ensure that the phenotype (i.e., stabilization of Guk1-7) was not acquiredduring the screening process, we assessed the stability of Guk1-7-GFP for each ofthe fifteen gene deletions we identified through Sanger sequencing. To do so, weindividually retransformed the Guk1-7-GFP containing plasmid into each knockoutstrain from our pre-pooled knockout collection. Results of this phenotypic valida-tion were considered positive (denoted as P in Table 2.3) if Guk1-7-GFP was atleast 15% more stable in the deletion strain than in wild type cells (Figure 2.12C).Strains that did not meet this criterion were classified as negative (denoted as Nin Table 2.3). Surprisingly, we failed to observe any stabilization of our modelsubstrate in our most frequently identified hits (e.g., tda2∆), which may have beensusceptible to the acquisition of secondary mutations (Table 2.1). Among the val-idated hits, we identified the N-end rule E3 ligase Ubr1, which was previouslyshown to target cytosolic misfolded proteins for degradation [74, 76, 101–104],and the prefoldin chaperone subunit Gim 3 (Table 2.3, Figure 2.12C).46!"#$%&'"(#)**()+(+#,)-"*."/."0 10 200 21003000310230023104$#&5$%"5(*%-$&/(/,67"-8,92:;(&/(!"#$8,92(&/(!"#$8,92:;(&/%&'($!"!"##!$%&'()* )#%$+",-"&-"./!01!!"##!$%&'()* )#%$+",-"&-"()* )#%$+",-"&-"!"##!$%&'01!2"#"-'"3<-$/*+)-6"5(="$*%(>?(.)##".%&)/@ABC(D-"*)-%E(BFGH(I-JK;LBM@ABC(*"#".%(%)D(20NO5"/%&+=(10(>?(*%-$&/*(7=(7$-.)5"(*"P,"/.&/QO*)#$%"(&/5&'&5,$#(.)#)/&"*!"D"$%(%R)(6)-"(-),/5*()+(*%"D*(&&(:(&'# ## ####$$$#8,92:;(&/%)*+,Figure 2.4: FACS-based screen. (A) Schematic of FACS-based screen. (B)Flow cytometry validation of 170 colonies isolated from the FACS screen.Relative loss of fluorescence of Guk1-GFP or Guk1-7-GFP in wild type cellsis noted as a comparison.47Table 2.3: Summary of FACS screen validationStandardNameSystematicNameNumber of TimesBarcode Identified bySanger SequencingResult ofPhenotypicValidation†TDA2 YER071C 22 NYAK1 YJL141C 10 NRIM15 YFL033C 5 PUBR1 YGR184C 2 PYOR364W YOR364W 1 PGIM3 YNL153C 1 PSLI15 YBR156C 1 NFAU1 YER183C 1 NVHR1 YIL056W 1 NBRA7 YER056C 1 PASP1 YDR321W 1 NMAL11 YGR289C 1 NMUP3 YHL036W 1 PPOL4 YCR014C 1 NPPH22 YOL188C 1 N†P: positiveN: negative2.3.5 Ubr1 Stabilizes Guk1 Missense MutantWe identified the E3 ubiquitin ligase Ubr1 in our screen for factors responsible fordegradative protein quality control of misfolded cytosolic proteins destabilized bymissense alleles. In addition to its role as the E3 ligase of the N-end rule pathway,Ubr1 has also been shown to target misfolded cytoplasmic proteins for degrada-tion [74, 101, 102]. In CHX chase experiments, Guk1-7-GFP levels were approxi-mately 10% higher in ubr1∆ cells compared to wild type (P = 0.0008) at two hours48and remained significantly higher after four hours (P = 0.00003) (Figure 2.5A).To confirm that the stability observed was directly caused by the loss of Ubr1, weperformed addback experiments whereby the wild type Ubr1 was expressed from aplasmid in ubr1∆ cells. We observed that Guk1-7-GFP levels were similar betweenthe UBR1 cells containing a control empty vector and ubr1∆ cells with the Ubr1expressing plasmid, confirming that the phenotype observed could be attributed tothe absence of Ubr1 (Figure 2.5B). To further validate our findings, we performedthe same addback experiments but this time included a mutant form of Ubr1, whichcontains a point mutation in the RING domain producing an inactive ligase [74].Guk1-7-GFP levels in the Ubr1 (C1220S) expressing cells were indistinguishablefrom those with a control plasmid, lending further support to Ubr1 having a directrole in controlling Guk1-7 stability (Figure 2.5C). We next assessed the impor-tance of Ubr1 on a second unstable allele of Guk1 (T290G, hereinafter referredto as Guk1-11) that contained a single missense mutation. This mutant was gen-erated by site directed mutagenesis and was selected based on its instability, as atwo hour incubation at 37◦C in the presence of cycloheximide typically resultedin approximately 60% loss of fluorescence. Consistent with our previous results,an absence of UBR1 led to a significantly reduced clearance of this second modelsubstrate (P = 0.00035) (Figure 2.5D). The relative fluorescence of Guk1-GFP wasnot significantly different between wild type and ubr1∆ cells (Figure 2.13A). Theseresults indicate Ubr1 participates in the clearance of these model misfolded sub-strates, although other factors are also involved.Ubr1 has been shown to act in concert with the nuclear E3 ligase San1 to targetmisfolded cytoplasmic proteins for degradation [74, 101]. To test whether Ubr1also acts with San1 in the degradation of Guk1-7-GFP, we performed flow cy-tometry experiments in the single ubr1∆ and san1∆ deletion strains along with adouble ubr1∆ san1∆ deletion. Guk1-7-GFP was not markedly more stable uponthe deletion of SAN1, although levels were slightly higher in ubr1∆ san1∆ cells incomparison to ubr1∆ cells (Figure 2.13B). These results indicate that San1 doesnot play a major role in the turnover of Guk1-7. To confirm that our assay was ca-pable of detecting an effect with San1, we ran the same assay using the previouslycharacterized Ubr1 and San1 substrate Pro3-1 [101]. In this case we were able toobserve a significant stabilization of Pro3-1 in san1∆ cells, which was even more49! "#!"#$!"#$%!"#$!&'$%%&'$!"#$%%&'$!&'$%((" )#(*((+,-*.'.+/0!0123145671!89!:4:5:6;<:=1!>?8,2@AB((#(*((" )+,-*.'.+/0!0123145671!89!:4:5:6;<:=1!>?8,2@A(%&'$(C!DE(!"#$(C!DE(!"#$(C!%&'$+,-*.'.+/0!0123145671!89!:4:5:6;(!"#$!C!DE(!"#$C!%&'$(!"#$(C!FG2*!>%*""(HA(#(*((( " )BBBBBBB BBBBBBBB$(I(*I("I(J1;65:K1!+,-*.**.+/0!9;,821@31431!:4514@:5L%&'$ !"#$<:=1!>?8,2@ABBBB!BB!BB!B B!BFigure 2.5: Ubr1 promotes Guk1-7-GFP degradation. (A) Wild type andubr1∆ cells expressing Guk1-7-GFP were incubated with CHX at 25◦C or37◦C and samples were analysed by flow cytometry at the indicated timepoints. The results represent the means and standard deviations of three inde-pendent experiments. P values were calculated with an unpaired Student’s ttest, *, ** and *** denote P< 0.05, 0.005, and 0.0005, respectively. (B)UBR1and ubr1∆ cells expressing Guk1-7-GFP along with an empty vector (EV)control or UBR1 were incubated at 37◦C and samples were collected at the in-dicated time points for flow cytometry analysis. Results represent the meansand standard deviations of three independent experiments. P values were cal-culated with a one-way ANOVA and post-hoc Tukey HSD to assess signifi-cance, ** denotes P < 0.005. (C) ubr1∆ cells coexpressing Guk1-7-GFP andan empty vector control or either UBR1 or UBR1 (C1220S) were incubatedat 37◦C with CHX and samples were collected at the indicated time points.(D) Wild type or ubr1∆ cells expressing Guk1 (T290G) fused to GFP wereincubated with CHX at 37◦C for two hours before being analyzed by flow cy-tometry. The results represent the relative fluorescence intensities from threeindependent experiments (with standard deviations). P values were calculatedwith a one-way ANOVA and post-hoc Tukey HSD to assess significance, ***denotes P < 0.0005.50pronounced in the double ubr1∆ san1∆ strain (Figure 2.13C). Together, these datasuggest that San1 does not play a role alongside Ubr1 in targeting Guk1-7-GFP fordegradation, indicating that other E3 ligases may be involved in the proteasome-mediated degradation of this substrate.2.3.6 Gim3 Impairs Guk1-7-GFP DegradationPrefoldin is a hetero-oligomeric protein complex composed of six subunits rangingin size from 14–23 kDa [41]. Conserved in archaea and eukaryotes, but absent inprokaryotes, the prefoldin hexamer forms a “jellyfish-like” structure with N- andC-terminal coiled-coil regions of each subunit forming “tentacles” that emanatefrom a central region [42]. Misfolded substrates are transferred for folding fromprefoldin to the TRiC/CCT chaperonin in an ATP-independent manner through di-rect binding of the two chaperone complexes [41]. In addition to its role in aidingnascent proteins, such as actin and tubulin, to attain their functional conformations,the prefoldin chaperone complex has been shown to prevent huntingtin and alpha-synuclein aggregate formation [43, 44]. Having identified the prefoldin subunitGim3 in our screen, we decided to further examine its potential role in degradativeprotein quality control. In CHX chase experiments, Guk1-7-GFP levels were ap-proximately 25% higher in the gim3∆ strain compared to wild type (P = 0.0057)(Figure 2.6A). To ensure that the stabilization was specifically caused by the ab-sence of Gim3, we expressed in gim3∆ cells the wild-type GIM3 from a plasmid,which rescued the degradation of the model substrate (Figure 2.6B). While degra-dation of Guk1-7-GFP is not fully inhibited in gim3∆ cells, levels are markedlyhigher than in the wild type strain, indicating that Gim3 is required for the normalturnover of our model substrate. We next wished to see if Gim3 works together withUbr1. In this case we preferred a model substrate that is misfolded as the result ofa single point mutation (Guk1-11) to eliminate or minimize potential confoundingfactors caused by multiple destabilizing mutations. The double ubr1∆ gim3∆ strainshowed increased Guk1-11 stability compared to single deletion strains, howeverthe substrate was still degraded by over 50% (Figure 2.6C). This data indicates thatpotentially other E3 ligases or chaperones are required for complete proteolysisto occur. In addition, this would suggest that Ubr1 and Gim3 work partially in51parallel or in independent pathways to target the assessed misfolded substrate fordegradation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igure 2.6: Absence of Gim3 reduces Guk1-7 turnover.52Figure 2.6: (Previous page) Absence of Gim3 reduces Guk1-7 turnover. (A)Wild type and gim3∆ cells expressing Guk1-7-GFP were incubated with CHXat 25◦C or 37◦C and samples were analysed by flow cytometry at the indicatedtime points. The results represent the means and standard deviations of threeindependent experiments and the asterix denotes significance of P< 0.05. (B)Gim3 addback experiment. GIM3 or gim3∆ cells expressing Guk1-7-GFP andeither an empty vector (EV) control or GIM3. The results represent the meansand standard deviations of three independent experiments of the relative flu-orescence intensity after a two hour CHX incubation at 37◦C. P values werecalculated with a one-way ANOVA and post-hoc Tukey HSD to assess signifi-cance, ** denotes P< 0.005. (C) Wild type, ubr1∆, gim3∆, and ubr1∆ gim3∆cells expressing Guk1 (T290G) fused to GFP were incubated at 37◦C withCHX for two hours and then analysed by flow cytometry. P values were cal-culated with a one-way ANOVA and Holm multiple comparison to assesssignificance, * and ** denote P < 0.05 and 0.01, respectively. (D) Protea-some activity assay. Gim3 or gim3∆ cells expressing Deg1-GFP under theCup1 promoter were incubated at 30◦C in the presence of CHX and sampleswere collected at the indicated time points. Deg1-GFP was immunoblottedwith an anti-GFP antibody. The results represent the mean and standard de-viation of three independent experiments. (E) GIM3 or gim3∆ cells express-ing Guk1-7-GFP, GFP alone, or a control empty vector were grown at 25◦C.Guk1-7-GFP was immunoprecipitated with GFP-Trap beads and eluted sam-ples were immunoblotted with anti-ubiquitin and anti-GFP antibodies.The TRiC/CCT chaperonin cooperates with prefoldin in folding a number ofcellular proteins and has been shown to interact with proteasome subunits, suggest-ing that it may be involved in proteasome maturation [194]. Therefore, one possi-bility is that the stabilizing effect of Gim3 on Guk1-7 could be indirect, a result ofdecreased proteasome function. We tested for compromised proteasome functionin the gim3∆ strain using the constitutive Deg1-GFP proteasome substrate [184].We found that there was no significant difference in the degradation of Deg1-GFPin gim3∆ cells compared to the wild type strain at all time points tested, with theexception of the thirty minute sample (P = 0.77, P = 0.22, P = 0.04, P = 0.07 forthe 10, 20, 30, and 60 minute time points, respectively) (Figure 2.6D). Hence, thereduced turnover of misfolded protein observed in gim3∆ cells is unlikely causedby an impaired proteasome.53We next determined whether an absence of GIM3 could affect ubiquitinationof our misfolded model substrate. GFP tagged Guk1-7 was pulled down fromcells grown at 25◦C, where it remained mostly soluble, and then ubiquitin levelswere detected by immunoblotting. After normalizing the quantity of ubiquitin tothat of eluted Guk1-7, ubiquitination levels were essentially unchanged, with un-der 5% less ubiquitinated model substrate in gim3∆ cells compared to wild type(Figure 2.6E). This experiment indicates that the absence of Gim3 did not impairubiquitination of our model substrate, in agreement with Gim3 functioning inde-pendently of Ubr1.2.3.7 Gim3 Facilitates the Clearance of Insoluble Guk1 andMaintains Guk1-7 SolubilityWe next sought to evaluate the impact an absence of Gim3 has on Guk1-7 localiza-tion. Fluorescence microscopy performed on wild type and gim3∆ strains showedthat while there was no difference in wild type Guk1-GFP localization betweenthe two strains, Guk1-7-GFP formed cytoplasmic puncta in 93% of gim3∆ cellswhen incubated at 37◦C, and additional faint and diffuse cytoplasmic GFP wasalso visible (n = 200) (Figure 2.7A). Cells contained on average 1.5 puncta, whichwere typically located next to the nucleus. In contrast, only sixteen percent ofwild type cells contained Guk1-7-GFP puncta (n = 200). These puncta were nolonger present when we expressed Gim3 from a plasmid in gim3∆ cells and thediffuse cytoplasmic GFP signal was also not present, similar to that observed inGIM3 cells (Figure 2.7B). We decided to examine this phenomenon more closelyby performing a time course microscopy experiment incubating cells at 37◦C, butin the absence of the translation inhibitor CHX which we had been using up to thispoint and which may interfere with aggregate formation [195]. Within five minutesnumerous Guk1-7-GFP containing puncta were detected within the cytoplasm ofGIM3 cells with those in the gim3∆ strain being slightly delayed and visible af-ter 10 minutes (Figure 2.7C). While puntca remained visible in both strains after45 minutes at 37◦C, those in the gim3∆ strain appeared to coalesce slower than inGIM3 (after 15–20 minutes in GIM3 compared to 20–25 minutes in gim3∆) andremained visibly brighter. Some diffuse cytoplasmic Guk1-7-GFP signal was alsopresent in gim3∆ cells up to 25 minutes after shifting to the increased growth tem-54perature, but was only present in the first 10 minutes for GIM3 cells. While thenumber of puncta did not differ between Gim3 containing or deleted cells, the in-tensity of the gim3∆ puncta remained brighter for longer. These results suggest thatGim3may play a role in maintaining Guk1-7-GFP solubility at higher temperaturesto facilitate substrate degradation.These Guk1-7-GFP puncta observed in the time course experiment in bothGIM3 and gim3∆ cells are reminiscent of the Q-bodies described by Frydmanand colleagues [136]. To determine whether this is indeed the case, we examinedGuk1-GFP and Guk1-7-GFP colocalization in GIM3 cells with two cytosolic ag-gregate markers: Hsp104 and Hsp42. Hsp104 is an aggregate-specific chaperonethat has a diffuse cytoplasmic and nuclear localization pattern at 25◦C but formspuncta when incubated at 37◦C (Figure 2.8A, enlarged Figure 2.8C). Hsp104-mCherry and Guk1-7-GFP puncta colocalized in 100% of cells at 37◦C (n = 100).We then examined Guk1-7-GFP colocalization with the small heat shock proteinHsp42, which is required for peripheral aggregate formation during physiologicalheat stress [137]. As with Hsp104, Hsp42-mCherry formed puncta when incu-bated at 37◦C, but not at 25◦C (Figure 2.8B, enlarged Figure 2.8D). Guk1-7-GFPcolocalized in all Hsp42-mCherry puncta. However, in 34% of the cells exam-ined (n = 100), we find an average of 1.4 Guk1-7-GFP puncta per cell that do notcolocalize with Hsp42. Overall, Hsp42-free Guk1-7-GFP puncta represented 9%of all puncta observed in the one hundred cells examined. Together, the Hsp104and Hsp42 colocalization data suggest that at 37◦C Guk1-7-GFP forms cytosolicinclusions similar to Q-bodies.55! "!"#$%&'()*"+,-./ ,-./ ,-./ ,-./01./ 01./ 01./ 01./!"#$ %&'$ !"#$ %&'$*2345*"+ *234515*"+,-./ 01./ 01./ 01./!"#$ %&'$6789 6789 6789 ! !"#$#$%&'():%;<%*234515*"+=>/:%;<%#? - 4? 4- ,? ,- 0? @-!"#$*234515*"+=>/#$%&'()%&'$*234515*"+=>/#$%&'():%;<%:%;<%ABC7D)701$/Figure 2.7: Gim3 facilitates clearance of insoluble Guk1-7.56Figure 2.7: (Previous page) Gim3 facilitates clearance of insoluble Guk1-7.(A) GIM3 or gim3∆ cells expressing Guk1-7-GFP were grown at 25◦C andthen incubated at either 25◦C or 37◦C for 2 hours in the presence of CHX be-fore fixation and imaging. (B)GIM3 and gim3∆ cells expressing Guk1-7-GFPalong with an empty vector control or GIM3 were incubated at 25◦C or 37◦Cfor two hours in the presence of CHX before fixation and imaging. (C)GIM3 or gim3∆ cells expressing Guk1-7-GFP were incubated at 25◦C andthen shifted to 37◦C. Samples were collected at the indicated time points andthen fixed before imaging. For all images, the scale bar represents 5 µm anddotted lines demark cell boundaries.To verify the importance of Gim3 in maintaining Guk1-7 solubility, we exam-ined the sedimentation of the mutated protein after centrifugation. Twice as muchGuk1-7 is found in the NP-40 insoluble pellet fraction at 25◦C in gim3∆ cellscompared to wild type GIM3 cells (Figure 2.9A). There was also more Guk1-7 inthe pellet of gim3∆ than wild type cells after incubating cells at 37◦C. We thenperformed immunoprecipitation experiments to test whether Gim3 could directlyinteract with Guk1-7 as a potential mechanism for maintaining Guk1-7 solubil-ity. From cell extracts incubated at 25◦C, Guk1-7-GFP can pull down a TAP-tagged form of Gim3 whereas no interaction was detected between Gim3-TAPand Guk1-GFP (Figure 2.9B). We verified this interaction in an independent ex-periment (Figure 2.14A). Once again, a lower temperature was used for pulldownexperiments to avoid losing Guk1-7 in the insoluble pellet fraction. These resultssuggest that Gim3 could maintain Guk1-7 in a more soluble state through physicalinteraction, potentially acting as a holdase. Holdases are a type of molecular chap-erone that bind to misfolded proteins in an ATP-independent manner to preventprotein aggregation, but they do not directly refold their substrates [47]. Consis-tent with these findings, we tested the viability of the guk1-7 strain over a range oftemperatures (25◦C to 37◦C), in the presence or absence of GIM3, and found thatin both cases viability largely decreased between 32◦C and 33◦C with no growth attemperatures of 34◦C or above (Figure 2.14B). These results indicate that, whereasdegradation of poorly soluble Guk1-7 was delayed, temperature-dependent lethal-ity is not rescued in gim3∆ cells.57!!"# $%&'()* +&,-&./0$)123456#(&,,789:#;<:#89:#;<:#.=>25./0.=>25<5./0"!"# $%&'()* +&,-&./0$)14856#(&,,789:#;<:#89:#;<:#.=>25./0.=>25<5./0./0$)123456#(&,,7 +&,-&#$./0$)14856#(&,,7 +&,-&Figure 2.8: Guk1-7-GFP puncta colocalize with Q-body markers. (A) Cellswith Hsp104 endogenously tagged with mCherry and ectopically expressingGuk1-GFP or Guk1-7-GFP were grown at 25◦C and then incubated at 25◦C or37◦C for 30 minutes before fixation and imaging. Scale bar represents 5 µm.(B) Hsp42-mCherry cells ectopically expressing Guk1-GFP or Guk1-7-GFPwere grown at 25◦C prior to incubation at 25◦C or 37◦C for 30 minutes. Cellswere then fixed before imaging. The scale bar represents 5 µm. (C) Enlargedimages from cells collected as in A. Scale bar represents 2.5 µm. (D) Enlargedimages from cells collected as in B. Scale bar represents 2.5 µm.58! "!"#"$%$"#&'()#*$%$+*#"',)#+$%$!*#+'*-#*$%$!+#"'./00/12 3 .+(45+(45,*45,*45!"#$%&'$678!9*96:.678!96:.;<=1>$?/@1AB6C<,92D.E.F$6:.GF$2D.E.F$6:.GF$6:.2D.6:..H8!CI=71JB$K8LMN(-(-(-(-JB$K8LMN,*(-,*(-(-Figure 2.9: Gim3 helps maintain Guk1-7 solubility. (A) GIM3 or gim3∆Guk1-7-GFP expressing cells were grown at 25◦C or shifted to 37◦C for 20min. The ratio of the pellet fraction to total cell lysate is noted and repre-sents the mean and standard deviation of three independent experiments. (B)Guk1-7-GFP was immunoprecipitated from Gim3-TAP expressing cells incu-bated at 25◦C and then immunoblotted with anti-TAP, anti-GFP, or anti-Pgk1antibodies.2.3.8 Gim3 Has a General Effect Towards Thermally DestabilizedProteinsTo see if the effect of Gim3 on Guk1-7 solubility was specific to this prefoldin sub-unit, or common to all prefoldin subunits, we performed fluorescence microscopywith the other prefoldin mutant strains. Guk1-7-GFP puncta were visible in allof the prefoldin deletions, albeit to varying degrees, suggesting that they all playa role in Guk1-7 solubility (Figure 2.10A). Fifty-six per cent of gim1∆ cells con-tained puncta, whereas only 26% of gim4∆ did (n = 50, each). While the numberof puncta per cell only differed slightly between prefolin strains, either faint or nocytoplasmic Guk1-7-GFP was visible in gim2∆, gim4∆, gim5∆, and gim6∆ strainswhile markedly present in gim1∆ and gim3∆ cells. To better quantify the effect, wemeasured Guk1-7 levels by flow cytometry and found that only deletions of GIM1and GIM3, and to a lesser extent GIM5, retarded the degradation of the model sub-strate (Figure 2.10B). Not surprisingly, gim1∆ and gim3∆were the only strains that,in addition to puncta, also had a diffuse cytoplasmic Guk1-7-GFP signal visible byfluorescence microscopy. All together, these results suggest that while deletion of59individual members of the prefoldin complex impacted degradation of the modelsubstrate, some (i.e., Gim1 and Gim3) may play a more important role.!"!"#$!"#%!"#&!"#'!"#(!"#)!"#$%&'()$%&'(*$%+,-.&%$/0123450 1.))6.7671(.-0)80980:;<$;:#'(*$%+%'=,>0-"0?@ABCD<EABC&+EA!"#)&+EA!"#(&+EA!"#$&+EA!"#%&+EA!"#'&+EA!"#&&+EA:F :F <GF &GF &DFHHFDGFDIF,0-80932"06.768011)6J43K6#(9832L.08K)36/01234506'(*$%+%'=,671(.-0)80980649309)43M*+,&-./$-./'!"#&-./$-./'*+,&012$3$-./'*+,&-./$012'3$:;:$;:D;:$'(*$%+%'=,L.08K)3>0-"0?@AD<EA &+EA &+EA &+EA &+EA*+,&-./$-./'!"#&-./$-./'*+,&012$3$-./'*+,&-./$012'3$,0-80932"06.768011)6J43K#(9832:F $:F +&F HIF G:FN6NNN6NFigure 2.10: Thermosensitive alleles are stabilized by prefoldin subunits.60Figure 2.10: (Previous page) Thermosensitive alleles are stabilized byprefoldin subunits. (A) Individual prefoldin subunit mutants expressingGuk1-7-GFP were incubated at 37◦C in the presence of CHX for 2 hours.The percentage of cells with puncta was calculated from 30–50 GFP posi-tive cells. Scale bar represents 5 µm. (B) Four thermosensitive alleles wereexpressed as GFP fusion proteins in the six prefoldin deletion strains. Cellswere incubated with CHX at 25◦C and 37◦C for 2 hours and fluorescence in-tensity was measured by flow cytometry. (C) Guk1-7-GFP was expressed inwild type, gim3∆, tcp1-1, or tcp4-1 cells and incubated with CHX for twohours at either 25◦C or 37◦C before flow cytometry analysis. (D) Wild type,gim3∆, tcp1-1, and tcp4-1 cells expressing Guk1-7-GFP were incubated withCHX for 2 hours at 25◦C or 37◦C prior to fixation and imaging. Scale barrepresents 5 µm.We next wished to explore whether prefoldin can stabilize additional proteinsthat are destabilized by missense mutations other than Guk1-7. We selected a num-ber of cytoplasmic thermosensitive alleles, which we have previously shown to bedegraded in a proteasome dependent manner, and created C-terminal GFP fusionsto test by flow cytometry [101]. Ugp1 is the UDP-glucose pyrophophorylase in S.cerevisiae and is involved in the oxidative stress response [196, 197]. The Ugp1-3allele contains two silent and two missense mutations and temperature sensitivelethality can be restored by Ubr1 deletion [101]. Ugp1-3-GFP was only modestlystabilized by all prefoldin deletions (including gim3∆) with the largest effect seenin gim1∆ and gim6∆ strains (Figure 2.8B). Glutamyl tRNA synthetase (Gus1) at-taches amino acids to cognate tRNA and the Gus1-3 allele contains seven missensemutations [101, 198, 199]. Unlike Guk1-7, Gus1-3 was most stabilized by gim2∆and gim6∆ strains. Delta 1-pyrroline-5-carboxylate reductase (Pro3) converts delta1-pyrroline-5-carboxylate to proline in the final step of the proline biosynthesispathway [200]. Pro3-1 has four missense mutations and its temperature sensitivelethality is restored in a double deletion of Ubr1 and San1 [101]. Of all the alle-les tested, Pro3-1 was the most stabilized by the prefoldin deletions, with gim1∆,gim2∆, and gim3∆ strains having the largest effect. Gim5 also stabilized Pro3-1, but to a lesser extent. The gim4∆ strain had the smallest effect on substratestabilization of all the deletions tested. These data suggest that while individual61prefoldin subunit deletions may differentially affect substrate stability, they canstabilize a range of substrates misfolded due to missense mutations.Given that the TRiC/CCT chaperonin folds client proteins delivered to it bythe prefoldin complex, we sought to see if it also had a role in stabilizing ourGuk1-7-GFP substrate. Using temperature sensitive alleles of the TRiC/CCT sub-units Tcp1 and Tcp4, we performed CHX chase experiments and found Guk1-7-GFP levels to be significantly higher compared to wild type, similar to those foundin gim3∆ cells (Figure 2.10C). Fluorescence microscopy showed that Guk1-7-GFPforms cytoplasmic puncta in 48% of tcp1-1 cells (n = 100) and 60% of tcp4-1 cells(n = 100) when incubated at 37◦C (Figure 2.10D). In cells where puncta were ob-served, an average of 1.8, 2.4, and 1.3 puncta per cell were found for the gim3∆,tcp1-1, and tcp4-1 strains, respectively. These data would suggest a possible rolefor TRiC/CCT chaperonin in addition to prefoldin in maintaining the solubility ofour Guk1-7-GFP model substrate.2.4 DiscussionClassically, degradative quality control pathways have been identified and charac-terized using model substrates. In this study, we have established Guk1-7 as a novelmodel protein quality control substrate whose stability is temperature dependentand is degraded by the proteasome. The mutant protein also forms Q-body likeinclusions when shifted to the non-permissive temperature that co-localize withHsp104, as well as Hsp42. We developed a flow cytometry assay to assess proteinstability and then performed a FACS-based screen to isolate factors important forcytosolic protein homeostasis. We identified the E3 ubiquitin ligase Ubr1 and theprefoldin chaperone subunit Gim3. Gim3 promotes Guk1-7-GFP degradation andinfluences its solubility, but not ubiquitination. We also showed that in additionto Guk1-7, prefoldin can stabilize a number of temperature sensitive proteins thatmisfold as the result of missense mutations.Protein degradation is generally assayed by pulse-chase metabolic labelling, orby using protein synthesis inhibitors coupled with downstream biochemical analy-sis [201]. More recently, fluorescently tagged proteins have been used to monitorprotein stability [74, 174]. This development means that it is now feasible to per-62form high-throughput genome-wide screens using flow cytometry to identify fac-tors that influence protein stability or abundance. Flow cytometry confers a num-ber of advantages compared to stability assays using endogenous or ectopicallyexpressed fluorescently tagged substrates as the method is quantitative, measure-ments are performed in vivo, thousands of cells can be analyzed in under an hour,and for most purposes no additional processing or cell lysis is required. Whiletemperature sensitive alleles have been used in suppressor screens to identify pro-tein quality control components such as San1 and Ubr1, these screens require thatthe model substrate be functional at the restrictive temperature [70, 101]. Perhapsmost importantly, in addition to the relative speed and precision flow cytometryprovides over Western blotting methods, the assay is sensitive enough to discernpartial effects (e.g., stabilization of Guk1-7 in ubr1∆) that might not be detected bytraditional Western blotting methods and can be used as a screening tool that doesnot rely on protein function.Limiting the damaging effects of misfolded proteins appears to have influencedprotein evolution as the most conserved proteins are those with the highest transla-tion rate and as a result face the greatest risk of incurring mistranslation errors [2].As missense mutations represent more than half of all mutations in the HGMDand mistranslation-induced misfolding is a potential mechanism for pathologiesindependent of genomic alterations, we anticipate that understanding the proteinquality control pathways that recognize and triage proteins misfolded as the resultof missense mutations will gain in importance [158]. A recent study by Sahni etal. [159] found two-thirds of the disease associated missense alleles they tested tohave disrupted protein-protein interactions compared to the wild type allele, andapproximately 30% of mutant proteins displayed increased binding to componentsof the protein homeostasis network. While nascent polypeptides that misfold dur-ing translation are rapidly degraded, our model substrate has a half life of over fourhours at the permissive temperature of 25◦C and approximately 90 minutes at 37◦C.Our lab has previously reported a panel of temperature sensitive alleles of essentialcytosolic genes in S. cerevisiae and showed that just under half of these alleles havehalf lives of three hours or less [101]. At the non-permissive temperature of 37◦C,Guk1-7’s half life is similar to that of the cytoplasmic model substrate ∆ssCPY*(∼1 hr) and GFP-Ubc9-2 (∼40 min) [74, 136]. Interestingly, by contrast, nuclear63temperature sensitive proteins such as Cdc68-1, Sir4-9, Cdc13-1 and Sir3-8, allrecognized by the E3 ubiquitin ligase San1, have significantly shorter half livesthan the cytoplasmic alleles we have identified [70]. There are a number of poten-tial explanations for why a slower turnover rate is observed. First, the cytoplasmicproteins used in our study and that of Escusa-Toret et al. [136] are constitutivelyexpressed at high levels compared to the low abundance endogenously expressednuclear proteins examined by Gardner et al. [70]. As a single protein in the cellis represented by a spectrum of folding states, it may be that highly abundant pro-teins prone to misfolding may only have a fraction of their cellular pool misfoldedin such a state as to be recognized and degraded at one time. Second, temperaturesensitive alleles of natural proteins, as opposed to engineered model substrates,have evolved in a cellular context replete with chaperones and protein homeostasismachinery. Given that most chaperones are cytoplasmic, our model substrates havethe potential to be recognized and interact with a number of chaperones undergo-ing refolding cycles before being targeted for degradation. This would be reflectedby a slower turnover rate. Finally, we identified the ubiquitin E3 ligase Ubr1 in ourscreen. Ubr1 alone, or as a double mutant in combination with Gim3 or San1, wasnot sufficient to completely stop degradation of our model substrate. This wouldsuggest that some misfolded proteins require the activity of a number of E3 ligasesfor their disposal.While performing fluorescence microscopy we observed that the model sub-strate Guk1-7-GFP forms Q-body like inclusions in the cytoplasm. A similar phe-nomenon was described for the temperature sensitive Ubc9-2 allele by Escusa-Toret et al. [136]. They speculate that Q-body formation is a rapid early responsedeployed by the cell to manage misfolded proteins. It would be interesting to see ifthis inclusion formation can explain the longer half life of our misfolded substrate.A major question is whether proteins sequestered in Q-bodies get redirected to thenucleus for degradation with the help of San1 (as in the case of Pro3-1), or doproteasomes co-localize to Q-bodies as they do with JUNQ inclusions providing ameans for substrate disposal in the cytoplasm.In addition to Ubr1, we identified the prefoldin chaperone subunit Gim3 inour screen. We demonstrate that Gim3 was necessary for maintaining Guk1-7solubility and interacted with our missense allele, but not the wild type protein.64In contrast to other reports, we did not find Gim3 to influence ubiquitination ofour model substrate, suggesting that it acts independent of substrate ubiquitination(Figure 2.11). This discrepancy could be explained as being due to previous studiesbeing performed under conditions of proteasome inhibition, or due to the natureof the substrate [202]. Previous reports have demonstrated that knocking downprefoldin subunits results in increased ubiquitination of alpha-synuclein and largeinclusion formation, as well as aggregation of huntingtin, an amyloidogenic IPODsubstrate [43, 44, 135, 136]. Whereas Abe et al. [202]examined the ubiquitinationstatus of the proteome either in the soluble or pellet fraction under proteasomeinhibition, we focused our attention to the soluble fraction of a single substrate.!!"#$%&'(%)*+),'-.+(/ 0!123)456&78#(.8'9"%&):8;&%,'-.+(84<+=3:>//&%/#9+'(Figure 2.11: Model for stabilization of temperature sensitive alleles by Gim3.(A) Proposed model for how Gim3 promotes degradation of temperature sen-sitive alleles destabilized by missense mutations.As the structure of the prefoldin complex has no evidence for a nucleotidebinding site and therefore lacks ATP-regulated functionality, it is tempting to spec-ulate that prefoldin may act as a holdase [42]. Interestingly, only the mutated andnot wild type Guk1 requires Gim3 to remain soluble. In addition, mutations thataffect chaperonin components also impaired Guk1-7 degradation. These results65indicate that in addition to maintaining misfolded proteins soluble, prefoldin alsohanded them to the chaperonin for refolding. More work will be required to clearlydemonstrate whether Gim3 acts as a holdase to prevent protein aggregation to en-hance substrate accessibility for ubiquitin-proteasome mediated degradation. Sev-eral other chaperone or co-chaperone proteins have been shown to be importantfor promoting the degradation of cytosolic misfolded proteins in yeast. Sse1 wasshown to help mediate the degradation of the tumor suppressor VHL and is requiredfor the recognition of misfolded proteins by Ubr1 [50, 74]. The Ydj1 J-domaincontaining Hsp40 mediates both the degradation of ER proteins with exposed mis-folded cytosolic domains and the Rsp5 mediated degradation of cytosolic proteinsafter heat shock [52, 53]. In contrast Sis1, another J-domain containing Hsp40,was shown to be important for the relocalization of cytosolic misfolded proteinsto the nucleus [75]. Fes1, an Hsp70 nucleotide exchange factor, was also shownto be important for the degradation of cytosolic misfolded proteins and does soby interacting with the misfolded proteins bound to Hsp70 and triggering their re-lease [54, 167, 203]. By demonstrating a role for Gim3 in substrate solubility, ourwork adds to a growing body of evidence suggesting that prefoldin is important forpreventing potentially toxic protein aggregation [44, 204, 205]. In addition to ourtemperature sensitive alleles, prefoldin has been shown to inhibit human amyloid-beta fibrillation and prevents aggregation of huntingtin [44, 204]. This underscoresthe potential importance the prefoldin chaperone complex has in maintaining pro-tein homeostasis.662.5 Supplemental Data!"!"#$%&%!'()*++,)-"./!"#$%&,0. 12,3456)7 !"#$%& 0. 12 38&6)7!"#$%&,0.,,3456)7 !"#$%&,0.,,38&6)7!"#$%&'(9 4 :94559&5$99!"#$%&%!'( 456)!"#$%!'(,456)!"#$%!'(,8&6)!"#$%&%!'(,8&6)20;*,3<-"=>7(*=?*./@A*,-B,0.0/0@+!"#$%&%!'()*++,)-"./'"#()*+,$-./ &'01 #%2$#'+"-=*>?*.?*,0./*.>0/C,3@D"D7989E999F9E999G9E999!"#$456).H$9I!"#$8&6).H$49!"#$%&456).H$9$!"#$%&8&6).H$FIFigure 2:12: Guk1-7-GFP flow cytometry.67Figure 2.12: (Previous page) Guk1-7-GFP flow cytometry. (A) Box plot ofquantification for fluorescence microscopy images in Figure 2.2A. Correctedtotal cell fluorescence was calculated by subtracting the mean fluorescence ofbackground readings from the integrated density. n = 108, 120, 101, and 168for Guk1 25◦C, Guk1 37◦C, Guk1-7 25◦C, and Guk1-7 37◦C, respectively.(B) Wild type cells expressing Guk1-GFP or Guk1-7-GFP on a plasmid andexpressed from their endogenous promoters were incubated at 25◦C or 37◦Cwith CHX. Samples were collected at the indicated time points and analyzedby flow cytometry. (C) Flow cytometry validation experiments for the deletionstrains identified by barcode sequencing. Cells expressing Guk1-7-GFP wereincubated with CHX at 25◦C or 37◦C for two hours prior to flow cytometryanalysis. Note that expression of YOR364W and RIM15 from a plasmid (i.e.,add back experiments) failed to rescue the phenotype indicating that an addi-tional mutation may have caused stabilization of the model substrate. Dele-tions of UBR1 and GIM3 were further analyzed in this work but not MUP3and BRA7.68! "#!"#$ %&'$!"#$%&'"()*+,-)./(0#*12"34"54"(&5%"53&%6 789:899895353!"#$()*$%&'$()*$!"#$+,-$%&'$+,-$!"#$%&'"(#133(10()*+,-;-)./(0#*12"34"54"(,8998<989=(==(=53=(==(==(!"#$%&'"(#133(10(/21>-,-)./(0#*12"34"54"(!"#$()*$%&'$()*$!"#$+,-$%&'$+,-$,8998<989=(==(==(==(==(==(=Figure 2.13: Ubr1 does not act with San1 in the degradation of Guk1-7-GFP.(A) Guk1-GFP was expressed in wild type or ubr1∆ cells and incubated withcycloheximide for 2 hours at 25◦C or 37◦C prior to performing flow cytom-etry. The results represent the relative fluorescence intensities and standarddeviations from three independent experiments. Statistical significance wastested using an unpaired two tailed Student’s t-test. (B) Guk1-7-GFP wasexpressed in wild type, ubr1∆, san1∆, and ubr1∆ san1∆ cells and incubatedwith cycloheximide for 2 hours at 25◦C or 37◦C prior to performing flow cy-tometry. The results represent the average and standard deviations from threeindependent experiments. Statistical significance was tested using a one-wayANOVA and a Tukey HSD post-hoc test. *, **, and ns denote P < 0.05,P < 0.01, and not significant, respectively. (C) Pro3-1-GFP expressing cellswere grown and treated as in B. Samples were analysed using a one-wayANOVA followed by Tukey’s post hoc test, ** denotes P < 0.01.69!"!"#$%&'()*((+,-./012.01././012.3(45)*(645)*(3(45)*(645&'(7859,:;<6&)$,-=1=)*()$,-=)*()*(>?#%@5ABC%D8)!?0=&'(2.EF 0/EF 0-EF 02EF00EF 0GEF 0.EF 0HEF 01EF!"#$!%&'()*$!%&'!"#$!%&'+,()*$!%&'+,Figure 2.14: Guk1-7-GFP Gim3 interaction and viability assay.70Figure 2.14: (Previous page) Guk1-7-GFP Gim3 interaction and viability as-say. (A) Guk1-7-GFP was immunoprecipitated from wild type or Gim3-TAPexpressing cells incubated at 25◦C and then immunoblotted with anti-TAP,anti-GFP, or anti-Pgk1 antibodies. (B) Viability assay. Wild type, gim3∆,guk1-7, or double guk1-7, gim3∆ cells were streaked on rich media plates andincubated for two days at the indicated temperatures.71Chapter 3Recurrent BackgroundMutations inWHI2 AlterProteostasis and ImpairDegradation of CytosolicMisfolded Proteins inSaccharomyces cerevisiae3.1 IntroductionProtein homeostasis (proteostasis) is maintained by an extensive protein qualitycontrol network that promotes and mediates protein folding by molecular chap-erones and prevents the accumulation of misfolded proteins by targeting them fordegradation via the ubiquitin proteasome system or autophagy [1]. The proteostaticbalance can be challenged by exposure to a range of intrinsic or extrinsic stressors,which require the cell to mount an adequate response, most notably by regulatingthe expression of protein quality control network elements in a concerted manner.Inadequate management of misfolded proteins can have deleterious consequences,72such as aggregation, which is characteristic of some neurodegenerative diseasesthat include Alzheimer’s, Parkinson’s, and ageing [11].Proteasomal degradation of misfolded cytosolic proteins is mediated by sev-eral quality control E3 ubiquitin ligases, which typically work in concert withother chaperone proteins to recognize their substrates [178, 203]. For instance,Hsp110 Sse1, which acts as a nucleotide exchange factor, was shown to promoteubiquitination by the Ubr1 E3 ligase in yeast [74]. As well, we proposed that theYdj1 Hsp40 co-chaperone acts as a substrate adaptor for the Rsp5 E3 ligase uponacute heat stress [52]. In other cases, chaperone proteins are also required to pro-mote proteolysis. The Hsp40 co-chaperone Sis1 for example, is necessary for thetranslocation of misfolded cytosolic proteins to the nucleus where most protea-somes reside [140]. We also recently showed that the yeast prefoldin subunit Gim3is required to promote proteolysis of cytosolic proteins misfolded due to missensemutations by preventing their aggregation [206]. Therefore, although chaperoneproteins primarily promote polypeptide folding and assembly, they may also playa key role in the clearance of misfolded proteins. Understandably, the relation-ship between the folding and degradation machineries is complex. For example,the structurally related chaperone regulatory proteins Bag1 and Bag2, respectivelypromote and inhibit the degradation of cytosolic misfolded proteins by the CHIPE3 quality control ligase [85, 86, 207]. Therefore, a major challenge is to un-derstand how changes in the intricate protein quality control network can perturbproteostasis, for instance by shifting the balance between folding and proteolysis.Temperature sensitive alleles of essential genes in S. cerevisiae are invaluablemodel substrates that can be employed to characterize components of the proteinquality control machinery [70, 101, 135, 136, 146, 206]. We previously identi-fied the E3 ubiquitin ligase Ubr1 from a genetic screen for factors involved indegradative protein quality control of Guk1-7, a thermally unstable mutant alleleof the guanylate kinase Guk1 [206]. Ubr1 activity alone, however, was not suffi-cient to account for the bulk of substrate degradation. Therefore, we performeda targeted flow cytometry based screen using a panel of E3 mutant strains. Us-ing this approach, we identified a surprising number of yeast strains with impaireddegradation. However, following whole genome sequencing we identified numer-ous secondary mutations in the stress response geneWHI2, which were responsible73for the impaired proteolysis of the misfolded model substrate. We linked this phe-notype to a deficiency of the Msn2/Msn4 transcription factor response that alteredthe cell’s capacity to adeptly degrade cytosolic misfolded proteins.3.2 Methods3.2.1 Yeast Strains, Media, and Growth ConditionsThe S. cerevisiae strains used in this study are listed in Table 3.1 and Table 3.2.Yeast strains were cultured in synthetic media with 2% dextrose (lacking the appro-priate amino acids for plasmid selection) or YPD (1% yeast extract, 2% peptone,2% dextrose) and grown at 25◦C with shaking unless indicated otherwise. Whennot specified otherwise, cultures in log phase were obtained by diluting overnightsaturated cultures grown at 25◦C to an OD600 = 0.2 and grown for 4–6 hours untillog phase OD600 = 0.8–1.0 was reached.Table 3.1: Yeast strains used in Chapter 3Strain ID Alias Genotype SourceYTM 408 BY4741 his3∆1, leu2∆0, ura3∆0, met15∆0OpenBiosystemsCollectionYTM 409 BY4742 his3∆1, leu2∆0, ura3∆0, lys2∆0OpenBiosystemsCollectionYTM 445 ssa1-45 his3∆11, leu2∆3, ura3∆52, trp1∆1 T. MayorYTM 639 rsp5-1 his3∆1, leu2∆, ura3∆0, met15∆0,RSP5::rsp5-1-KanMX T. MayorYTM 660 ydj1∆ his3∆1, leu2∆, ura3∆0, ydj1∆::KanMX T. MayorYTM 1867 asi1∆ Tetrad 3a his3∆1, leu2∆0, ura3∆0, MET15,LYS2, asi1∆::KanMX4, whi2-1 This thesisYTM 1868 asi1∆ Tetrad 3b his3∆1, leu2∆0, ura3∆0, met15∆0,lys2∆0, asi1∆::KanMX4, WHI2 This thesisYTM 1869 ASI1 Tetrad 3c his3∆1, leu2∆0, ura3∆0,MET15, LYS2, whi2-1 This thesisContinued on next page74Strain ID Alias Genotype SourceYTM 1870 ASI1 Tetrad 3d his3∆1, leu2∆0, ura3∆0,met15∆0, lys2∆0, WHI2 This thesisYTM 1857 ASI1 Tetrad 1a his3∆1, leu2∆0, ura3∆0, MET15,lys2∆0, whi2-1 This thesisYTM 1856 ASI1 Tetrad 4c his3∆1, leu2∆0, ura3∆0, MET15,lys2∆0, WHI2 This thesisYTM 1871 asi1∆ Tetrad 3a/ BY4741his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,whi2-1/WHI2This thesisYTM 1872 asi1∆ Tetrad 3a/ asi1∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0,met15∆0/MET15, LYS2/lys2∆0,asi1∆::KanMX4/asi1∆::KanMX4,whi2-1/whi2-1This thesisYTM 1873 asi1∆ Tetrad 3a/ das1∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,DAS1/das1∆::KanMX4, whi2-1/whi2-2This thesisYTM 1874 asi1∆ Tetrad 3a/ fap1∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,FAP1/fap1∆::KanMX4, whi2-1/whi2-3This thesisYTM 1875 asi1∆ Tetrad 3a/ hrt3∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,HRT3/hrt3∆::KanMX4, whi2-1/whi2-4This thesisYTM 1876 asi1∆ Tetrad 3a/ hul5∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,HUL5/hul5∆::KanMX4, whi2-1/whi2-5This thesisYTM 1877 asi1∆ Tetrad 3a/ ufd2∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,UFD2/ufd2∆::KanMX4, whi2-1/whi2-6This thesisContinued on next page75Strain ID Alias Genotype SourceYTM 1878 asi1∆ Tetrad 3a/ ufd4∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,UFD4/ufd4∆::KanMX4, whi2/whi2-7This thesisYTM 1879 asi1∆ Tetrad 3b/ BY4741his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,WHI2/WHI2This thesisYTM 1880 asi1∆ Tetrad 3b/ asi1∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0,met15∆0/MET15, LYS2/lys2∆0,asi1∆::KanMX4/asi1∆::KanMX4,WHI2/whi2-1This thesisYTM 1881 asi1∆ Tetrad 3b/ das1∆is3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,DAS1/das1∆::KanMX4, WHI2/whi2-2This thesisYTM 1882 asi1∆ Tetrad 3b/ fap1∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,FAP1/fap1∆::KanMX4, WHI2/whi2-3This thesisYTM 1883 asi1∆ Tetrad 3b/ hrt3∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,HRT3/hrt3∆::KanMX4, WHI2/whi2-4This thesisYTM 1884 asi1∆ Tetrad 3b/ hul5∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,HUL5/hul5∆::KanMX4, WHI2/whi2-5This thesisYTM 1885 asi1∆ Tetrad 3b/ ufd2∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,UFD2/ufd2∆::KanMX4, WHI2/whi2-6This thesisYTM 1886 asi1∆ Tetrad 3b/ ufd4∆his3∆1/his3∆1, leu2∆0/leu2∆0,ura3∆0/ura3∆0, met15∆0/MET15,LYS2/lys2∆0, asi1∆::KanMX4/ASI1,UFD4/ufd4∆::KanMX4, WHI2/whi2-7This thesisContinued on next page76Strain ID Alias Genotype SourceYTM 1744 msn2∆ his3∆1, leu2∆0, ura3∆0, met15∆0,msn2∆::KanMX4OpenBiosystemsCollectionYTM 1745 msn4∆ his3∆1, leu2∆0, ura3∆0, met15∆0,msn4∆::KanMX4OpenBiosystemsCollectionYTM 1691 whi2∆ his3∆1, leu2∆0, ura3∆0, met15∆0,whi2∆::KanMX4OpenBiosystemsCollectionYTM 1690 glo4∆ his3∆1, leu2∆0, ura3∆0, met15∆0,glo4∆::KanMX4OpenBiosystemsCollection3.2.2 PlasmidsPlasmids used in this study are listed in Table 3.3. Guk1-GFP (BPM453), Guk1-7-GFP (BPM458), and Guk1-7-His6 (BPM717) expressed from the GPD1 promoterin pRS313 were generated in a previous study [206]; Guk1-7-GFP was subclonedwith ApaI and SacI sites in pRS315 to generate BPM609 and with XhoI and SacIIsites in pRS316 to generate BPM781. To generate the E3 ligase addback plasmids(BPM748, ASI1; BPM749, DAS1; BPM750, FAP1; BPM751, HRT3; BPM752,HUL5; BPM753, UFD2; BPM754, UFD4), the open reading frames and approx-imately 500 bp of both endogenous 5’ and 3’UTR was PCR amplified from ge-nomic DNA (BY4741) and inserted in pRS316 using XhoI and XmaI sites for allbut HUL5 where SacII and XhoI sites were used. The WHI2 (BPM863, BPM914)addback plasmids were generated as for the E3 ligases except ligated into pRS315or pRS316 using SacI and XmaI sites, respectively.3.2.3 Flow CytometryCells in log phase were treated with 100 µg/mL cycloheximide and incubated ateither 25◦C or 37◦C as indicated. GFP fluorescence was measured for 50,000 cellsusing a FACSCalibur flow cytometer. Median GFP fluorescence values were ob-tained using FlowJo software. For chase experiments, percentage remaining values77Table 3.2: E3 ligase collection used for screeningSystematic Standard Well Systematic Standard WellName Name No. Name Name No.YMR258C ROY1 A1 YHL010C ETP1 D1YOL013C HRD1 A2 YLR368W MDM30 D2YOL054W PSH1 A3 YER116C SLX8 D3YML068W ITT1 A4 YLR352W n/a D4YDR049W VMS1 A5 YAL002W VPS8 D5YDR131C n/a A6 YDR360W TFB3 D6YDR143C SAN1 A7 YLR024C UBR2 D7YHR115C DMA1 A8 YMR119W ASI1 D8YKL010C UFD4 A9 YNL008C ASI3 D9YKL034W TUL1 A10 YGL003C CDH1 D10YJL149W DAS1 A11 YMR247C RKR1 D11YLR247C IRC20 A12 YNL023C FAP1 D12YNL230C ELA1 B1 YJL157C FAR1 E1YKR017C HEL1 B2 YDL013W SLX5 E2YDR265W PEX10 B3 YBR203W COS111 E3YDR306C n/a B4 YKL059C MPE1 E4YDR313C PIB1 B5 YER068W NOT4 E5YIL001W n/a B6 YMR026C PEX12 E6YCR066W RAD18 B7 YJL210W PEX2 E7YJR036C HUL4 B8 YOR191W ULS1 E8YDL190C UFD2 B9 YDR255C RMD5 E9YBR062C n/a B10 YGL131C SNT2 E10YNL116W DMA2 B11 YLR005W SSL1 E11YBR280C SAF1 B12 YDR103W STE5 E12YIL030C DOA10 C1 YDR266C HEL2 F1YBR114W RAD16 C2 YOL138C RTC1 F2YDR457W TOM1 C3 YBR158W AMN1 F3YGL141W HUL5 C4 YJR052W RAD7 F4YGR184C UBR1 C5 YDR132C n/a F5YDL074C BRE1 C6 YLR108C n/a F6YLR224W n/a C7 YMR080C NAM7 F7YLR097C HRT3 C8 YPL046C ELC1 F8YNL311C SKP2 C9 YJR090C GRR1 F9YDR219C MFB1 C10 YJL204C RCY1 F10YLR427W MAG2 C11YOR080W DIA2 C1278Table 3.3: Plasmids used in Chapter 3PlasmidID NameAuxotrophicMarkerPlasmidType SourceBPM 42 pRS316 Ura CEN/ARS RJD CollectionBPM 45 pRS313 His CEN/ARS RJD CollectionBPM 49 pRS315 Leu CEN/ARS RJD CollectionBPM 390 PYDJ1-YDJ1 Ura CEN/ARS E. CraigBPM 453 PGPD-Guk1-GFP His CEN/ARS T. MayorBPM 458 PGPD-Guk1-7-GFP His CEN/ARS T. MayorBPM 559 PSSA1-SSA1 Ura CEN/ARS T. MayorBPM 573 PRSP5-RSP5 Ura CEN/ARS T. MayorBPM 575 PRSP5-RSP5(C777A) Ura CEN/ARS T. MayorBPM 609 PGPD-Guk1-7-GFP Leu CEN/ARS T. MayorBPM 708 PCUP1-Deg1-GFP Ura CEN/ARS T. MayorBPM 718 PGPD-Guk1-7-GFP Ura CEN/ARS T. MayorBPM 748 PASI1-ASI1 Leu CEN/ARS This thesisBPM 749 PDAS1-DAS1 Leu CEN/ARS This thesisBPM 750 PFAP1-FAP1 Leu CEN/ARS This thesisBPM 751 PHRT3-HRT3 Leu CEN/ARS This thesisBPM 752 PHUL5-HUL5 Leu CEN/ARS This thesisBPM 753 PUFD2-UFD2 Leu CEN/ARS This thesisBPM 754 PUFD4-UFD4 Leu CEN/ARS This thesisBPM 914 PWHI2-WHI2 Ura CEN/ARS This thesis79were calculated by normalizing the median GFP fluorescence intensity values foreach time point to the initial t = 0 measurement. To calculate the relative loss of flu-orescence for single time-point measurements, the difference of GFP fluorescencevalues for samples incubated at 25◦C and 37◦C was normalized to that of the 25◦Csample. To perform multiple strain comparisons, the relative loss of fluorescencevalues (as calculated above) for each strain was normalized to that of the wild typeBY4741 strain.3.2.4 SequencingWhole-genome sequencing and library preparation was performed at the NextGenSequencing facility at the Biodiversity Research Centre of the University of BritishColumbia. Yeast cells were grown overnight to saturation in YPD at 25◦C andgenomic DNA was extracted using standard protocols [208]. Barcoded librariesfor each strain were created according to Illumina protocols (Illumina 2011, allrights reserved) and 100 bp paired end fragments were sequenced by pooling allsix libraries and run on a single lane of an Illumina HiSeq2000. The short-readaligner BWAwas used to map sequence reads to the yeast reference genome S288Cversion R64 (Saccharomyces Genome Database, SDG) [209]. Single-nucleotidevariants (SNVs) were identified using the SAMtools toolbox and then each SNVwas annotated with a custom-made Perl script using gene data downloaded fromSDG on January 21, 2014 [210]. IGV viewer was used to visually inspect readalignments in the regions of candidate SNVs [211, 212].3.2.5 WHI2 Plate AssayYeast cultures were grown overnight at 25◦C in 5 mL YPD to OD600 = 1–2 andthen diluted to OD600 = 0.2 in 5 mL YPD and left to grow for 2 hours at 25◦C.1 mL was kept as an untreated control and the remaining 4 mL of culture wastreated with 200 mM acetic acid for 4 hours at 25◦C. Treated and untreated cultureswere serially diluted fivefold in 1X PBS and plated on solid media. Plates wereincubated at 30◦C for 2 days before being imaged with a Gel Doc XR+ System(Bio-Rad).803.2.6 Turnover AssayCells transformed with a Deg1-GFP containing plasmid were grown to saturationovernight at 30◦C, diluted to OD600 = 0.2 and then incubated for 3 hours at 30◦C.Deg1-GFP expression was induced for 4 hours at 30◦C with 100 µM copper sul-phate and then 100 µg/mL cycloheximide was added with samples collected at theindicated time points. Cells were lysed with glass beads in lysis buffer (50 mMTris-HCl, pH 7.5, 1% Tx-100, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 1 mMPMSF, 1X protease inhibitor mix (Roche)). Protein concentrations were measuredusing the DC Protein Assay (Bio-Rad) and normalized prior to resolving equalvolumes by SDS-PAGE. Membranes were immunoblotted with mouse anti-GFP(Roche, 1:2,500) and rabbit anti-Pgk1 (Acris Antibodies, 1:10,000) primary anti-bodies and secondary antibodies (Mandel Scientific, 1:10,000). Membranes werescanned and analyzed with an Odyssey Infrared imaging system (LI-COR).3.2.7 Solubility AssayCells expressing Guk1-7-GFP in log phase were incubated at either 25◦C or 37◦Cfor 20 minutes. Cells were lysed with glass beads in native lysis buffer (20 mMHEPES, pH 7.5, 0.5% NP-40, 200 mM NaCl, 1X protease inhibitor mix (Roche),1 mM 1,10 phenanthroline, 1 mM EDTA) and centrifuged at 2,000 g for 5 minutesat 4◦C. Protein concentrations were determined using the DC Protein Assay (Bio-Rad) and normalized to 0.5 µg/µL. Samples were then fractionated into solubleand pellet fractions by centrifuging at 16,000 g for 10 minutes at 4◦C. The pelletfraction was washed twice with native lysis buffer prior to being resuspended in 1XSDS buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 3% glycerol). Equal volumes oftotal cell lysate, soluble, and pellet fractions were resolved by SDS-PAGE. Mem-branes were immunoblotted with mouse anti-GFP (Roche, 1:2,500) and secondaryantibodies (Mandel Scientific, 1:10,000).3.2.8 Guk1-7-GFP UbiquitinationCells expressing ectopic Guk1-7-GFP, Guk1-GFP, or a control empty vector(pRS313), were grown to log phase and then lysed with glass beads in nativelysis buffer (20 mM HEPES, pH 7.5, 0.5% NP-40, 200 mM NaCl, 1X protease81inhibitor mix (Roche), 1 mM 1,10 phenanthroline, 1 mM EDTA, 10 mM iodoac-etamide). GFP-tagged Guk1-7 was pulled down with GFP-Trap coupled agarosebeads (Chromotek; 10 µL per 3 mg of lysate) for 2 hours at 4◦C. Beads werewashed three times in lysis buffer before samples were eluted with 3X SDS buffer.Equal volumes of samples were resolved by SDS-PAGE. Membranes were im-munoblotted with mouse anti-GFP (Roche, 1:2,500), rabbit anti-Pgk1 (Acris Anti-bodies, 1:10,000), and mouse anti-ubiquitin (Millipore, 1:2,500) primary antibod-ies and secondary antibodies (Mandel Scientific, 1:10,000).3.2.9 Cellular Thermal Shift Assay (CETSA)Cells expressing Guk1-7-His6 were grown to log phase and then lysed with glassbeads in 200 µL native lysis buffer. The soluble fraction was collected by spinningat 16,000 g for 10 minutes at 4◦C on a benchtop centrifuge. Protein concentrationwas determined by the DC Protein Assay (Bio-Rad) and samples were normalizedto 2 µg/mL in native lysis buffer and 50 µL aliquots were distributed into PCR striptubes. Samples were heated using a CETSA PCR Program (25◦C, 3:00; 30–50◦Cgradient, 10:00; 25◦C, 1:00) on a thermocycler. The resulting soluble fraction wascollected by centrifugation at 16,000 g for 10 minutes at 4◦C and one third volumeof 3X SDS buffer was added to samples prior to resolving equal volumes by SDS-PAGE. Membranes were immunoblotted with a mouse anti-His primary antibody(Ablab, 1:2,500) and a secondary antibody (Mandel Scientific, 1:10,000).3.2.10 Statistical AnalysisData are presented as mean± SD unless otherwise stated. Comparisons were madeusing the two-tailed Student’s t-test and differences were considered significant at ap-value of< 0.05. When indicated, multiple strains were compared with a one-wayANOVA and post-hoc Tukey HSD to assess significance.823.3 Results3.3.1 Multiple Strains From the Yeast Knockout Collection DisplayImpaired ProteostasisTo monitor the stability of the Guk1-7 mutant by flow cytometry in yeast cells,we previously generated a C-terminal GFP fusion protein ectopically expressedfrom the constitutive GPD promoter [206]. As we reported, Guk1-7-GFP lev-els are ∼50% and ∼85% lower after incubating cells at 37◦C in the presence ofthe translation inhibitor cycloheximide for two and four hours, respectively (Fig-ure 3.1A). To identify another E3 ubiquitin ligase responsible for the degradation ofthe Guk1-7-GFP model substrate, we screened a collection of 70 non-essential E3ligase deletion strains that were individually transformed with a CEN/ARS plasmidencoding the Guk1-7-GFP fusion. Cultures were grown at 25◦C and then dividedand incubated in the presence of CHX for two hours at 25◦C and 37◦C beforeperforming flow cytometry analysis (Figure 3.1B). For each deletion strain, therelative difference in median GFP fluorescence intensities from samples incubatedat 25◦C and 37◦C was normalized to that of the wild type strain, to calculate a rel-ative loss of Guk1-7-GFP fluorescence (Figure 3.1B). The collection was screenedtwice and strains that had a relative loss of Guk1-7-GFP fluorescence value of 0.75or lower in at least one of the two rounds were selected for further validation. Atotal of 20 strains met this criterion and were further analysed by flow cytometryin three independent experiments (Figure 3.1C). In agreement with our previousfindings, deleting UBR1 led to a 25% lower averaged loss of Guk1-7-GFP fluores-cence compared to that of wild type cells [206]. Surprisingly, we identified twelveE3 ligase deletion strains with a greater impairment in Guk1-7-GFP degradationthan that observed in ubr1∆ cells. Of these, seven strains had an averaged rela-tive loss of Guk1-7-GFP fluorescence value of 0.5 or lower. These results indicatethat an unusually high number of strains from our yeast knockout collection havea reduced capacity to eliminate misfolded cytosolic proteins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igure 3.1: Flow cytometry based screen for E3 ligases targetingGuk1-7-GFP for degradation.84Figure 3.1: (Previous page) Flow cytometry based screen for E3 ligases tar-geting Guk1-7-GFP for degradation. a) CHX chase assay. Wild type cells ex-pressing ectopic Guk1-GFP or Guk1-7-GFP were incubated with CHX for 4hours at 25◦C or 37◦C and samples were collected at the indicated time points.Results represent the mean and standard deviation of three independent ex-periments. *** and ns denote p < 0.005 and not significant, respectively. b)E3 ligase screen. Seventy non-essential E3 ligase deletion strains express-ing Guk1-7-GFP were incubated with CHX at 25◦C or 37◦C for 2 hours andthen analyzed by flow cytometry. Red line demarks strains with a relativeloss of fluorescence value of 0.75 or lower. c) Triplicate validation. The top20 strains were selected for further validation by flow cytometry with exper-iments performed as in b. Data points in red correspond to strains displayingGuk1-7-GFP stabilization levels higher than that of ubr1∆. d) Cycloheximidechase assay. Wild type or asi1∆ cells expressing Guk1-7-GFP were incubatedwith CHX at 25◦C and 37◦C for two hours prior to flow cytometry analysis.Results represent the mean and standard deviation of three independent ex-periments. * and *** denote p < 0.05 and p < 0.005, respectively. e) Wildtype and asi1∆ cells co-expressing Guk1-7-GFP and an empty vector (EV)or ASI1 were treated as in d. ns and ** denote not significant and p < 0.01,respectively.3.3.2 A Secondary Mutation inWHI2 Co-Segregates with IncreasedGuk1-7-GFP StabilityWe confirmed the results of our screen by performing CHX chase experiments withcells lacking ASI1, a member of the nuclear inner membrane Asi ubiquitin ligasecomplex (Figure 3.1D) [213]. To determine whether the impaired turnover of themodel substrate was caused by the absence of ASI1, we co-expressed Guk1-7-GFPwith ASI1 under its endogenous promoter or with a control empty vector (EV). Ad-dition of the wild type ASI1 did not re-establish normal model substrate degradationlevels (Figure 3.1E). We obtained similar results with the six remaining E3 ligasedeletions that displayed stabilization values comparable with ASI1 (DAS1, FAP1,HRT3, HUL5, UFD2, and UFD4) (Figure 3.2 and Figure 3.3). This data impliedthat the decrease in Guk1-7-GFP degradation conferred by these strains could notbe attributed to the absence of the assessed E3 ubiquitin ligase, but rather to that of85another factor, such as a background mutation or potentially an epigenetic factor.Similarly, we previously isolated several hits in a genome wide screen for factorsinvolved in degradative protein quality control (e.g., YER071C and YJL141C) thatwe could not confirm after addback experiments expressing the deleted gene froma plasmid, indicating that this phenomenon was not limited to E3 ligase mutantcells [206].We next sought to determine whether the observed phenotype was caused by asingle background mutation. Therefore, we performed tetrad analysis on the hap-loid spores obtained from backcrossing the MATa asi1∆ strain to wild type MATal-pha BY4742 cells. No discernable difference in growth rate was seen across thedissected spores. We then expressed Guk1-7 in seven sets of tetrads for furtheranalysis. While both the KanMX deletion cassette, conferring kanamycin resis-tance, and the impaired degradation phenotype measured by flow cytometry seg-regated in the expected 2:2 ratio, the two did not appear to be linked, as is shownby a representative tetrad set (Figure 3.4A). We obtained similar data with das1∆cells (Figure 3.5). Data from CHX chase experiments confirmed that Guk1-7-GFPlevels were significantly higher in tetrad c (ASI1) compared to those in the parentalwild type and tetrad d strains after a two hour (p = 0.001 and p = 0.0023) and fourhour (p = 0.025 and p = 0.028) incubation at 37◦C (Figure 3.4B). These experi-ments indicate that the stabilization phenotype in the asi1∆ strain was likely due tobackground mutations at a single locus.86!"#$%&'()%*+,- !"#$%&'()%./,- 0)#)&"12%3&45"2%*+,- 0)#)&"12%3&45"2%./,-!"#$6)47)2&%18%"2"&"5!9":)%;<1=43>? * @?+?A??BBBBBBBB6)47)2&%18%"2"&"5!9":)%;<1=43>? * @?+?A?? %"&$BBBBBBBBBB9":)%;<1=43>? * @?+?A?? '()*6)47)2&%18%"2"&"5!23BBBBBB6)47)2&%18%"2"&"5!9":)%;<1=43>? * @?+?A?? '+,-BBBBBBBBB+%!.6)47)2&%18%"2"&"5!9":)%;<1=43>? * @?+?A??BBBBBBBBBB6)47)2&%18%"2"&"5!+%!/9":)%;<1=43>? * @?+?A??2323BBBBBB! "# $% &Figure 3.2: Guk1-7-GFP degradation in E3 ligase deletion strains. Cyclohex-imide chase assay. Wild type or the corresponding E3 ligase deletion strainexpressing ectopic Guk1-7-GFP were incubated in the presence of CHX ateither 25◦C or 37◦C for four hours and samples were analysed by flow cy-tometry at the indicated time points. Results represent the mean and standarddeviation of three independent experiments. P values were calculated witha two-tailed unpaired Student’s t-test (*, **, ***, and ns denote p < 0.05,0.01, 0.005, and not significant, respectively). a) das1∆, b) fap1∆, c) hrt3∆,d) hul5∆, e) ufd2∆, f) ufd4∆.87! "!"!!!"#!$"!!!"#$%&'(%&'$%&'(%&'$%&!"#$)*++++# $!"!!!"#!$"!!(")$%&'(*&+$%&'(*&+$%&(")$)*++++,-./%&'(012/%&'(012/%&,-./!"!!!"#!$"!!)*++++% &!"!!!"#!$"!!,345%&'(0675%&'(0675%&,345)*++++3(!8%&'(6*%8%&'(6*%8%&3(!8!"!!!"#!$"!!)*++++!"!!!"#!$"!!3(!9%&'(6*%9%&'(6*%9%&3(!9)*++++'%(!)*+%,(-..,-&,/012343/56,&(0-7%.#%8#%Figure 3.3: Guk1-7-GFP stability is not a direct effect of E3 ligase deletion.Wild type and E3 ligase deletion strains expressing Guk1-7-GFP along withan empty vector (EV) control or corresponding E3 gene under its endogenouspromoter and terminator were incubated with CHX at 25◦C and 37◦C for twohours prior to flow cytometry analysis. Results represent three independentexperiments. P values were calculated with a one-way ANOVA and post-hoc Tukey HSD to assess significance (** and ns denotes p < 0.01 and notsignificant, respectively). a) das1∆ b) fap1∆ c) hrt3∆ d) hul5∆ e) ufd2∆ f)ufd4∆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igure 3.4: Mutations inWHI2 segregate with the Guk1-7-GFP stability phe-notype.89Figure 3.4: (Previous page) Mutations in WHI2 segregate with theGuk1-7-GFP stability phenotype. a) Backcross and phenotypic segrega-tion. The MATa asi1∆ strain was backcrossed with the wild type MATalphaBY4742 to produce sets of tetrads. Growth was assessed by culturing cells onYPD and 2:2 KanMX deletion marker segregation was observed by spottingonto YPD+G418 plates. This segregation pattern was compared to tetrads ex-pressing Guk1-7-GFP and analyzed by flow cytometry following incubationwith CHX at 25◦C and 37◦C for 2 hours. Results represent three indepen-dent experiments and p values were calculated with a one-way ANOVA andpost-hoc Tukey HSD to assess significance, ** denotes p < 0.01. b) CHXchase assay. Tetrad c and d, produced from the asi1∆ backcross, expressingGuk1-7-GFP were incubated with CHX at 25◦C and 37◦C four hours. Sam-ples were analysed by flow cytometry at the indicated time points. The resultsrepresent the mean and standard deviation of three independent experiments.P values were calculated with a two-tailed unpaired Student’s t-test (*, **,and ns denote p < 0.05, 0.01, and not significant, respectively). c) Comple-mentation test. MATa E3 ligase deletion strains were mated with MATalphawild type BY4742 and asi1∆ cells from tetrad a and b. The resulting diploidsexpressing Guk1-7-GFP were incubated with CHX for two hours at 25◦C and37◦C and analysed by flow cytometry. d) Whole genome sequencing of wildtype, asi1∆, and four asi1∆ backcross tetrad strains revealed a single base pairdeletion in the coding sequence ofWHI2 co-segregates with the Guk1-7-GFPstability phenotype. Arrow head denotes nucleotide base deleted in the asi1∆strain and derivatives.Next, we performed a complementation test to determine whether the sec-ondary mutations responsible for the stability phenotype observed in the sevenE3 ligase deletion strains are in the same locus, or different loci. Heterozygousdiploids were produced by mating a wild type strain (BY4742) and each of theseven E3 ligase deletions to two haploids, derived from the asi1∆ backcross shown:one, contained the secondary mutation (tetrad a) and the other, did not (tetradb). Guk1-7-GFP levels were indistinguishable between heterozygous diploids pro-duced from mating tetrad a and BY4741 and diploids produced from crossingtetrad b with any of the E3 deletion mutants, or the wild type BY4741 (Fig-ure 3.4C). Conversely, all heterozygous diploids derived from mating E3 ligasedeletions with tetrad a (harbouring the secondary mutation) demonstrated increased90!!"#$%&'"(#)**()+(,-./010,23(+#-)4"*5"65" /788798!"#$ !"#$ %&'$ %&'$"8879/78()*+:(;<,-.+:(;<,-.+: /012== ==6*!"#$%&'"(#)**()+(,-./010,23(+#-)4"*5"65"Figure 3.5: das1∆ tetrad analysis and WHI2 addback. a) Analysis of onetetrad obtained from backcrossing the MATa das1∆ strain with the wild typeMATalpha BY4742. Tetrad spores expressing Guk1-7-GFP were incubatedwith CHX at 25◦C and 37◦C for two hours prior to flow cytometry analysis.b) Wild type and glo4∆ cells co-expressing Guk1-7-GFP and an empty con-trol vector (EV) orWHI2 were incubated with CHX at 25◦C and 37◦C for twohours and then analysed by flow cytometry. Results represent three indepen-dent experiments and p values were calculated with a one-way ANOVA andpost-hoc Tukey HSD to assess significance (** and ns denote p < 0.01 andnot significant, respectively).Guk1-7-GFP stability, thereby indicating that they belong to the same complemen-tation group, and suggests that the secondary mutations present in each strain arein the same gene.To identify the locus containing the secondary mutation, we performed whole-genome sequencing on four haploid tetrads and their parental wild type and asi1∆strains. Secondary mutations in the genes GLO4 and WHI2, which are approxi-mately 3000 bp apart on chromosome fifteen, co-segregated with the strains har-bouring the increased Guk1-7-GFP stability phenotype. Interestingly, secondarymutations in the general stress response geneWHI2 have been identified previouslyin yeast knockout collections and genome evolution studies [214–216]. We identi-fied a single nucleotide deletion in the coding sequence of WHI2. This mutation,hereinafter referred to as whi2-sc1, produces a frameshift introducing a prematurestop codon and likely results in a loss ofWHI2 function (Figure 3.4D). In contrast,the coding sequence of GLO4, a mitochondrial glyoxalase, contained a single mis-sense mutation.913.3.3 Guk1-7-GFP Degradation is Impaired Owing to SecondaryMutations inWHI2To determine whether the mutation in WHI2 caused the observed stabilization, weexpressed the wild type ORF from a plasmid in cells derived from the backcross.Whereas the addition of an empty vector did not rescue the phenotype, addition ofWHI2 re-established normal Guk1-7-GFP degradation levels (Figure 3.6A). More-over, we observed a similar impairment in the degradation of the Guk1-7-GFPmodel substrate in whi2∆ cells, that could be rescued by the expression of WHI2(Figure 3.6B). Intriguingly, we found that glo4∆ cells had a similar reduction inGuk1-7-GFP degradation (Figure 3.5B). Subsequent Sanger sequencing of a PCRproduct amplified from the WHI2 locus of glo4∆ cells identified two point muta-tions that produce a premature stop codon. These results suggest that the effectobserved in glo4∆ cells is attributed to a loss of WHI2 function, not of GLO4, andthat loss of WHI2 function is sufficient to strongly impair degradation of a mis-folded cytosolic model substrate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igure 3.6: Absence of WHI2 leads to Guk1-7-GFP stability.93Figure 3.6: (Previous page) Absence ofWHI2 leads to Guk1-7-GFP stability.a) ASI1 tetrads c and d co-expressing Guk1-7-GFP and a control empty vec-tor (EV) or WHI2 were incubated with CHX at 25◦C and 37◦C for two hoursand then analysed by flow cytometry. Results represent three independentexperiments and p values were calculated with a one-way ANOVA and post-hoc Tukey HSD to assess significance (ns and ** denote not significant andp< 0.01, respectively). b) Wild type and whi2∆ cells expressing Guk1-7-GFPalong with an empty vector (EV) control or WHI2 were treated and analysedas in a. c) WHI2 function assay. Diluted overnight cultures of wild type orE3 ligase deletion strains expressing either an empty control vector or WHI2were treated with 200 mM acetic acid for four hours prior to serial dilutionand spotting onto synthetic drop out plates. Images were taken after two daysof growth at 30◦C. d) Mutations in WHI2 were identified by Sanger sequenc-ing of a PCR amplicon spanning 100 bp up and downstream of the start andstop codons. For each strain, the mutations identified are as listed and, thepredicted protein length is depicted in red. Black boxes denote the C-terminalmismatch extensions.We next sought to confirm that WHI2 was also mutated in the other E3 lig-ase mutant strains in which Guk1-7-GFP degradation was impaired. Mutations inWHI2 sensitize cells to exposure to acetic acid, which lends itself to a convenientassay for Whi2 function [214]. The whi2∆ and all seven E3 ligase deletion strainswere sensitive to acetic acid treatment (Figure 3.6C). ExpressingWHI2 from a plas-mid under its endogenous promoter restored cell viability in all strains, confirmingdata from the complementation test suggesting that all strains contain secondarymutations in the same locus (Figure 3.4C). We proceeded to sequence the entireWHI2 gene, including approximately one hundred base pairs upstream and down-stream of the start and stop codons, in all twenty of the top E3 ligase deletionstrains from our screen. Whereas the ubr1∆ and six other strains had no apparentmutations, we identifiedWHI2 mutations in a total of eleven strains (Figure 3.6D).These consist of: dma1∆, elc1∆, etp1∆, cos111∆, and all seven strains that failedin addback experiments: asi1∆, das1∆, fap1∆, hrt3∆, hul5∆, ufd2∆, and ufd4∆.In two cases, pex2∆ and tom1∆, we were unable to obtain unambiguous sequenc-ing results after two independent genomic extractions and sequencing runs. Of theWHI2 mutations identified, nine are predicted to produce truncated proteins result-94ing from the introduction of a premature stop codon. Three of the nine also containadditional C-terminal extensions (ranging from 4 to 31 amino acids in length) asthe result of frameshift mutations. The mutations are relatively evenly dispersedalong the length of the protein with the exception of a mutation free region, sev-enty amino acids in length, found approximately three quarters of the way into theprotein. Whereas the different WHI2 mutations led to varying degrees of impairedGuk1-7-GFP degradation, we did not see a clear correlation between the severityof the Guk1-7-GFP stabilisation phenotype and the predicted Whi2 length in thesestrains (Figure 3.6D).3.3.4 Reduced Proteostasic Capacity inWHI2Mutants is Linked toMsn2Exposure to stressors such as heat, oxidative or osmotic shock, and nutrient star-vation results in the transcriptional activation of approximately 200 genes in yeast[146]. Activation of this general stress response is mediated by binding of thepartially-redundant zinc finger transcription factors Msn2 and Msn4 to STREs inthe promoters of stress-response genes [146, 217]. Under non-stress conditions,Msn2 is sequestered in the cytoplasm and upon exposure to stress, Msn2 translo-cates to the nucleus [218, 219]. To determine whether Guk1-7-GFP stability inWHI2 mutants is linked to reduced Msn2/Msn4 activity, we assessed Guk1-7-GFPlevels in single deletions. An absence of MSN2, but not MSN4, led to a significantincrease in Guk1-7-GFP compared to wild type (p = 0.003) with levels similar tothose seen in whi2∆ cells (Figure 3.7A). These data would therefore suggest thatdecreased Guk1-7-GFP degradation is associated to a general impairment of stressresponse factors acting downstream of Msn2.Intriguingly, while performing WHI2 addback experiments we noticed a pro-nounced decrease in Guk1-7-GFP stability when adding a second plasmid bearingthe auxotrophic marker leucine. To further investigate this observation, we trans-formed wild type and whi2-sc1 cells with a plasmid expressing Guk1-7-GFP thatcontained one of the following selection markers: histidine, uracil, or leucine. Con-sistent with our previous data, loss of Guk1-7-GFP fluorescence was 53% lower inwhi2-sc1 cells compared to wild type when the histidine marker was used (Fig-ure 3.7B). However, when whi2-sc1 cells containing the leucine selection marker95were grown in synthetic media without additional leucine, Guk1-7-GFP degrada-tion was mostly impaired and levels were approximately two fold higher. As well,uracil selection resulted in an intermediate phenotype. Leucine was previouslyshown to activate the TORC1 kinase complex that can also inhibit Msn2/4 [220–222]. One possibility is that Whi2 is only required to maintain Msn2 active whenTORC1 is stimulated in the presence of high levels of exogenous leucine. In sup-port of this view, the addition of increasing amounts of leucine restored the im-paired Guk1-7-GFP degradation in whi2-sc1 cells (Figure 3.7C). These data sug-gest that mutations in WHI2 only impair proteostasis in conditions where Whi2 isrequired to maintain Msn2 active.3.3.5 MutantWHI2 Impairs Guk1-7-GFP Degradation by ReducingSubstrate UbiquitinationTo determine how an absence of WHI2 results in increased Guk1-7-GFP stabi-lization we first needed to clarify what aspect of protein quality control is alteredin the mutants. We first compared the thermodynamic stability of ectopically ex-pressed Guk1-7 in cellular lysates by CETSA. Solubility decreased rapidly at tem-peratures above 42◦C in extracts from both wild type and whi2-sc1 strains (Fig-ure 3.8A). While not marked, slightly more Guk1-7 remained soluble in whi2-sc1lysates compared to wild type at 46.5◦C and 48.8◦C (p = 0.04 and p = 0.032). Bycontrast, Guk1-7 was slightly, but not significantly, less soluble in whi2-sc1 cellsgrown at 25◦C or following a short twenty-minute incubation at 37◦C (p = 0.18 andp = 0.07) (Figure 3.8B). Together these data suggest that Whi2 does not markedlyinfluence Guk1-7-GFP degradation by increasing its thermal stability or inducingits aggregation.It is possible that mutations in WHI2 might generally alter the ubiquitin pro-teasome system. However, using the known proteasome substrate Deg1-GFP, wefound no significant difference in degradation in whi2-sc1 cells compared to wildtype (p = 0.31, 0.4, 0.7, 0.52 for 10, 20, 30, and 60 minute time points, respec-tively) (Figure 3.8C). We next asked whether an absence of WHI2 could affectGuk1-7-GFP ubiquitination. Ubiquitin levels were measured following pulldownof Guk1-GFP and Guk1-7-GFP from cultures grown at 25◦C. Normalizing theubiquitin signal to the amount of GFP tagged substrate eluted revealed approxi-96mately 30% less ubiquitinated Guk1-7-GFP in whi2-sc1 cells compared to wildtype (Figure 3.8D). Together these data suggest that mutated WHI2 could impairGuk1-7-GFP degradation by decreasing substrate ubiquitination.!"#!!"#$"!%& !"#$ %&'$ %&'('()*+,-(.)/00./1.234$5652781)3/9(0:((;:(;0<<<'()*+,-(.)/00./1234$565278.1)3/9(0:(;:( $"!!"#!!. !"!#. !"$. $=>>,+,/;*).)(3:,;(.*>>(>.?@ABCDE)*+$ !"#$,&-.'()*+,-(.)/00./1234$565278.1)3/9(0:(;:( $"!!"#!F,0+,>,;( D(3:,;(G9*:,))*+$ !"#$,&-.=3H/+9/IJ,:.K*94(9Figure 3.7: Msn2 is linked to reduced proteostatic capacity inWHI2mutants.a) Wild type, whi2∆, msn2∆, and msn4∆ cells expressing Guk1-7-GFP wereanalysed by flow cytometry following a two hour incubation at 25◦C and 37◦Cin the presence of CHX. P values were calculated with a one-way ANOVA andpost-hoc Tukey HSD to assess significance (*, **, and ns denote p < 0.05,0.01, and not significant, respectively). b) Guk1-7-GFP was expressed fromCEN/ARS plasmids with histidine, uracil, or leucine auxotrophic markers inwild type or whi2-sc1 cells. Cultures were incubated with CHX for two hoursat 25◦C or 37◦C before being analysed by flow cytometry. c) Wild type andwhi2-sc1 cells were co-transformed with Guk1-7-GFP and pRS315 (LEU2).Cultures were grown in synthetic drop out media containing different amountsof leucine and then incubated for two hours in the presence of CHX at 25◦Cand 37◦C before flow cytometry analysis.97! "! " # "$%&'%()*+,-.+,)*+,-.+,!"#$%&'$()*+./0123240.54.0623240657-0/232.0/5-*0)232.0*5!(89(:;<&:(2=+,>!"#$%&'$()*+-/0/ -?0) --06 -.07 4)0* 470* 4606#(:@(A<2$B2CAC<C;% ?//*//-- -6 4- 46!(89(:;<&:(2=+,>!"#$%&'$()*+!C8(2=8CA0>D(E?FGH##EI?/ ?/ )/ -/ 7/ / ?/ )/ -/ 7/!"#$ %&'$()*+#(:@(A<2$B2CAC<C;% ?//*/// )/ 4/ 7/!C8(2=8CA0># $)*/?*/?//.**/-.CA9&< GH##EI?J#K2GH#LK2GH#J#K2GH#LK2M'N:2=ID;> %&'$()*+!"#$G&I?F.FGH#G&I?FGH#O89<P2Q(@<$:RRFigure 3.8: whi2∆ promotes Guk1-7-GFP stability through reduced ubiqui-tination. a) Cellular thermal shift assay of Guk1-7 fused to a six histidinetag in lysates of WHI2 and whi2-sc1 cells grown at 25◦C. One representativeanti-His western blot is shown. The graph represents the means and standarddeviations of Guk1-7 levels from three independent experiments. b) WHI2and whi2-sc1 cells expressing Guk1-7-GFP were grown at 25◦C or shifted to37◦C for 20 min. Total cell lysate (T), soluble (S), and pellet fractions (P)were immunoblotted with an anti-GFP antibody. One representative blot isshown and the ratio of soluble fraction to total cell lysate is noted and rep-resents the mean and standard deviation of three independent experiments.c) Proteasome degradation assay. WHI2 and whi2-sc1 cells expressing Deg1-GFP under the Cup1 promoter were incubated with CHX at 30◦C and sampleswere collected at the indicated time points. Membranes were immunoblottedwith an anti-GFP antibody and an anti-Pgk1 antibody as a loading control.The graph represents the mean and standard deviation of three independentexperiments. d) Guk1-GFP or Guk1-7-GFP were immunoprecipitated withGFP-Trap beads from lysates of WHI2 or whi2-sc1 cells grown at 25◦C andexpressing a control empty vector, Guk1-GFP, or Guk1-7-GFP. Samples wereeluted and immunoblotted with anti-ubiquitin, anti-GFP, and anti-Pgk1 anti-bodies.983.3.6 Essential E3 Ligase Rsp5 and Molecular Chaperones Ydj1 andSsa1 are Required for Guk1-7-GFP DegradationConsidering that the majority of E3 ligase mutants we tested had negligible effectson Guk1-7-GFP degradation, we wanted to know what other quality control fac-tors might play a role in addition to Ubr1. One limitation of the targeted screenis that it was restricted to non-essential genes encoding for known or putative E3ligases. The essential E3 ligase Rsp5 is required for the increase in ubiquitinationobserved following acute heat stress and confers increased thermotolerance whenoverexpressed [52, 223]. Thus, we next sought to determine whether Rsp5 couldalso play a role in the degradation of cytosolic proteins misfolded due to missensemutation in mild heat shock conditions. We performed cycloheximide chase as-says to test whether the temperature sensitive mutant allele rsp5-1 had an effecton Guk1-7-GFP stability. Guk1-7-GFP levels were significantly higher in rsp5-1cells compared to wild type cells after two and four hours at 37◦C (p = 0.002 andp = 0.001, respectively) (Figure 3.9A). To confirm that this enhanced stabilizationwas the direct consequence of a loss of RSP5 function, we performed addback ex-periments whereby wild type RSP5, or a catalytically inactive form (C777A), wasexpressed from a plasmid in rsp5-1 cells. Following a four hour incubation at 37◦C,Guk1-7-GFP levels in rsp5-1 cells expressing Rsp5 were similar to wild type cellscontaining a control empty plasmid. Likewise, rsp5-1 cells containing an emptyvector control or expressing the catalytically inactive Rsp5 (C777A) mutant hadnearly equivalent Guk1-7-GFP levels that were significantly higher than those ob-served in the wild type strain (p = 0.003 and p = 0.006, respectively) (Figure 3.9B).These data confirm that the reduced turnover in rsp5-1 cells could be directly at-tributed to the absence of Rsp5 and indicate that Rsp5 has a role in promoting thedegradation of Guk1-7-GFP. It is also possible that the role of Rsp5 is indirect asRsp5 is required for Msn2/4 and Hsf1 mRNA export or mRNA processing duringsome stress conditions [224, 225].We next assessed whether Ydj1, which is an Hsp40 chaperone known to asso-ciate with Rsp5 to ubiquitinate misfolded proteins following heat shock, may alsoparticipate in the turnover of the model substrate [52]. An absence of YDJ1 re-sulted in Guk1-7-GFP levels 42% higher than wild type (p = 0.001) (Figure 3.9C).An addback experiment expressing YDJ1 from a plasmid in ydj1∆ cells resulted99in Guk1-7-GFP levels comparable to those in the wild type strain. Strikingly, theimpairment of the model substrate turnover was more pronounced in ydj1∆ than inrsp5-1. Therefore, an absence of YDJ1 is likely causing a broader impact than justimpairing Rsp5 function. We next asked whether Ssa1, a member of the Hsp70family of molecular chaperones shown to be required for the degradation of modelcytosolic proteins, was also required for Guk1-7-GFP degradation [203]. Loss ofGuk1-7-GFP fluorescence was strikingly low in ssa1-45 cells, a temperature sensi-tive mutant of SSA1, compared to that observed in wild type cells after a two hourincubation at 37◦C (p = 0.001) (Figure 3.9D). ssa1-45 cells expressing a wild typecopy of SSA1 had Guk1-7-GFP levels similar to those of wild type cells suggestingthat Ssa1 has a role in promoting Guk1-7-GFP degradation. Together, these datasuggest that Hsp70 and Hsp40 chaperones play a part in promoting Guk1-7-GFPdegradation.3.4 DiscussionTemperature sensitive alleles have proven to be fruitful model substrates used toelucidate the existence and function of protein quality control pathways. In thisstudy, we set out to identify the E3 ubiquitin ligase, or ligases, responsible for thetemperature dependent degradation of the thermally unstable model protein qual-ity control substrate Guk1-7. Screening a yeast deletion collection using a flowcytometry based approach resulted in the identification of a number of putative E3ligase hits. Further validation, however, suggested that the phenotype observedwas produced by an indirect effect. Subsequent complementation analysis andwhole genome sequencing revealed secondary mutations in the general stress re-sponse factor encoded by the WHI2 gene that account for the observed increasein Guk1-7-GFP stability. WHI2 promotes Guk1-7-GFP degradation via substrateubiquitination with no effect on solubility or thermal stability.We previously identified the E3 ubiquitin ligase Ubr1 from a genetic screen forfactors involved in degradative protein quality control of Guk1-7 [206]. Ubr1 ac-tivity alone, however, was not sufficient to account for the bulk of substrate degra-dation. In some cases, the nuclear E3 ligase San1 and cytosolic ligase Ubr1 havebeen shown to act in parallel to degrade cytosolic substrates [74, 101]. We found100! "# $!"#$"%&'()'*%*&*+,-*."'/0(1#234 5 6474844!"#$'579:!"#$';<9:%&'$()'579:%&'$()';<9:===%2======== ===>",+&*?"',(22'()'@1A8B<B@C!'),1(#"2$"%$"48D44D7""*)E'FG&&+)(,$E'FG&&+)(,$E'""*)=== ==>",+&*?"',(22'()'@1A8B<B@C!'),1(#"2$"%$"8D44D74-./)E'FG012)E'FG012)E'-./)==== %2!"#$"%&'()'*%*&*+,-*."'/0(1#234 5 6474844!"#$'579:'E'FG!"#$';<9:'E'FG%&'$()'579:'E'FG%&'$()';<9:'E'FG%&'$()'579:'E'!"#$%&'$()';<9:'E'!"#$%&'$()'579:'E'!"#$'/:<<<H3'%&'$()';<9:'E'!"#$'/:<<<H3==== ==Figure 3.9: A role for essential E3 ligases and molecular chaperones inGuk1-7-GFP degradation. a) RSP5 and rsp5-1 cells expressing Guk1-7-GFPwere incubated with CHX for a total of four hours with samples collectedat the indicated time points. P values were calculated using a two-tailed un-paired Student’s t-test (*, **, ***, ns denotes p < 0.05, 0.01, 0.005, andnot significant, respectively). b) RSP5 cells expressing Guk1-7-GFP and acontrol empty vector as well as rsp5-1 cells expressing Guk1-7-GFP and ei-ther a control empty vector, RSP5, or RSP5 (C777A) were incubated withCHX and grown for four hours at 25◦C and 37◦C. Samples were collected atthe indicated time points and analysed by flow cytometry. c) ydj1∆ cells co-expressing Guk1-7-GFP and an empty vector control or YDJ1 were incubatedat 37◦C with CHX for two hours before being analysed by flow cytometry.P values were calculated with a one-way ANOVA and post-hoc Tukey HSDto assess significance (** and ns denote p < 0.01 and not significant, respec-tively). d) SSA1 and ssa1-45 cells expressing Guk1-7-GFP were analysed byflow cytometry after being incubated with CHX at 25◦C and 37◦C for twohours. P values were calculated with a two-tailed unpaired Student’s t-test(*** denotes p < 0.005).101no role for San1, and no additive effect with the double ubr1∆ san1∆ mutant inthe degradation of the Guk1-7 substrate. Therefore, in this study we wanted toidentify the E3 ubiquitin ligases, in addition to Ubr1, that are responsible for theproteasomal degradation of Guk1-7. We identified a number of potential hits inour screen which included: ASI1, DAS1, FAP1, HRT3, HUL5, UFD2, and UFD4.Asi1 is a RING domain family member localized to the inner nuclear membraneand is part of the Asi complex that acts as a branch of the ERAD degradation path-way independent from Hrd3 and Doa10 [80, 226]. Both Das1 and Hrt3 are putativeF-box SCF ubiquitin ligases [227, 228]. A homologue of the human transcriptionfactor NF-X1, Fap1 also confers resistance to rapamycin by acting as a ligand forFKBP12 [229]. Hul5 is a member of the HECT ubiquitin ligase family. Involved incytoplasmic protein quality control of short-lived misfolded proteins, Hul5 is alsonecessary for the increased ubiquitination observed as part of the heat shock qualitycontrol response [109]. Ufd2 is both an E3 and E4 enzyme with mutants being hy-persensitive to protein misfolding stressors [230]. Finally, Ufd4, like Hul5, is alsoa member of the HECT family of E3 ligases and physically interacts with Ubr1 toincrease processivity of ubiquitin chain formation in the N-end rule pathway [231].Guk1 is found in both the cytoplasmic and nuclear compartments of yeast cells asassessed by GFP tagging and fluorescence microscopy [154, 206]. It was, there-fore, not entirely unexpected for some hits to be nuclear proteins. However, basedon the ascribed functions for some of the ligases, and the nature of our substrate,it was puzzling to have identified DAS1, FAP1, and HRT3 in our screen. While anabsence of seven genes resulted in a significant stabilization our model substrate,we were surprised to have identified so many of them in our screen. Accordingly,the rescue experiments we performed indicated that deletions of these E3 ligaseswere not the cause of the observed phenotype (Guk1-7-GFP stabilization), whichwas likely caused by another background mutation.We found compelling evidence that the impairment of the degradation of themisfolded reporter was caused by mutations in the background of the assessedstrains that belong to the same complementation group (Figure 3.4C). Indeed, weidentified secondary mutations in the coding sequence of the general stress re-sponse gene WHI2 in eleven of twenty E3 ligase deletion strains tested (∼15% ofthe E3 mutant stains we assessed) (Figure 3.6D). Mutations in WHI2 have been102reported in laboratory based evolution studies and are speculated to provide a fa-vorable fitness advantage under certain environments or in combination with othercompensatory mutations [215, 232]. However, we did not observe striking differ-ences when comparing growth of haploid cells following tetrad analysis. To ourknowledge, ours is the first report of WHI2 mutations among E3 ubiquitin lig-ase deletion strains. Nevertheless, the presence of WHI2 mutations is unlikelyrestricted to E3 ligase mutant cells. Accordingly, three other studies have reportedthe presence of secondary mutations in the WHI2 locus of strains from the yeastdeletion collection [216, 233, 234]. In the most recent study, approximately ∼30%of all strains sequenced carried unique mutations in WHI2 and/or five other genes.Most of the mutations identified were frameshift or nonsense mutations suggestinga loss of function phenotype. Finally, it was found that serially passaging whi2∆strains under conditions with a prolonged stationary phase resulted in an increasedabundance of the deletion strain relative to a wild type control. This suggests thatsecondary mutations in these genes might be found at higher frequencies as theresult of selecting for mutants that delay the onset of the stationary phase underlaboratory growth conditions.As part of the general stress response, Whi2 forms a complex with the plasmamembrane phosphatase Psr1 and zinc finger transcription factor Msn2 [235]. Uponexposure to stress, Msn2 translocates to the nucleus where it can bind to STRE inthe promoters of stress responsive genes [147]. We found that like WHI2, an ab-sence ofMSN2 led to reduced turnover of Guk1-7-GFP. This finding suggests thatthe increase in Guk1-7-GFP stability observed inWHI2 mutants is likely mediatedby genes regulated by Msn2. Studying the general stress response, mediated byMsn2/4 signalling, has gained renewed importance in light of a recent study ex-amining the role of Hsf1 in the transcriptional response to heat shock [146]. Theauthors of that study concluded that in yeast, the majority of genes induced by heatshock are activated through Msn2/4 activity and not by Hsf1, as was previouslybelieved [146].While performing WHI2 addback experiments, we observed that Guk1-7-GFPstability depended upon adequate levels of leucine being present in the media.Amino acids, especially leucine, have been shown to regulate the TOR signalingpathway [236]. Target of rapamycin (TOR) is a conserved serine threonine protein103kinase which as part of the TORC1 complex promotes growth by linking proteinsynthesis to extrinsic signals such as nutrient levels and environmental stresses.Treating cells with the antifungal rapamycin mimics nutrient starvation and stress,thereby inhibiting TORC1 and allowing a number of stress and growth responsetranscription factors (such as Msn2) to translocate into the nucleus [237]. We pro-pose a model in whichWhi2 is required to maintain Msn2 functional when TORC1is active (e.g., in the presence of high concentrations of leucine in the media). Un-der these conditions, mutations inWHI2 would result in Msn2 remaining phospho-rylated and an inhibition of the downstream induction of stress responsive genes,thereby preventing degradation of cytosolic misfolded proteins such as Guk1-7.In cases where leucine levels are low (e.g., in LEU2 cells deprived of exogenousleucine) an absence or decrease in TORC1 activity allows Msn2 to remain activein a WHI2 independent manner. In these conditions, Guk1-7-GFP is degradedwith similar dynamics as in wild type cells. Our findings of a link between thegeneral stress response and the degradation of our model substrate might help ex-plain some puzzling results we obtained from a previous screen we conducted withGuk1-7-GFP (see Chapter 2) [206]. In that screen, a number of hits were for dele-tions in YAK1 and RIM15, but we were unable to validate the data after restreakingthe deletion strains from the knockout collection. Perhaps these strains are morelikely to accumulate WHI2 mutations for compensatory reasons, which leads to areduction in Guk1-7 degradation. The kinases Yak1 and Rim15 translocate intothe nucleus from the cytoplasm when TORC1 is inhibited and have been shown todirectly phosphorylate Msn2 in vitro, leading to the induction of Msn2 dependentgenes [238, 239]. There is one striking point that remains unanswered; Msn2 isthought to be mostly active after a stress to induce expression of stress responsegenes. In our conditions, translation of newly expressed genes would be preventedby cycloheximide. Therefore, the impairment of the turnover of our model sub-strate is either the result of an imbalance of the proteostatic network prior to thestress (i.e., Msn2 basal activity is also required in unstressed conditions), or stressinduced mRNA are also directly required for the proper triage and degradation ofmisfolded proteins (e.g., by mediating the formation of stress granules).Given that the majority of non-essential E3 ligases tested had negligible effectson Guk1-7-GFP degradation, we then asked whether the essential E3 ligase Rps5104or Hsp40/70 chaperones might play a role in this process. Rsp5, an E3 ligase ofthe NEDD4 (neural precursor cell expressed, developmentally downregulated 4)family ubiquitinates cytosolic misfolded proteins following heat shock [52]. Wefound that an absence of Rsp5 resulted in Guk1-7-GFP stabilization similar to thatseen in ubr1∆ cells. One possibility is that Rsp5 directly ubiquitinates misfoldedGuk1-7-GFP. Interestingly, WHI2 was identified as a multicopy suppressor of atemperature sensitive allele of RSP5 and rescues the general stress response phe-notype of Rsp5 mutants [235]. Moreover, Rsp5 is required for the nuclear export ofMsn2/4 mRNA under stress conditions [225]. Therefore, another possibility is thatthe observed impaired turnover of Guk1-7-GFP in rsp5-1 is indirect and causedby reduced Msn2 activity. Hsp70 and its Hsp40 co-chaperones have previouslybeen implicated in degradative protein quality control [48, 74, 167, 240]. Here weprovide yet another example of a misfolded cytoplasmic protein whose proteaso-mal degradation is mediated by Ssa1 and Ydj1. It will be important to determinewhether the reduced turnover of misfolded proteins in WHI2 and MSN2 defectivecells is also mediated by reduce levels of Ydj1 and Ssa1. While in a number ofcases Ssa1 and Ydj1 have been shown to target substrates to the nuclear E3 ligaseSan1, we found no role for San1 in the degradation of the Guk1-7 substrate [48].Together our data suggests that a number of cytosolic E3 ubiquitin ligases andmolecular chaperones potentially work in parallel to target misfolded substratesfor degradation. While the need for multiple E3 ligases has been reported be-fore, besides substrates targeted by Ubr1 and San1, few such examples have beenreported [100, 167]. Future studies will be needed to address how a variety of mis-folded substrates, bound by the same Ssa1 and Ydj1 chaperones, are specificallyrecognized and ubiquitinated by one or more E3 ligases.105Chapter 4Conclusion4.1 Chapter SummariesIn Chapter 2 we established novel model substrates to further characterize howcytosolic misfolded proteins are targeted for degradation. We first focused on amutant allele of the yeast guanylate kinase Guk1 and demonstrated that the mutantprotein displays temperature dependent stability and has decreased NP-40 solubil-ity compared to the wild type protein. We employed a GFP fusion approach thatenabled us to combine observations based on microscopy, biochemical, and flowcytometry methods. At the elevated temperature of 37◦C Guk1-7-GFP formedCytoQ-like inclusions that co-localized with the general aggregate marker Hsp104and the CytoQ specific marker Hsp42. In addition, whereas soluble Guk1-7-GFPwas ubiquitinated, the wild type Guk1 was not. We developed a flow cytome-try assay to monitor protein stability and then performed a flow cytometry basedscreen to isolate factors that promote Guk1-7 proteasomal degradation. We iden-tified the E3 ubiquitin ligase Ubr1 and the prefoldin chaperone subunit Gim3. Wefurther characterized how the absence of GIM3 influenced Guk1-7-GFP stability.While an absence of GIM3 did not impair proteasomal function or the ubiquiti-nation of Guk1-7-GFP, it led to delayed degradation and the accumulation of themodel substrate in cellular inclusions. Interestingly, while Gim3 interacted withGuk1-7, no interaction was found with the wild type Guk1 protein. This suggeststhat the interaction occurs as the result of protein misfolding and that Gim3 is not106required for Guk1 to attain its native conformation. Prefoldin is known to deliverproteins to the chaperonin complex for folding. We showed that Guk1-7-GFP alsoformed aggregates in temperature sensitive mutant strains of two essential chaper-onin subunits suggesting a possible role for the chaperonin complex in maintainingsubstrate solubility. Finally, we demonstrated that in addition to Guk1-7, prefoldincan also stabilize other misfolded cytosolic proteins containing missense muta-tions. By identifying a role for Gim3 to maintain solubility of mutant proteins, ourwork adds to a growing body of evidence suggesting that prefoldin is importantfor preventing potentially toxic protein aggregation and underscores its potentialimportance in maintaining protein homeostasis. As Gim3 is dispensable for thefolding of the wild type Guk1, our work also illustrates how complex the rela-tionship between chaperones and their client proteins is, and that it is an adaptiveprocess.In Chapter 3 we followed on from work in Chapter 2 and performed a secondtargeted flow cytometry based genetic screen to identify the E3 ubiquitin ligase,or ligases, responsible for the proteasomal degradation of the thermally unstablemodel protein quality control substrate, Guk1-7. Attempts to validate a numberof putative E3 ligase hits pointed to Guk1-7-GFP stability being the result of anindirect effect. Whole genome sequencing revealed secondary mutations in thegeneral stress response gene WHI2 in a number of hits obtained from the screen.We then demonstrated that an absence of WHI2 was responsible for the observedimpairment in the proteolytic degradation of Guk1-7. We propose a link betweenmutations in WHI2 to a deficiency in the Msn2/4 transcriptional response therebyaltering the cells capacity to degrade misfolded cytosolic proteins.4.2 General Discussion4.2.1 Using Temperature Sensitive Alleles as Model Protein QualityControl SubstratesTemperature sensitive alleles have proven to be fruitful model substrates used toelucidate the existence and function of protein quality control pathways. Our labpreviously identified a panel of temperature sensitive alleles of essential genes en-107coding for cytosolic proteins in Saccharomyces cerevisiae [101]. A large fractionof mutant proteins underwent proteasome-mediated degradation when incubatedat an elevated temperature of 37◦C, whereas the wild type proteins were stable.Approximately one third of the unstable alleles were found to be substrates of theE3 ubiquitin ligase Ubr1. Using four nuclear temperature sensitive mutant proteins(encoded by the cdc13-1, cdc68-1, sir3-8, and sir4-9 alleles), Gardner and col-leagues identified the nuclear protein quality control E3 ligase San1 [70]. As is thecase with Ubr1, the wild type proteins were stable and not targeted for degradationby San1. However, our approach remains distinctive, as different model substrateshave been employed to characterize cytosolic quality control.The Guk1-7 allele used in this thesis was produced by mutagenesis of the wildtype sequence by error prone PCR [186]. Temperature sensitive mutations are of-ten missense mutations that preserve the function of the essential protein at normalgrowth temperatures (permissive) but become non-functional at higher tempera-tures (non-permissive). As missense mutations represent more than half of all mu-tations in the HGMD we anticipate that understanding the protein quality controlpathways that recognize and triage proteins misfolded as the result of missensemutations will gain in importance [158]. Moreover, because most proteins are onlymarginally stable, it is predicted that most amino acid substitutions are not neutralwith respect to protein stability and approximately 70% of rare human missensealleles are predicted to be mildly deleterious [241, 242]. These predictions areunderscored by a recent study that found of the human disease associated mis-sense alleles that were tested, approximately 30% displayed increased binding tospecific components of the protein homeostasis network. The majority of alleles,however, resulted in disrupted protein-protein interactions [159]. The human mu-tation ORFeome created as part of this study contains 2,890 human mutant ORFsfrom 1,140 genes and is the most extensive human mutation collection created todate. It should be noted that while mutant ORFs in the above collection typicallycontain a single nucleotide change, the temperature sensitive alleles used in thisthesis contain a minimum of four nucleotide changes resulting in at least two non-silent amino acid changes per protein, with the exception of the Guk1-11 mutant.Whether proteins destabilized by multiple mutations are recognized and handled bythe protein quality control network differently to proteins containing a single mu-108tation remains to be tested, and should be taken into consideration while designingexperiments for projects with potential medical or therapeutic applications.4.2.2 Flow Cytometry: An Ideal Method for Identifying andCharacterizing Protein Quality Control FactorsProtein degradation is generally assayed by pulse-chase metabolic labeling, or byusing protein synthesis inhibitors coupled with downstream biochemical analy-sis [201]. Prior to starting the work described in this thesis, fluorescently taggedproteins had already been used to monitor protein stability by flow cytometry. Hecket al. used a genomically integrated CPY‡-GFP substrate to screen a collection ofdeletion strains of ubiquitin pathway genes to identify the parallel requirement forUbr1 and San1 E3 ligases in the degradation of some misfolded cytoplasmic sub-strates [74]. Years prior, a GFP-tagged Hmg2g had been used to study degradationin the ER [243].As mentioned earlier in this thesis, flow cytometry has a number of advantagescompared to stability assays as measurements are performed in vivo, thousandsof cells can be analysed in under an hour, and for most purposes no additionalprocessing or cell lysis is required. Perhaps most importantly, in addition to therelative speed and precision flow cytometry provides over Western blotting meth-ods, the assay we have described is sensitive enough to discern partial effects. Forinstance, we found ubr1∆ leads to minor but statistically significant stabilization ofGuk1-7-GFP (15% more protein after a two hour incubation at 37◦C in compari-son to wild type cells) that was observed in a consistent manner in multiple experi-ments. Using Western blots, we had previously missed that Ubr1 plays a role in theturnover of the Guk1-7 mutant protein [101]. Another advantage of the GFP-basedapproach is that it can be used as a screening tool that does not rely on a func-tional model substrate. As mentioned earlier, Ura3 fusion proteins have been usedto study Ubr1 and cytoplasmic protein quality control pathways [98, 167]. Theseassays rely on a functional Ura3 protein as they are conducted in auxotrophic yeaststrains that do not produce uracil. Were our studies to have been based on a func-tional Guk1 protein, we would have missed Gim3 in our screen. Because our novelmodel substrates do not need to meet a minimum functional threshold required forcell viability, our assay is far more sensitive and is amenable to detecting protein109quality control components that would be missed by traditional methods. A po-tential issue arising from the use of GFP fusions for protein degradation studiesis that GFP itself might influence stability of the fusion protein. This has beenreported previously for the yeast tandem affinity purification (TAP) tag collection.A comparison of protein half-lives between tagged and untagged versions of thesame protein showed that the TAP tag versions were degraded more rapidly [244].While we did not perform CHX chase assays comparing GFP to His tag versions ofGuk1-7, we did compare the solubility differences between these tags. We foundthat Guk1-7 was much less soluble with the smaller six histidine tag, potentiallyproviding a better reflection of the true solubility of this substrate when untagged.This data suggests that at the very least, GFP influences the solubility of Guk1-7,but we did not confirm whether degradation rates are also influenced. Despite thepotential drawbacks of relying on a GFP fusion protein, we believe that the workpresented in this thesis demonstrates the benefits of using a flow cytometry basedapproach to studying protein homeostasis networks and identifying protein qualitycontrol components.4.2.3 Triage Decisions: Simply a Matter of Kinetic Partitioning?One of the major unanswered questions in the protein quality control field is whatare the mechanisms involved in the changeover from chaperone assisted refoldingto targeted degradation of terminally misfolded proteins? One possibility is thatkinetic partitioning could dictate the order of sequential events, in which the E3ligase and associated cofactors would have lower Kon compared to components ofthe folding machinery. In this scenario, efficient refolding would occur when therate constant of folding (Kfold) is faster than that of chaperone rebinding to the fold-ing intermediate (Kon). In this thesis we have demonstrated that an absence of theprefoldin chaperone subunit Gim3 leads to the aggregation of our model misfoldedsubstrate and is accompanied by delayed degradation. This would suggest thatprefoldin chaperones, which lack ATP dependent chaperone activity, are importantfor enabling and/or maintaining substrate solubility such that substrates may be tar-geted for degradation by the ubiquitin proteasome machinery instead of enteringreiterative cycles of chaperone binding. Another strategy in protein quality control110might be that certain E3 ligases only recognize and bind to substrates when theyare in a substrate-chaperone complex and, therefore, an absence of the chaperonecofactor abolishes substrate recognition in these cases. By exchanging ADP forATP, the nucleotide exchange factor Fes1 releases misfolded proteins from Hsp70increasing their susceptibility to proteasomal degradation [54]. Sse1, another NEFof the Hsp110 family, can bind directly to hydrophobic patches on misfolded sub-strates which might assist in maintaining substrate solubility, aid in protein refold-ing, or shield substrates from interacting with the ubiquitination machinery [54].An example of substrate competition is the nuclear E3 ligase San1, which directlyinteracts with short hydrophobic stretches on misfolded substrates [69]. In vitro,Sse1 binding inhibits ubiquitination of San1 substrates, potentially as the resultof competitive binding [53]. Together, the data would suggest a model wherebykinetic partitioning, protein abundance, and intracellular localization converge todictate protein triage decisions in protein quality control.4.2.4 The Importance Of, and Difficulty In, Maintaining ProteostasisMany stresses disrupt protein folding prompting transcriptional responses to in-crease the chaperone and proteostatic capacity of the cell in order to maintain cellviability. These stress response mechanisms act to restore protein homeostatic bal-ance by matching levels of protein quality control factors with the protein foldingrequirements of the cell. Moreover, the relationship between protein folding anddegradation is underscored by the observation that an increase in folding capac-ity is almost always accompanied by an increase in the degradation machinery [1].Regulatory mechanisms are required to adequately respond to the pressure of in-creasing loads of misfolded proteins as excess capacity is not inherent to the sys-tem. A number of experimental observations support the hypothesis that foldingcapacity is tightly regulated. For example, exposure to stresses that induce proteinmisfolding elicit a stress response that decreases translation of non-essential pro-tein products but induces gene expression of molecular chaperones and other cyto-protective components. In Caenorhabditis elegans, expressing an unstable mutantprotein prone to aggregate resulted in decreased stability of other proteome mem-bers. This suggests that excess folding capacity is not present as the system is111unable to respond to both the increase in a single species of misfolded protein andto maintain the remainder of the proteome [245]. As the system fails to produce anadequate response in some cases, one could reasonably question why the cell doesnot simply begin with a higher basal folding capacity? A possible answer is thatnot only would it be costly to the cell to produce additional chaperone proteins butalso increased chaperone levels can themselves be detrimental to cellular functions.For instance, abnormally high levels of Hsp70 in Drosophila melanogaster cells orlarvae can interfere with growth, development, or survival to adulthood [246, 247].Second, many proteins rely on conformational changes that involve transitions be-tween low energy states in order to perform their functions [248]. These transitionsmay expose sensitive binding surfaces or pass through less stable intermediates.Were the proteostasis network to have an excessive folding capacity, a number ofthese states might be shielded, thereby impeding or eliminating many cellular func-tions. Finally, while HSF1 mediated signaling is vital for the heat shock responsein mammals, increased expression of some heat shock proteins is associated withthe propagation of some cancers and the emergence of drug resistant viruses [249].These examples illustrate how important proper regulation of proteostasis is tomaintaining proteome integrity and cellular and organism viability.In this thesis we describe how an absence of a single non-essential chaper-one subunit (Gim3) and mutations in the general stress response gene WHI2 canprofoundly impact the cell’s capacity to respond to higher levels of misfolded pro-teins. Notably, WHI2 mutations are responsible for the impaired proteolysis ofthe Guk1-7-GFP protein quality control substrate. We linked this phenotype to adeficiency of the Msn2/4 transcription factor response that altered the ability forcells to adequately degrade cytosolic misfolded proteins. Interestingly, our experi-ments were performed in the presence of cycloheximide that prevents translation ofnewly synthesized proteins in response to increased temperatures and levels of mis-folded proteins. Our work indicates that potentially small changes to the balanceof the protein homeostasis network may impact the cell’s ability to adapt rapidly tochange, such as before the cell can mount a transcriptional response. These find-ings present a starting point from which we can begin to understand how changesin the protein quality control network can disturb proteostasis. Understanding howthe proteostatic network responds to stress is increasingly becoming important for112our understanding of disease and for drug discovery.4.3 Future DirectionsIn this thesis, we have developed a flow cytometry based approach to screen forprotein quality control factors that promote proteasome mediated degradation ofunstable model substrates. Work presented in this thesis also presents a number ofinteresting avenues for future research.4.3.1 Flow Cytometry Screens for E3 Ligases Targeting HumanDisease AllelesSahni et al. created the human mutation ORFeome, a resource of cloning vectorscontaining human germline mutations associated with Mendelian diseases [159].We have requested a number of these ORFs, specifically selecting from those thatdemonstrated an interaction with the chaperone machinery, suggestive of decreasedstability. At the same time, we also acquired the DsRed/EGFP vector developedfor global protein stability analysis [174]. The plan is to create EGFP fusions withthe mutant and wild type human ORFs. The constructs would be tested initiallyusing the CETSA assay as was used in Chapters 2 and 3 to identify those thathave the lowest thermal stability. Once an ideal substrate has been identified ascreen using flow cytometry to monitor changes in the EGFP/DsRed ratio couldbe performed to identify the E3 ubiquitin ligases required to target this diseaseassociated protein for degradation. The screen could be performed using a panelof short hairpin RNA (shRNAs) that specifically target and down regulate levels ofhuman E3 ligases or by using a genome-wide clustered regularly interspaced shortpalindromic repeats (CRISPR-Cas9) approach as developed by the laboratory ofDr. Moffat at the University of Toronto [250].4.3.2 Characterizing the Role of the E3 Ligase Ubr1 in CytoplasmicProtein Quality ControlUbr1 was first identified and characterized as the E3 ligase of the N-end rule, apathway whereby the half-life of a protein correlates with the identity of its N-terminal amino acid residue [97]. A number of studies now suggest that Ubr1113may play a role in protein degradation independent of the N-end rule [74, 99–101].Furthermore, previous work from the Mayor lab and work presented in this the-sis demonstrates that Ubr1 targets proteins destabilized by missense mutations fordegradation [101, 206]. How Ubr1 recognizes these substrates, and whether it doesso through a mechanism independent from the N-end rule are both questions thatremain unanswered. With the exception of Guk1-11, the alleles we have used toassess Ubr1 function contain a number of mutations that could potentially com-plicate the analysis of Ubr1 substrate binding. It is necessary therefore to create anew set of alleles by site directed mutagenesis that contain a single destabilizingmutation. Going forward, these new alleles that are stabilized by a Ubr1 deletionwould be used to address a number of questions.Ubr1 recognizes N-end rule substrates through two domains: the UBR box andthe ClpS domain [98]. We have generated overexpression plasmids containing thefull length Ubr1 protein with point mutations in either the UBR box or ClpS do-main. My hypothesis is that the UBR box is responsible for mediating substratedegradation. To further assess the role of the UBR box (or the ClpS domain), Iwould use dipeptides to block Type I and Type II binding sites thereby inhibitingN-end rule activity, and possibly degradation of the misfolded model substrates. Ifmutations in either of these domains show no effect on the degradation of modelsubstrates, Ubr1 truncations would be created to define the region involved in sub-strate recognition. It is also possible that through proteolytic cleavage misfoldedproteins are recognized as N-end rule substrates. To assess whether this is the case,dual N-and C-terminally tagged temperature sensitive alleles could be created thathave an N-terminal HA tag and a C-terminal His tag, similar to experiments previ-ously performed [74]. In the absence of cleavage, both tags should remain presentafter pulling down the ubiquitinated misfolded substrate. Currently, we have iden-tified a limited number of Ubr1 model substrates. It would be helpful to identifywhich misfolded proteins are normally targeted by Ubr1. We could express a Ubr1mutant that cannot recognize the misfolded model substrate (developed above).Using stable isotope labelling with amino acids in cell culture (SILAC) we couldthen identify which proteins are no longer ubiquitinated in the presence of the Ubr1mutant in comparison to cells expressing the wild type Ubr1 using mass spectrom-etry. Validation could be performed using the flow cytometry assay developed in114this thesis and pulldown analysis.There is currently great interest in understanding how proteostasis networksmaintain proteome integrity. In this thesis we describe the development of a flowcytometry based assay to exploit novel model substrates to study proteostasis. Us-ing this approach we identified the prefoldin subunit Gim3 and the general stressresponse factor Whi2 and characterized their roles in promoting protein homeosta-sis. This work underscores the complexity of the systems required to maintainproteostasis and the development of novel model substrates provides a valuableresource for future studies of protein quality control.115Bibliography[1] W. E. Balch, R. I. Morimoto, A. Dillin, and J. W. Kelly. Adaptingproteostasis for disease intervention. Science, 319(5865):916–9, 2008.[2] D. A. Drummond and C. O. Wilke. The evolutionary consequences oferroneous protein synthesis. Nat Rev Genet, 10(10):715–24, 2009.[3] A. L. Goldberg. Protein degradation and protection against misfolded ordamaged proteins. Nature, 426(6968):895–9, 2003.[4] K. A. Geiler-Samerotte, M. F. Dion, B. A. Budnik, S. M. Wang, D. L.Hartl, and D. A. Drummond. Misfolded proteins impose adosage-dependent fitness cost and trigger a cytosolic unfolded proteinresponse in yeast. Proc Natl Acad Sci U S A, 108(2):680–5, 2011.[5] H. Olzscha, S. M. Schermann, A. C. Woerner, S. Pinkert, M. H. Hecht,G. G. Tartaglia, M. Vendruscolo, M. Hayer-Hartl, F. U. Hartl, and R. M.Vabulas. Amyloid-like aggregates sequester numerous metastable proteinswith essential cellular functions. Cell, 144(1):67–78, 2011.[6] Y. E. Kim, F. Hosp, F. Frottin, H. Ge, M. Mann, M. Hayer-Hartl, and F. U.Hartl. Soluble oligomers of polyq-expanded huntingtin target a multiplicityof key cellular factors. Mol Cell, 2016.[7] F. Chiti and C. M. Dobson. Protein misfolding, functional amyloid, andhuman disease. Annu Rev Biochem, 75:333–66, 2006.[8] T. J. Kamerzell and C. R. Middaugh. The complex inter-relationshipsbetween protein flexibility and stability. J Pharm Sci, 97(9):3494–517,2008.[9] T. W. Mu, D. S. Ong, Y. J. Wang, W. E. Balch, 3rd Yates, J. R., L. Segatori,and J. W. Kelly. Chemical and biological approaches synergize toameliorate protein-folding diseases. Cell, 134(5):769–81, 2008.116[10] F. U. Hartl and M. Hayer-Hartl. Converging concepts of protein folding invitro and in vivo. Nat Struct Mol Biol, 16(6):574–81, 2009.[11] R. I. Morimoto. Proteotoxic stress and inducible chaperone networks inneurodegenerative disease and aging. Genes Dev, 22(11):1427–38, 2008.[12] A. Ben-Zvi, E. A. Miller, and R. I. Morimoto. Collapse of proteostasisrepresents an early molecular event in caenorhabditis elegans aging. ProcNatl Acad Sci U S A, 106(35):14914–9, 2009.[13] A. H. Elcock. Molecular simulations of cotranslational protein folding:fragment stabilities, folding cooperativity, and trapping in the ribosome.PLoS Comput Biol, 2(7):e98, 2006.[14] V. Albanese, S. Reissmann, and J. Frydman. A ribosome-anchoredchaperone network that facilitates eukaryotic ribosome biogenesis. J CellBiol, 189(1):69–81, 2010.[15] F. Brandt, L. A. Carlson, F. U. Hartl, W. Baumeister, and K. Grunewald.The three-dimensional organization of polyribosomes in intact human cells.Mol Cell, 39(4):560–9, 2010.[16] H. Otto, C. Conz, P. Maier, T. Wolfle, C. K. Suzuki, P. Jeno, P. Rucknagel,J. Stahl, and S. Rospert. The chaperones mpp11 and hsp70l1 form themammalian ribosome-associated complex. Proc Natl Acad Sci U S A, 102(29):10064–9, 2005.[17] S. Preissler and E. Deuerling. Ribosome-associated chaperones as keyplayers in proteostasis. Trends Biochem Sci, 37(7):274–83, 2012.[18] F. Willmund, M. del Alamo, S. Pechmann, T. Chen, V. Albanese, E. B.Dammer, J. Peng, and J. Frydman. The cotranslational function ofribosome-associated hsp70 in eukaryotic protein homeostasis. Cell, 152(1-2):196–209, 2013.[19] A. Koplin, S. Preissler, Y. Ilina, M. Koch, A. Scior, M. Erhardt, andE. Deuerling. A dual function for chaperones ssb-rac and the nac nascentpolypeptide-associated complex on ribosomes. J Cell Biol, 189(1):57–68,2010.[20] M. P. Mayer and B. Bukau. Hsp70 chaperones: cellular functions andmolecular mechanism. Cell Mol Life Sci, 62(6):670–84, 2005.117[21] H. H. Kampinga and E. A. Craig. The hsp70 chaperone machinery: Jproteins as drivers of functional specificity. Nat Rev Mol Cell Biol, 11(8):579–92, 2010.[22] E. B. Sarbeng, Q. Liu, X. Tian, J. Yang, H. Li, J. L. Wong, and L. Zhou. Afunctional dnak dimer is essential for the efficient interaction with hsp40heat shock protein. J Biol Chem, 290(14):8849–62, 2015.[23] P. Walsh, D. Bursac, Y. C. Law, D. Cyr, and T. Lithgow. The j-proteinfamily: modulating protein assembly, disassembly and translocation.EMBO Rep, 5(6):567–71, 2004.[24] H. Raviol, H. Sadlish, F. Rodriguez, M. P. Mayer, and B. Bukau.Chaperone network in the yeast cytosol: Hsp110 is revealed as an hsp70nucleotide exchange factor. EMBO J, 25(11):2510–8, 2006.[25] J. Cuellar, J. Martin-Benito, S. H. Scheres, R. Sousa, F. Moro,E. Lopez-Vinas, P. Gomez-Puertas, A. Muga, J. L. Carrascosa, and J. M.Valpuesta. The structure of cct-hsc70 nbd suggests a mechanism for hsp70delivery of substrates to the chaperonin. Nat Struct Mol Biol, 15(8):858–64, 2008.[26] M. Taipale, D. F. Jarosz, and S. Lindquist. Hsp90 at the hub of proteinhomeostasis: emerging mechanistic insights. Nat Rev Mol Cell Biol, 11(7):515–28, 2010.[27] Y. E. Kim, M. S. Hipp, A. Bracher, M. Hayer-Hartl, and F. U. Hartl.Molecular chaperone functions in protein folding and proteostasis. AnnuRev Biochem, 82:323–55, 2013.[28] A. J. Caplan, A. K. Mandal, and M. A. Theodoraki. Molecular chaperonesand protein kinase quality control. Trends Cell Biol, 17(2):87–92, 2007.[29] C. Dekker, P. C. Stirling, E. A. McCormack, H. Filmore, A. Paul, R. L.Brost, M. Costanzo, C. Boone, M. R. Leroux, and K. R. Willison. Theinteraction network of the chaperonin cct. EMBO J, 27(13):1827–39, 2008.[30] M. A. Kabir, W. Uddin, A. Narayanan, P. K. Reddy, M. A. Jairajpuri,F. Sherman, and Z. Ahmad. Functional subunits of eukaryotic chaperonincct/tric in protein folding. J Amino Acids, 2011:843206, 2011.[31] T. Lopez, K. Dalton, and J. Frydman. The mechanism and function ofgroup ii chaperonins. J Mol Biol, 427(18):2919–30, 2015.118[32] F. Russmann, M. J. Stemp, L. Monkemeyer, S. A. Etchells, A. Bracher, andF. U. Hartl. Folding of large multidomain proteins by partial encapsulationin the chaperonin tric/cct. Proc Natl Acad Sci U S A, 109(52):21208–15,2012.[33] S. Reissmann, L. A. Joachimiak, B. Chen, A. S. Meyer, A. Nguyen, andJ. Frydman. A gradient of atp affinities generates an asymmetric powerstroke driving the chaperonin tric/cct folding cycle. Cell Rep, 2(4):866–77,2012.[34] L. A. Joachimiak, T. Walzthoeni, C. W. Liu, R. Aebersold, and J. Frydman.The structural basis of substrate recognition by the eukaryotic chaperonintric/cct. Cell, 159(5):1042–55, 2014.[35] M. W. Melville, A. J. McClellan, A. S. Meyer, A. Darveau, and J. Frydman.The hsp70 and tric/cct chaperone systems cooperate in vivo to assemble thevon hippel-lindau tumor suppressor complex. Mol Cell Biol, 23(9):3141–51, 2003.[36] A. Y. Yam, Y. Xia, H. T. Lin, A. Burlingame, M. Gerstein, and J. Frydman.Defining the tric/cct interactome links chaperonin function to stabilizationof newly made proteins with complex topologies. Nat Struct Mol Biol, 15(12):1255–62, 2008.[37] A. Bouhouche, A. Benomar, N. Bouslam, T. Chkili, and M. Yahyaoui.Mutation in the epsilon subunit of the cytosolic chaperonin-containingt-complex peptide-1 (cct5) gene causes autosomal recessive mutilatingsensory neuropathy with spastic paraplegia. J Med Genet, 43(5):441–3,2006.[38] Y. Inoue, H. Aizaki, H. Hara, M. Matsuda, T. Ando, T. Shimoji,K. Murakami, T. Masaki, I. Shoji, S. Homma, Y. Matsuura, T. Miyamura,T. Wakita, and T. Suzuki. Chaperonin tric/cct participates in replication ofhepatitis c virus genome via interaction with the viral ns5b protein.Virology, 410(1):38–47, 2011.[39] A. G. Trinidad, P. A. Muller, J. Cuellar, M. Klejnot, M. Nobis, J. M.Valpuesta, and K. H. Vousden. Interaction of p53 with the cct complexpromotes protein folding and wild-type p53 activity. Mol Cell, 50(6):805–17, 2013.[40] H. Zhou, M. Xu, Q. Huang, A. T. Gates, X. D. Zhang, J. C. Castle, E. Stec,M. Ferrer, B. Strulovici, D. J. Hazuda, and A. S. Espeseth. Genome-scale119rnai screen for host factors required for hiv replication. Cell Host Microbe,4(5):495–504, 2008.[41] I. E. Vainberg, S. A. Lewis, H. Rommelaere, C. Ampe, J. Vandekerckhove,H. L. Klein, and N. J. Cowan. Prefoldin, a chaperone that delivers unfoldedproteins to cytosolic chaperonin. Cell, 93(5):863–73, 1998.[42] R. Siegert, M. R. Leroux, C. Scheufler, F. U. Hartl, and I. Moarefi.Structure of the molecular chaperone prefoldin: unique interaction ofmultiple coiled coil tentacles with unfolded proteins. Cell, 103(4):621–32,2000.[43] M. Takano, E. Tashiro, A. Kitamura, H. Maita, S. M. Iguchi-Ariga,M. Kinjo, and H. Ariga. Prefoldin prevents aggregation of alpha-synuclein.Brain Res, 1542:186–94, 2014.[44] E. Tashiro, T. Zako, H. Muto, Y. Itoo, K. Sorgjerd, N. Terada, A. Abe,M. Miyazawa, A. Kitamura, H. Kitaura, H. Kubota, M. Maeda, T. Momoi,S. M. Iguchi-Ariga, M. Kinjo, and H. Ariga. Prefoldin protects neuronalcells from polyglutamine toxicity by preventing aggregation formation. JBiol Chem, 288(27):19958–72, 2013.[45] A. Shiber, W. Breuer, M. Brandeis, and T. Ravid. Ubiquitin conjugationtriggers misfolded protein sequestration into quality-control foci whenhsp70 chaperone levels are limiting. Mol Biol Cell, 2013.[46] D. W. Summers, K. J. Wolfe, H. Y. Ren, and D. M. Cyr. The type ii hsp40sis1 cooperates with hsp70 and the e3 ligase ubr1 to promote degradationof terminally misfolded cytosolic protein. PLoS One, 8(1):e52099, 2013.[47] Q. Wang, Y. Liu, N. Soetandyo, K. Baek, R. Hegde, and Y. Ye. A ubiquitinligase-associated chaperone holdase maintains polypeptides in solublestates for proteasome degradation. Mol Cell, 42(6):758–70, 2011.[48] C. J. Guerriero, K. F. Weiberth, and J. L. Brodsky. Hsp70 targets acytoplasmic quality control substrate to the san1p ubiquitin ligase. J BiolChem, 2013.[49] G. C. Meacham, C. Patterson, W. Zhang, J. M. Younger, and D. M. Cyr.The hsc70 co-chaperone chip targets immature cftr for proteasomaldegradation. Nat Cell Biol, 3(1):100–5, 2001.120[50] A. J. McClellan, M. D. Scott, and J. Frydman. Folding and quality controlof the vhl tumor suppressor proceed through distinct chaperone pathways.Cell, 121(5):739–48, 2005.[51] A. K. Mandal, P. A. Gibney, N. B. Nillegoda, M. A. Theodoraki, A. J.Caplan, and K. A. Morano. Hsp110 chaperones control client fatedetermination in the hsp70-hsp90 chaperone system. Mol Biol Cell, 21(9):1439–48, 2010.[52] N. N. Fang, G. T. Chan, M. Zhu, S. A. Comyn, A. Persaud, R. J. Deshaies,D. Rotin, J. Gsponer, and T. Mayor. Rsp5/nedd4 is the main ubiquitinligase that targets cytosolic misfolded proteins following heat stress. NatCell Biol, 16(12):1227–37, 2014.[53] S. H. Park, N. Bolender, F. Eisele, Z. Kostova, J. Takeuchi, P. Coffino, andD. H. Wolf. The cytoplasmic hsp70 chaperone machinery subjectsmisfolded and endoplasmic reticulum import-incompetent proteins todegradation via the ubiquitin-proteasome system. Mol Biol Cell, 18(1):153–65, 2007.[54] N. K. Gowda, G. Kandasamy, M. S. Froehlich, R. J. Dohmen, andC. Andreasson. Hsp70 nucleotide exchange factor fes1 is essential forubiquitin-dependent degradation of misfolded cytosolic proteins. Proc NatlAcad Sci U S A, 110(15):5975–80, 2013.[55] R. Minami, A. Hayakawa, H. Kagawa, Y. Yanagi, H. Yokosawa, andH. Kawahara. Bag-6 is essential for selective elimination of defectiveproteasomal substrates. J Cell Biol, 190(4):637–50, 2010.[56] C. M. Pickart and M. J. Eddins. Ubiquitin: structures, functions,mechanisms. Biochim Biophys Acta, 1695(1-3):55–72, 2004.[57] R. J. Deshaies and C. A. Joazeiro. Ring domain e3 ubiquitin ligases. AnnuRev Biochem, 78:399–434, 2009.[58] D. M. Wenzel and R. E. Klevit. Following ariadne’s thread: a newperspective on rbr ubiquitin ligases. BMC Biol, 10:24, 2012.[59] M. B. Metzger, V. A. Hristova, and A. M. Weissman. Hect and ring fingerfamilies of e3 ubiquitin ligases at a glance. J Cell Sci, 125(Pt 3):531–7,2012.121[60] C. B. Lucking, A. Durr, V. Bonifati, J. Vaughan, G. De Michele, T. Gasser,B. S. Harhangi, G. Meco, P. Denefle, N. W. Wood, Y. Agid, and A. Brice.Association between early-onset parkinson’s disease and mutations in theparkin gene. N Engl J Med, 342(21):1560–7, 2000.[61] D. Finley. Recognition and processing of ubiquitin-protein conjugates bythe proteasome. Annu Rev Biochem, 78:477–513, 2009.[62] D. Finley, H. D. Ulrich, T. Sommer, and P. Kaiser. Theubiquitin-proteasome system of saccharomyces cerevisiae. Genetics, 192(2):319–60, 2012.[63] A. Buchberger, B. Bukau, and T. Sommer. Protein quality control in thecytosol and the endoplasmic reticulum: brothers in arms. Mol Cell, 40(2):238–52, 2010.[64] S. S. Vembar and J. L. Brodsky. One step at a time: endoplasmicreticulum-associated degradation. Nat Rev Mol Cell Biol, 9(12):944–57,2008.[65] A. Ruggiano, O. Foresti, and P. Carvalho. Quality control: Er-associateddegradation: protein quality control and beyond. J Cell Biol, 204(6):869–79, 2014.[66] P. Carvalho, V. Goder, and T. A. Rapoport. Distinct ubiquitin-ligasecomplexes define convergent pathways for the degradation of er proteins.Cell, 126(2):361–73, 2006. Carvalho, Pedro Goder, Veit Rapoport, Tom AGM052586/GM/NIGMS NIH HHS/United States Comparative StudyResearch Support, N.I.H., Extramural Research Support, Non-U.S. Gov’tUnited States Cell Cell. 2006 Jul 28;126(2):361-73.[67] V. Denic, E. M. Quan, and J. S. Weissman. A luminal surveillance complexthat selects misfolded glycoproteins for er-associated degradation. Cell,126(2):349–59, 2006. Denic, Vladimir Quan, Erin M Weissman, JonathanS Research Support, Non-U.S. Gov’t Research Support, U.S. Gov’t,Non-P.H.S. United States Cell Cell. 2006 Jul 28;126(2):349-59.[68] C. Wojcik and G. N. DeMartino. Intracellular localization of proteasomes.Int J Biochem Cell Biol, 35(5):579–89, 2003.[69] E. K. Fredrickson, J. C. Rosenbaum, M. N. Locke, T. I. Milac, and R. G.Gardner. Exposed hydrophobicity is a key determinant of nuclear qualitycontrol degradation. Mol Biol Cell, 22(13):2384–95, 2011.122[70] R. G. Gardner, Z. W. Nelson, and D. E. Gottschling. Degradation-mediatedprotein quality control in the nucleus. Cell, 120(6):803–15, 2005.[71] R. Ibarra, D. Sandoval, E. K. Fredrickson, R. G. Gardner, and G. Kleiger.The san1 ubiquitin ligase functions preferentially withubiquitin-conjugating enzyme ubc1 during protein quality control. J BiolChem, 291(36):18778–90, 2016.[72] J. C. Rosenbaum, E. K. Fredrickson, M. L. Oeser, C. M. Garrett-Engele,M. N. Locke, L. A. Richardson, Z. W. Nelson, E. D. Hetrick, T. I. Milac,D. E. Gottschling, and R. G. Gardner. Disorder targets misorder in nuclearquality control degradation: a disordered ubiquitin ligase directlyrecognizes its misfolded substrates. Mol Cell, 41(1):93–106, 2011.[73] P. S. Gallagher, S. V. Clowes Candadai, and R. G. Gardner. Therequirement for cdc48/p97 in nuclear protein quality control degradationdepends on the substrate and correlates with substrate insolubility. J CellSci, 127(Pt 9):1980–91, 2014.[74] J. W. Heck, S. K. Cheung, and R. Y. Hampton. Cytoplasmic protein qualitycontrol degradation mediated by parallel actions of the e3 ubiquitin ligasesubr1 and san1. Proc Natl Acad Sci U S A, 107(3):1106–11, 2010.[75] S. H. Park, Y. Kukushkin, R. Gupta, T. Chen, A. Konagai, M. S. Hipp,M. Hayer-Hartl, and F. U. Hartl. Polyq proteins interfere with nucleardegradation of cytosolic proteins by sequestering the sis1p chaperone. Cell,154(1):134–45, 2013.[76] R. Prasad, S. Kawaguchi, and D. T. Ng. A nucleus-based quality controlmechanism for cytosolic proteins. Mol Biol Cell, 21(13):2117–27, 2010.[77] S. B. Miller, C. T. Ho, J. Winkler, M. Khokhrina, A. Neuner, M. Y.Mohamed, D. L. Guilbride, K. Richter, M. Lisby, E. Schiebel, A. Mogk,and B. Bukau. Compartment-specific aggregases direct distinct nuclear andcytoplasmic aggregate deposition. EMBO J, 34(6):778–97, 2015.[78] E. Grossman, O. Medalia, and M. Zwerger. Functional architecture of thenuclear pore complex. Annu Rev Biophys, 41:557–84, 2012.[79] R. D. Jones and R. G. Gardner. Protein quality control in the nucleus. CurrOpin Cell Biol, 40:81–9, 2016.123[80] A. Khmelinskii, E. Blaszczak, M. Pantazopoulou, B. Fischer, D. J. Omnus,G. Le Dez, A. Brossard, A. Gunnarsson, J. D. Barry, M. Meurer,D. Kirrmaier, C. Boone, W. Huber, G. Rabut, P. O. Ljungdahl, andM. Knop. Protein quality control at the inner nuclear membrane. Nature,516(7531):410–3, 2014.[81] M. K. Sung, T. R. Porras-Yakushi, J. M. Reitsma, F. M. Huber, M. J.Sweredoski, A. Hoelz, S. Hess, and R. J. Deshaies. A conservedquality-control pathway that mediates degradation of unassembledribosomal proteins. Elife, 5, 2016.[82] P. Connell, C. A. Ballinger, J. Jiang, Y. Wu, L. J. Thompson, J. Hohfeld,and C. Patterson. The co-chaperone chip regulates protein triage decisionsmediated by heat-shock proteins. Nat Cell Biol, 3(1):93–6, 2001.[83] C. Graf, M. Stankiewicz, R. Nikolay, and M. P. Mayer. Insights into theconformational dynamics of the e3 ubiquitin ligase chip in complex withchaperones and e2 enzymes. Biochemistry, 49(10):2121–9, 2010.[84] L. Kundrat and L. Regan. Balance between folding and degradation forhsp90-dependent client proteins: a key role for chip. Biochemistry, 49(35):7428–38, 2010.[85] V. Arndt, C. Daniel, W. Nastainczyk, S. Alberti, and J. Hohfeld. Bag-2 actsas an inhibitor of the chaperone-associated ubiquitin ligase chip. Mol BiolCell, 16(12):5891–900, 2005.[86] Q. Dai, S. B. Qian, H. H. Li, H. McDonough, C. Borchers, D. Huang,S. Takayama, J. M. Younger, H. Y. Ren, D. M. Cyr, and C. Patterson.Regulation of the cytoplasmic quality control protein degradation pathwayby bag2. J Biol Chem, 280(46):38673–81, 2005.[87] S. Alberti, J. Demand, C. Esser, N. Emmerich, H. Schild, and J. Hohfeld.Ubiquitylation of bag-1 suggests a novel regulatory mechanism during thesorting of chaperone substrates to the proteasome. J Biol Chem, 277(48):45920–7, 2002.[88] N. Kettern, C. Rogon, A. Limmer, H. Schild, and J. Hohfeld. Thehsc/hsp70 co-chaperone network controls antigen aggregation andpresentation during maturation of professional antigen presenting cells.PLoS One, 6(1):e16398, 2011.124[89] K. M. Scaglione, E. Zavodszky, S. V. Todi, S. Patury, P. Xu,E. Rodriguez-Lebron, S. Fischer, J. Konen, A. Djarmati, J. Peng, J. E.Gestwicki, and H. L. Paulson. Ube2w and ataxin-3 coordinately regulatethe ubiquitin ligase chip. Mol Cell, 43(4):599–612, 2011.[90] T. M. Durcan and E. A. Fon. Ataxin-3 and its e3 partners: implications formachado-joseph disease. Front Neurol, 4:46, 2013.[91] N. R. Jana, P. Dikshit, A. Goswami, S. Kotliarova, S. Murata, K. Tanaka,and N. Nukina. Co-chaperone chip associates with expandedpolyglutamine protein and promotes their degradation by proteasomes. JBiol Chem, 280(12):11635–40, 2005.[92] Y. Imai, M. Soda, and R. Takahashi. Parkin suppresses unfolded proteinstress-induced cell death through its e3 ubiquitin-protein ligase activity. JBiol Chem, 275(46):35661–4, 2000.[93] J. Niwa, S. Ishigaki, N. Hishikawa, M. Yamamoto, M. Doyu, S. Murata,K. Tanaka, N. Taniguchi, and G. Sobue. Dorfin ubiquitylates mutant sod1and prevents mutant sod1-mediated neurotoxicity. J Biol Chem, 277(39):36793–8, 2002.[94] Y. C. Tsai, P. S. Fishman, N. V. Thakor, and G. A. Oyler. Parkin facilitatesthe elimination of expanded polyglutamine proteins and leads topreservation of proteasome function. J Biol Chem, 278(24):22044–55,2003.[95] C. Vives-Bauza and S. Przedborski. Mitophagy: the latest problem forparkinson’s disease. Trends Mol Med, 17(3):158–65, 2011.[96] C. Vives-Bauza, C. Zhou, Y. Huang, M. Cui, R. L. de Vries, J. Kim, J. May,M. A. Tocilescu, W. Liu, H. S. Ko, J. Magrane, D. J. Moore, V. L. Dawson,R. Grailhe, T. M. Dawson, C. Li, K. Tieu, and S. Przedborski.Pink1-dependent recruitment of parkin to mitochondria in mitophagy. ProcNatl Acad Sci U S A, 107(1):378–83, 2010.[97] A. Varshavsky. The n-end rule: functions, mysteries, uses. Proc Natl AcadSci U S A, 93(22):12142–9, 1996.[98] Z. Xia, A. Webster, F. Du, K. Piatkov, M. Ghislain, and A. Varshavsky.Substrate-binding sites of ubr1, the ubiquitin ligase of the n-end rulepathway. J Biol Chem, 283(35):24011–28, 2008.125[99] A. Stolz, S. Besser, H. Hottmann, and D. H. Wolf. Previously unknownrole for the ubiquitin ligase ubr1 in endoplasmic reticulum-associatedprotein degradation. Proc Natl Acad Sci U S A, 110(38):15271–6, 2013.[100] M. A. Theodoraki, N. B. Nillegoda, J. Saini, and A. J. Caplan. A networkof ubiquitin ligases is important for the dynamics of misfolded proteinaggregates in yeast. J Biol Chem, 287(28):23911–22, 2012.[101] F. Khosrow-Khavar, N. N. Fang, A. H. Ng, J. M. Winget, S. A. Comyn, andT. Mayor. The yeast ubr1 ubiquitin ligase participates in a prominentpathway that targets cytosolic thermosensitive mutants for degradation. G3(Bethesda), 2(5):619–28, 2012.[102] B. Bartel, I. Wunning, and A. Varshavsky. The recognition component ofthe n-end rule pathway. EMBO J, 9(10):3179–89, 1990.[103] F. Eisele and D. H. Wolf. Degradation of misfolded protein in thecytoplasm is mediated by the ubiquitin ligase ubr1. FEBS Lett, 582(30):4143–6, 2008.[104] N. B. Nillegoda, M. A. Theodoraki, A. K. Mandal, K. J. Mayo, H. Y. Ren,R. Sultana, K. Wu, J. Johnson, D. M. Cyr, and A. J. Caplan. Ubr1 and ubr2function in a quality control pathway for degradation of unfolded cytosolicproteins. Mol Biol Cell, 21(13):2102–16, 2010.[105] R. Sultana, M. A. Theodoraki, and A. J. Caplan. Ubr1 promotes proteinkinase quality control and sensitizes cells to hsp90 inhibition. Exp CellRes, 318(1):53–60, 2012.[106] C. S. Hwang, M. Sukalo, O. Batygin, M. C. Addor, H. Brunner, A. P.Aytes, J. Mayerle, H. K. Song, A. Varshavsky, and M. Zenker. Ubiquitinligases of the n-end rule pathway: assessment of mutations in ubr1 thatcause the johanson-blizzard syndrome. PLoS One, 6(9):e24925, 2011.[107] B. Medicherla and A. L. Goldberg. Heat shock and oxygen radicalsstimulate ubiquitin-dependent degradation mainly of newly synthesizedproteins. J Cell Biol, 182(4):663–73, 2008.[108] A. H. Ng, N. N. Fang, S. A. Comyn, J. Gsponer, and T. Mayor.System-wide analysis reveals intrinsically disordered proteins are prone toubiquitylation after misfolding stress. Mol Cell Proteomics, 2013.126[109] N. N. Fang, A. H. Ng, V. Measday, and T. Mayor. Hul5 hect ubiquitinligase plays a major role in the ubiquitylation and turnover of cytosolicmisfolded proteins. Nat Cell Biol, 13(11):1344–52, 2011.[110] B. Crosas, J. Hanna, D. S. Kirkpatrick, D. P. Zhang, Y. Tone, N. A.Hathaway, C. Buecker, D. S. Leggett, M. Schmidt, R. W. King, S. P. Gygi,and D. Finley. Ubiquitin chains are remodeled at the proteasome byopposing ubiquitin ligase and deubiquitinating activities. Cell, 127(7):1401–13, 2006.[111] D. S. Leggett, J. Hanna, A. Borodovsky, B. Crosas, M. Schmidt, R. T.Baker, T. Walz, H. Ploegh, and D. Finley. Multiple associated proteinsregulate proteasome structure and function. Mol Cell, 10(3):495–507,2002.[112] R. Dunn and L. Hicke. Domains of the rsp5 ubiquitin-protein ligaserequired for receptor-mediated and fluid-phase endocytosis. Mol Biol Cell,12(2):421–35, 2001.[113] P. Kaliszewski, T. Ferreira, B. Gajewska, A. Szkopinska, T. Berges, andT. Zoladek. Enhanced levels of pis1p (phosphatidylinositol synthase)improve the growth of saccharomyces cerevisiae cells deficient in rsp5ubiquitin ligase. Biochem J, 395(1):173–81, 2006.[114] M. S. Rodriguez, C. Gwizdek, R. Haguenauer-Tsapis, and C. Dargemont.The hect ubiquitin ligase rsp5p is required for proper nuclear export ofmrna in saccharomyces cerevisiae. Traffic, 4(8):566–75, 2003.[115] M. H. Bengtson and C. A. Joazeiro. Role of a ribosome-associated e3ubiquitin ligase in protein quality control. Nature, 467(7314):470–3, 2010.[116] S. Shao, K. von der Malsburg, and R. S. Hegde. Listerin-dependent nascentprotein ubiquitination relies on ribosome subunit dissociation. Mol Cell,2013.[117] K. von der Malsburg, S. Shao, and R. S. Hegde. The ribosome qualitycontrol pathway can access nascent polypeptides stalled at the sec61translocon. Mol Biol Cell, 26(12):2168–80, 2015.[118] O. Brandman, J. Stewart-Ornstein, D. Wong, A. Larson, C. C. Williams,G. W. Li, S. Zhou, D. King, P. S. Shen, J. Weibezahn, J. G. Dunn,S. Rouskin, T. Inada, A. Frost, and J. S. Weissman. A ribosome-boundquality control complex triggers degradation of nascent peptides andsignals translation stress. Cell, 151(5):1042–54, 2012.127[119] Q. Defenouillere, Y. Yao, J. Mouaikel, A. Namane, A. Galopier,L. Decourty, A. Doyen, C. Malabat, C. Saveanu, A. Jacquier, andM. Fromont-Racine. Cdc48-associated complex bound to 60s particles isrequired for the clearance of aberrant translation products. Proc Natl AcadSci U S A, 110(13):5046–51, 2013.[120] S. Shao, A. Brown, B. Santhanam, and R. S. Hegde. Structure andassembly pathway of the ribosome quality control complex. Mol Cell, 57(3):433–44, 2015.[121] R. Verma, R. S. Oania, N. J. Kolawa, and R. J. Deshaies. Cdc48/p97promotes degradation of aberrant nascent polypeptides bound to theribosome. Elife, 2:e00308, 2013.[122] J. J. Crowder, M. Geigges, R. T. Gibson, E. S. Fults, B. W. Buchanan,N. Sachs, A. Schink, S. G. Kreft, and E. M. Rubenstein. Rkr1/ltn1ubiquitin ligase-mediated degradation of translationally stalledendoplasmic reticulum proteins. J Biol Chem, 290(30):18454–66, 2015.[123] D. Lyumkis, S. K. Doamekpor, M. H. Bengtson, J. W. Lee, T. B. Toro,M. D. Petroski, C. D. Lima, C. S. Potter, B. Carragher, and C. A. Joazeiro.Single-particle em reveals extensive conformational variability of the ltn1e3 ligase. Proc Natl Acad Sci U S A, 110(5):1702–7, 2013.[124] J. Chu, N. A. Hong, C. A. Masuda, B. V. Jenkins, K. A. Nelms, C. C.Goodnow, R. J. Glynne, H. Wu, E. Masliah, C. A. Joazeiro, and S. A. Kay.A mouse forward genetics screen identifies listerin as an e3 ubiquitin ligaseinvolved in neurodegeneration. Proc Natl Acad Sci U S A, 106(7):2097–103, 2009.[125] D. Glick, S. Barth, and K. F. Macleod. Autophagy: cellular and molecularmechanisms. J Pathol, 221(1):3–12, 2010.[126] X. Chen and X. M. Yin. Coordination of autophagy and the proteasome inresolving endoplasmic reticulum stress. Vet Pathol, 48(1):245–53, 2011.[127] C. Kraft, A. Deplazes, M. Sohrmann, and M. Peter. Mature ribosomes areselectively degraded upon starvation by an autophagy pathway requiringthe ubp3p/bre5p ubiquitin protease. Nat Cell Biol, 10(5):602–10, 2008.[128] R. S. Marshall, F. Li, D. C. Gemperline, A. J. Book, and R. D. Vierstra.Autophagic degradation of the 26s proteasome is mediated by the dualatg8/ubiquitin receptor rpn10 in arabidopsis. Mol Cell, 58(6):1053–66,1282015. Marshall, Richard S Li, Faqiang Gemperline, David C Book, Adam JVierstra, Richard D T32 GM007133/GM/NIGMS NIH HHS/United StatesResearch Support, U.S. Gov’t, Non-P.H.S. United States Molecular cellNihms771991 Mol Cell. 2015 Jun 18;58(6):1053-66. doi:10.1016/j.molcel.2015.04.023. Epub 2015 May 21.[129] R. S. Marshall, F. McLoughlin, and R. D. Vierstra. Autophagic turnover ofinactive 26s proteasomes in yeast is directed by the ubiquitin receptor cue5and the hsp42 chaperone. Cell Rep, 16(6):1717–32, 2016. Marshall,Richard S McLoughlin, Fionn Vierstra, Richard D United States Cellreports Cell Rep. 2016 Aug 9;16(6):1717-32. doi:10.1016/j.celrep.2016.07.015. Epub 2016 Jul 28.[130] B. Ossareh-Nazari, C. A. Nino, M. H. Bengtson, J. W. Lee, C. A. Joazeiro,and C. Dargemont. Ubiquitylation by the ltn1 e3 ligase protects 60sribosomes from starvation-induced selective autophagy. J Cell Biol, 204(6):909–17, 2014.[131] I. Kim, S. Rodriguez-Enriquez, and J. J. Lemasters. Selective degradationof mitochondria by mitophagy. Arch Biochem Biophys, 462(2):245–53,2007.[132] D. Narendra, A. Tanaka, D. F. Suen, and R. J. Youle. Parkin-inducedmitophagy in the pathogenesis of parkinson disease. Autophagy, 5(5):706–8, 2009.[133] G. Bjorkoy, T. Lamark, A. Brech, H. Outzen, M. Perander, A. Overvatn,H. Stenmark, and T. Johansen. p62/sqstm1 forms protein aggregatesdegraded by autophagy and has a protective effect on huntingtin-inducedcell death. J Cell Biol, 171(4):603–14, 2005.[134] J. A. Johnston, C. L. Ward, and R. R. Kopito. Aggresomes: a cellularresponse to misfolded proteins. J Cell Biol, 143(7):1883–98, 1998.[135] D. Kaganovich, R. Kopito, and J. Frydman. Misfolded proteins partitionbetween two distinct quality control compartments. Nature, 454(7208):1088–95, 2008.[136] S. Escusa-Toret, W. I. Vonk, and J. Frydman. Spatial sequestration ofmisfolded proteins by a dynamic chaperone pathway enhances cellularfitness during stress. Nat Cell Biol, 15(10):1231–43, 2013.129[137] S. Specht, S. B. Miller, A. Mogk, and B. Bukau. Hsp42 is required forsequestration of protein aggregates into deposition sites in saccharomycescerevisiae. J Cell Biol, 195(4):617–29, 2011.[138] M. Haslbeck, N. Braun, T. Stromer, B. Richter, N. Model, S. Weinkauf, andJ. Buchner. Hsp42 is the general small heat shock protein in the cytosol ofsaccharomyces cerevisiae. EMBO J, 23(3):638–49, 2004.[139] D. Wotton, K. Freeman, and D. Shore. Multimerization of hsp42p, a novelheat shock protein of saccharomyces cerevisiae, is dependent on aconserved carboxyl-terminal sequence. J Biol Chem, 271(5):2717–23,1996.[140] L. Malinovska, S. Kroschwald, M. C. Munder, D. Richter, and S. Alberti.Molecular chaperones and stress-inducible protein-sorting factorscoordinate the spatiotemporal distribution of protein aggregates. Mol BiolCell, 23(16):3041–56, 2012.[141] J. Verghese, J. Abrams, Y. Wang, and K. A. Morano. Biology of the heatshock response and protein chaperones: budding yeast (saccharomycescerevisiae) as a model system. Microbiol Mol Biol Rev, 76(2):115–58,2012.[142] Y. Sanchez and S. L. Lindquist. Hsp104 required for inducedthermotolerance. Science, 248(4959):1112–5, 1990.[143] K. A. Morano, C. M. Grant, and W. S. Moye-Rowley. The response to heatshock and oxidative stress in saccharomyces cerevisiae. Genetics, 190(4):1157–95, 2012.[144] D. B. Berry and A. P. Gasch. Stress-activated genomic expression changesserve a preparative role for impending stress in yeast. Mol Biol Cell, 19(11):4580–7, 2008.[145] S. B. Ferguson, E. S. Anderson, R. B. Harshaw, T. Thate, N. L. Craig, andH. C. Nelson. Protein kinase a regulates constitutive expression of smallheat-shock genes in an msn2/4p-independent and hsf1p-dependent mannerin saccharomyces cerevisiae. Genetics, 169(3):1203–14, 2005.[146] E. J. Solis, J. P. Pandey, X. Zheng, D. X. Jin, P. B. Gupta, E. M. Airoldi,D. Pincus, and V. Denic. Defining the essential function of yeast hsf1reveals a compact transcriptional program for maintaining eukaryoticproteostasis. Mol Cell, 63(1):60–71, 2016.130[147] A. P. Gasch, P. T. Spellman, C. M. Kao, O. Carmel-Harel, M. B. Eisen,G. Storz, D. Botstein, and P. O. Brown. Genomic expression programs inthe response of yeast cells to environmental changes. Mol Biol Cell, 11(12):4241–57, 2000.[148] A. P. Schmitt and K. McEntee. Msn2p, a zinc finger dna-binding protein, isthe transcriptional activator of the multistress response in saccharomycescerevisiae. Proc Natl Acad Sci U S A, 93(12):5777–82, 1996.[149] A. Sadeh, D. Baran, M. Volokh, and A. Aharoni. Conserved motifs in themsn2-activating domain are important for msn2-mediated yeast stressresponse. J Cell Sci, 125(Pt 14):3333–42, 2012.[150] W. Gorner, E. Durchschlag, J. Wolf, E. L. Brown, G. Ammerer, H. Ruis,and C. Schuller. Acute glucose starvation activates the nuclear localizationsignal of a stress-specific yeast transcription factor. EMBO J, 21(1-2):135–44, 2002.[151] E. Durchschlag, W. Reiter, G. Ammerer, and C. Schuller. Nuclearlocalization destabilizes the stress-regulated transcription factor msn2. JBiol Chem, 279(53):55425–32, 2004.[152] A. Santhanam, A. Hartley, K. Duvel, J. R. Broach, and S. Garrett. Pp2aphosphatase activity is required for stress and tor kinase regulation of yeaststress response factor msn2p. Eukaryot Cell, 3(5):1261–71, 2004.[153] M. Jacquet, G. Renault, S. Lallet, J. De Mey, and A. Goldbeter. Oscillatorynucleocytoplasmic shuttling of the general stress response transcriptionalactivators msn2 and msn4 in saccharomyces cerevisiae. J Cell Biol, 161(3):497–505, 2003.[154] S. Ghaemmaghami, W. K. Huh, K. Bower, R. W. Howson, A. Belle,N. Dephoure, E. K. O’Shea, and J. S. Weissman. Global analysis of proteinexpression in yeast. Nature, 425(6959):737–41, 2003.[155] Y. Liu, S. Ye, and A. M. Erkine. Analysis of saccharomyces cerevisiaegenome for the distributions of stress-response elements potentiallyaffecting gene expression by transcriptional interference. In Silico Biol, 9(5-6):379–89, 2009.[156] J. Stewart-Ornstein, C. Nelson, J. DeRisi, J. S. Weissman, andH. El-Samad. Msn2 coordinates a stoichiometric gene expression program.Curr Biol, 23(23):2336–45, 2013.131[157] B. Chen, M. Retzlaff, T. Roos, and J. Frydman. Cellular strategies ofprotein quality control. Cold Spring Harb Perspect Biol, 3(8):a004374,2011.[158] P. D. Stenson, M. Mort, E. V. Ball, K. Shaw, A. Phillips, and D. N. Cooper.The human gene mutation database: building a comprehensive mutationrepository for clinical and molecular genetics, diagnostic testing andpersonalized genomic medicine. Hum Genet, 133(1):1–9, 2014.[159] N. Sahni, S. Yi, M. Taipale, J. I. Fuxman Bass, J. Coulombe-Huntington,F. Yang, J. Peng, J. Weile, G. I. Karras, Y. Wang, I. A. Kovacs,A. Kamburov, I. Krykbaeva, M. H. Lam, G. Tucker, V. Khurana,A. Sharma, Y. Y. Liu, N. Yachie, Q. Zhong, Y. Shen, A. Palagi,A. San-Miguel, C. Fan, D. Balcha, A. Dricot, D. M. Jordan, J. M. Walsh,A. A. Shah, X. Yang, A. K. Stoyanova, A. Leighton, M. A. Calderwood,Y. Jacob, M. E. Cusick, K. Salehi-Ashtiani, L. J. Whitesell, S. Sunyaev,B. Berger, A. L. Barabasi, B. Charloteaux, D. E. Hill, T. Hao, F. P. Roth,Y. Xia, A. J. Walhout, S. Lindquist, and M. Vidal. Widespreadmacromolecular interaction perturbations in human genetic disorders. Cell,161(3):647–60, 2015.[160] P. G. Richardson, B. Barlogie, J. Berenson, S. Singhal, S. Jagannath,D. Irwin, S. V. Rajkumar, G. Srkalovic, M. Alsina, R. Alexanian, D. Siegel,R. Z. Orlowski, D. Kuter, S. A. Limentani, S. Lee, T. Hideshima, D. L.Esseltine, M. Kauffman, J. Adams, D. P. Schenkein, and K. C. Anderson.A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl JMed, 348(26):2609–17, 2003.[161] G. Nalepa, M. Rolfe, and J. W. Harper. Drug discovery in theubiquitin-proteasome system. Nat Rev Drug Discov, 5(7):596–613, 2006.[162] S. Khare, A. S. Nagle, A. Biggart, Y. H. Lai, F. Liang, L. C. Davis, S. W.Barnes, C. J. Mathison, E. Myburgh, M. Y. Gao, J. R. Gillespie, X. Liu,J. L. Tan, M. Stinson, I. C. Rivera, J. Ballard, V. Yeh, T. Groessl, G. Federe,H. X. Koh, J. D. Venable, B. Bursulaya, M. Shapiro, P. K. Mishra,G. Spraggon, A. Brock, J. C. Mottram, F. S. Buckner, S. P. Rao, B. G. Wen,J. R. Walker, T. Tuntland, V. Molteni, R. J. Glynne, and F. Supek.Proteasome inhibition for treatment of leishmaniasis, chagas disease andsleeping sickness. Nature, 537(7619):229–233, 2016.[163] T. Kirkegaard, J. Gray, D. A. Priestman, K. L. Wallom, J. Atkins, O. D.Olsen, A. Klein, S. Drndarski, N. H. Petersen, L. Ingemann, D. A. Smith,132L. Morris, C. Bornaes, S. H. Jorgensen, I. Williams, A. Hinsby, C. Arenz,D. Begley, M. Jaattela, and F. M. Platt. Heat shock protein-based therapy asa potential candidate for treating the sphingolipidoses. Sci Transl Med, 8(355):355ra118, 2016.[164] K. Kuk and J. L. Taylor-Cousar. Lumacaftor and ivacaftor in themanagement of patients with cystic fibrosis: current evidence and futureprospects. Ther Adv Respir Dis, 9(6):313–26, 2015. Kuk, KellyTaylor-Cousar, Jennifer L Research Support, N.I.H., Extramural ResearchSupport, Non-U.S. Gov’t Review England Therapeutic advances inrespiratory disease Ther Adv Respir Dis. 2015 Dec;9(6):313-26. doi:10.1177/1753465815601934. Epub 2015 Sep 28.[165] A. R. Schoenfeld, E. J. Davidowitz, and R. D. Burk. Elongin bc complexprevents degradation of von hippel-lindau tumor suppressor gene products.Proc Natl Acad Sci U S A, 97(15):8507–12, 2000.[166] M. Knop, A. Finger, T. Braun, K. Hellmuth, and D. H. Wolf. Der1, a novelprotein specifically required for endoplasmic reticulum degradation inyeast. EMBO J, 15(4):753–63, 1996.[167] M. J. Maurer, E. D. Spear, A. T. Yu, E. J. Lee, S. Shahzad, andS. Michaelis. Degradation signals for ubiquitin-proteasome dependentcytosolic protein quality control (cytoqc) in yeast. G3 (Bethesda), 2016.[168] W. Seufert, B. Futcher, and S. Jentsch. Role of a ubiquitin-conjugatingenzyme in degradation of s- and m-phase cyclins. Nature, 373(6509):78–81, 1995.[169] J. Betting and W. Seufert. A yeast ubc9 mutant protein withtemperature-sensitive in vivo function is subject to conditional proteolysisby a ubiquitin- and proteasome-dependent pathway. J Biol Chem, 271(42):25790–6, 1996.[170] O. Shimomura, F. H. Johnson, and Y. Saiga. Extraction, purification andproperties of aequorin, a bioluminescent protein from the luminoushydromedusan, aequorea. J Cell Comp Physiol, 59:223–39, 1962.[171] R. Heim, D. C. Prasher, and R. Y. Tsien. Wavelength mutations andposttranslational autoxidation of green fluorescent protein. Proc Natl AcadSci U S A, 91(26):12501–4, 1994.133[172] D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J.Cormier. Primary structure of the aequorea victoria green-fluorescentprotein. Gene, 111(2):229–33, 1992.[173] R. Heim, A. B. Cubitt, and R. Y. Tsien. Improved green fluorescence.Nature, 373(6516):663–4, 1995.[174] H. C. Yen, Q. Xu, D. M. Chou, Z. Zhao, and S. J. Elledge. Global proteinstability profiling in mammalian cells. Science, 322(5903):918–23, 2008.[175] H. C. Yen and S. J. Elledge. Identification of scf ubiquitin ligase substratesby global protein stability profiling. Science, 322(5903):923–9, 2008.[176] A. Khmelinskii, P. J. Keller, A. Bartosik, M. Meurer, J. D. Barry, B. R.Mardin, A. Kaufmann, S. Trautmann, M. Wachsmuth, G. Pereira,W. Huber, E. Schiebel, and M. Knop. Tandem fluorescent protein timers forin vivo analysis of protein dynamics. Nat Biotechnol, 30(7):708–14, 2012.[177] J. F. Morley, H. R. Brignull, J. J. Weyers, and R. I. Morimoto. Thethreshold for polyglutamine-expansion protein aggregation and cellulartoxicity is dynamic and influenced by aging in caenorhabditis elegans.Proc Natl Acad Sci U S A, 99(16):10417–22, 2002.[178] S. A. Comyn, G. T. Chan, and T. Mayor. False start: cotranslational proteinubiquitination and cytosolic protein quality control. J Proteomics, 100:92–101, 2014.[179] F. U. Hartl, A. Bracher, and M. Hayer-Hartl. Molecular chaperones inprotein folding and proteostasis. Nature, 475(7356):324–32, 2011.[180] K. Schneider and A. Bertolotti. Surviving protein quality controlcatastrophes - from cells to organisms. J Cell Sci, 128(21):3861–9, 2015.[181] G. Kleiger and T. Mayor. Perilous journey: a tour of theubiquitin-proteasome system. Trends Cell Biol, 24(6):352–9, 2014.[182] N. N. Fang and T. Mayor. Hul5 ubiquitin ligase: good riddance to badproteins. Prion, 6(3):240–4, 2012.[183] J. Tyedmers, S. Treusch, J. Dong, J. M. McCaffery, B. Bevis, andS. Lindquist. Prion induction involves an ancient system for thesequestration of aggregated proteins and heritable changes in prionfragmentation. Proc Natl Acad Sci U S A, 107(19):8633–8, 2010.134[184] U. Lenk and T. Sommer. Ubiquitin-mediated proteolysis of a short-livedregulatory protein depends on its cellular localization. J Biol Chem, 275(50):39403–10, 2000.[185] S. Ben-Aroya, C. Coombes, T. Kwok, K. A. O’Donnell, J. D. Boeke, andP. Hieter. Toward a comprehensive temperature-sensitive mutant repositoryof the essential genes of saccharomyces cerevisiae. Mol Cell, 30(2):248–58, 2008.[186] S. Ben-Aroya, X. Pan, J. D. Boeke, and P. Hieter. Makingtemperature-sensitive mutants. Methods Enzymol, 470:181–204, 2010.[187] A. Berger, E. Schiltz, and G. E. Schulz. Guanylate kinase fromsaccharomyces cerevisiae. isolation and characterization, crystallizationand preliminary x-ray analysis, amino acid sequence and comparison withadenylate kinases. Eur J Biochem, 184(2):433–43, 1989.[188] J. Blaszczyk, Y. Li, H. Yan, and X. Ji. Crystal structure of unligatedguanylate kinase from yeast reveals gmp-induced conformational changes.J Mol Biol, 307(1):247–57, 2001.[189] Y. Shimma, A. Nishikawa, B. bin Kassim, A. Eto, and Y. Jigami. A defectin gtp synthesis affects mannose outer chain elongation in saccharomycescerevisiae. Mol Gen Genet, 256(5):469–80, 1997.[190] J. Schymkowitz, J. Borg, F. Stricher, R. Nys, F. Rousseau, and L. Serrano.The foldx web server: an online force field. Nucleic Acids Res, 33(WebServer issue):W382–8, 2005.[191] M. J. Gallagher, L. Ding, A. Maheshwari, and R. L. Macdonald. The gabaareceptor alpha1 subunit epilepsy mutation a322d inhibits transmembranehelix formation and causes proteasomal degradation. Proc Natl Acad Sci US A, 104(32):12999–3004, 2007.[192] A. L. Pey, F. Stricher, L. Serrano, and A. Martinez. Predicted effects ofmissense mutations on native-state stability account for phenotypicoutcome in phenylketonuria, a paradigm of misfolding diseases. Am J HumGenet, 81(5):1006–24, 2007.[193] D. Martinez Molina, R. Jafari, M. Ignatushchenko, T. Seki, E. A. Larsson,C. Dan, L. Sreekumar, Y. Cao, and P. Nordlund. Monitoring drug targetengagement in cells and tissues using the cellular thermal shift assay.Science, 341(6141):84–7, 2013.135[194] C. Guerrero, T. Milenkovic, N. Przulj, P. Kaiser, and L. Huang.Characterization of the proteasome interaction network using a qtax-basedtag-team strategy and protein interaction network analysis. Proc Natl AcadSci U S A, 105(36):13333–8, 2008.[195] C. Zhou, B. D. Slaughter, J. R. Unruh, F. Guo, Z. Yu, K. Mickey,A. Narkar, R. T. Ross, M. McClain, and R. Li. Organelle-basedaggregation and retention of damaged proteins in asymmetrically dividingcells. Cell, 159(3):530–42, 2014.[196] J. M. Daran, N. Dallies, D. Thines-Sempoux, V. Paquet, and J. Francois.Genetic and biochemical characterization of the ugp1 gene encoding theudp-glucose pyrophosphorylase from saccharomyces cerevisiae. Eur JBiochem, 233(2):520–30, 1995.[197] D. G. Yi and W. K. Huh. Udp-glucose pyrophosphorylase ugp1 is involvedin oxidative stress response and long-term survival during stationary phasein saccharomyces cerevisiae. Biochem Biophys Res Commun, 467(4):657–63, 2015.[198] M. Delarue. Aminoacyl-trna synthetases. Curr Opin Struct Biol, 5(1):48–55, 1995.[199] K. Galani, H. Grosshans, K. Deinert, E. C. Hurt, and G. Simos. Theintracellular location of two aminoacyl-trna synthetases depends oncomplex formation with arc1p. EMBO J, 20(23):6889–98, 2001.[200] M. C. Brandriss and D. A. Falvey. Proline biosynthesis in saccharomycescerevisiae: analysis of the pro3 gene, which encodes delta1-pyrroline-5-carboxylate reductase. J Bacteriol, 174(11):3782–8, 1992.[201] J. W. Yewdell, J. R. Lacsina, M. C. Rechsteiner, and C. V. Nicchitta. Outwith the old, in with the new? comparing methods for measuring proteindegradation. Cell Biol Int, 35(5):457–62, 2011.[202] A. Abe, K. Takahashi-Niki, Y. Takekoshi, T. Shimizu, H. Kitaura, H. Maita,S. M. Iguchi-Ariga, and H. Ariga. Prefoldin plays a role as a clearancefactor in preventing proteasome inhibitor-induced protein aggregation. JBiol Chem, 288(39):27764–76, 2013.[203] H. Lee do, M. Y. Sherman, and A. L. Goldberg. The requirements of yeasthsp70 of ssa family for the ubiquitin-dependent degradation of short-livedand abnormal proteins. Biochem Biophys Res Commun, 475(1):100–6,2016.136[204] M. Sakono, T. Zako, H. Ueda, M. Yohda, and M. Maeda. Formation ofhighly toxic soluble amyloid beta oligomers by the molecular chaperoneprefoldin. FEBS J, 275(23):5982–93, 2008.[205] K. M. Sorgjerd, T. Zako, M. Sakono, P. C. Stirling, M. R. Leroux, T. Saito,P. Nilsson, M. Sekimoto, T. C. Saido, and M. Maeda. Human prefoldininhibits amyloid-beta (abeta) fibrillation and contributes to formation ofnontoxic abeta aggregates. Biochemistry, 52(20):3532–42, 2013.[206] S. A. Comyn, B. P. Young, C. J. Loewen, and T. Mayor. Prefoldin promotesproteasomal degradation of cytosolic proteins with missense mutations bymaintaining substrate solubility. PLoS Genet, 12(7):e1006184, 2016.[207] F. Tsukahara and Y. Maru. Bag1 directly routes immature bcr-abl forproteasomal degradation. Blood, 116(18):3582–92, 2010.[208] D. Sambrook J., Russell. Molecular Cloning: A Laboratory Manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, 3 edition, 2001.[209] H. Li and R. Durbin. Fast and accurate long-read alignment withburrows-wheeler transform. Bioinformatics, 26(5):589–95, 2010.[210] H. Li, B. Handsaker, A. Wysoker, T. Fennell, J. Ruan, N. Homer, G. Marth,G. Abecasis, and R. Durbin. The sequence alignment/map format andsamtools. Bioinformatics, 25(16):2078–9, 2009.[211] J. T. Robinson, H. Thorvaldsdottir, W. Winckler, M. Guttman, E. S. Lander,G. Getz, and J. P. Mesirov. Integrative genomics viewer. Nat Biotechnol,29(1):24–6, 2011.[212] H. Thorvaldsdottir, J. T. Robinson, and J. P. Mesirov. Integrative genomicsviewer (igv): high-performance genomics data visualization andexploration. Brief Bioinform, 14(2):178–92, 2013.[213] O. Foresti, V. Rodriguez-Vaello, C. Funaya, and P. Carvalho. Qualitycontrol of inner nuclear membrane proteins by the asi complex. Science,346(6210):751–5, 2014.[214] W. C. Cheng, X. Teng, H. K. Park, C. M. Tucker, M. J. Dunham, and J. M.Hardwick. Fis1 deficiency selects for compensatory mutations responsiblefor cell death and growth control defects. Cell Death Differ, 15(12):1838–46, 2008.137[215] G. I. Lang, D. P. Rice, M. J. Hickman, E. Sodergren, G. M. Weinstock,D. Botstein, and M. M. Desai. Pervasive genetic hitchhiking and clonalinterference in forty evolving yeast populations. Nature, 500(7464):571–4,2013.[216] X. Teng, M. Dayhoff-Brannigan, W. C. Cheng, C. E. Gilbert, C. N. Sing,N. L. Diny, S. J. Wheelan, M. J. Dunham, J. D. Boeke, F. J. Pineda, andJ. M. Hardwick. Genome-wide consequences of deleting any single gene.Mol Cell, 52(4):485–94, 2013.[217] M. T. Martinez-Pastor, G. Marchler, C. Schuller, A. Marchler-Bauer,H. Ruis, and F. Estruch. The saccharomyces cerevisiae zinc finger proteinsmsn2p and msn4p are required for transcriptional induction through thestress response element (stre). EMBO J, 15(9):2227–35, 1996.[218] H. Garreau, R. N. Hasan, G. Renault, F. Estruch, E. Boy-Marcotte, andM. Jacquet. Hyperphosphorylation of msn2p and msn4p in response toheat shock and the diauxic shift is inhibited by camp in saccharomycescerevisiae. Microbiology, 146 ( Pt 9):2113–20, 2000.[219] W. Gorner, E. Durchschlag, M. T. Martinez-Pastor, F. Estruch,G. Ammerer, B. Hamilton, H. Ruis, and C. Schuller. Nuclear localizationof the c2h2 zinc finger protein msn2p is regulated by stress and proteinkinase a activity. Genes Dev, 12(4):586–97, 1998.[220] M. G. Buse and S. S. Reid. Leucine. a possible regulator of proteinturnover in muscle. J Clin Invest, 56(5):1250–61, 1975.[221] A. K. Said and D. M. Hegsted. Response of adult rats to low dietary levelsof essential amino acids. J Nutr, 100(11):1363–75, 1970.[222] S. C. Schriever, M. J. Deutsch, J. Adamski, A. A. Roscher, andR. Ensenauer. Cellular signaling of amino acids towards mtorc1 activationin impaired human leucine catabolism. J Nutr Biochem, 24(5):824–31,2013.[223] H. Shahsavarani, M. Sugiyama, Y. Kaneko, B. Chuenchit, andS. Harashima. Superior thermotolerance of saccharomyces cerevisiae forefficient bioethanol fermentation can be achieved by overexpression of rsp5ubiquitin ligase. Biotechnol Adv, 30(6):1289–300, 2012.[224] F. Cardona, A. Aranda, and M. del Olmo. Ubiquitin ligase rsp5p isinvolved in the gene expression changes during nutrient limitation insaccharomyces cerevisiae. Yeast, 26(1):1–15, 2009.138[225] Y. Haitani and H. Takagi. Rsp5 is required for the nuclear export of mrnaof hsf1 and msn2/4 under stress conditions in saccharomyces cerevisiae.Genes Cells, 13(2):105–16, 2008.[226] D. J. Omnus and P. O. Ljungdahl. Latency of transcription factor stp1depends on a modular regulatory motif that functions as cytoplasmicretention determinant and nuclear degron. Mol Biol Cell, 25(23):3823–33,2014.[227] B. M. Kus, C. E. Caldon, R. Andorn-Broza, and A. M. Edwards.Functional interaction of 13 yeast scf complexes with a set of yeast e2enzymes in vitro. Proteins, 54(3):455–67, 2004.[228] A. R. Willems, T. Goh, L. Taylor, I. Chernushevich, A. Shevchenko, andM. Tyers. Scf ubiquitin protein ligases and phosphorylation-dependentproteolysis. Philos Trans R Soc Lond B Biol Sci, 354(1389):1533–50, 1999.[229] J. Kunz, A. Loeschmann, M. Deuter-Reinhard, and M. N. Hall. Fap1, ahomologue of human transcription factor nf-x1, competes with rapamycinfor binding to fkbp12 in yeast. Mol Microbiol, 37(6):1480–93, 2000.[230] M. Koegl, T. Hoppe, S. Schlenker, H. D. Ulrich, T. U. Mayer, andS. Jentsch. A novel ubiquitination factor, e4, is involved in multiubiquitinchain assembly. Cell, 96(5):635–44, 1999.[231] C. S. Hwang, A. Shemorry, D. Auerbach, and A. Varshavsky. The n-endrule pathway is mediated by a complex of the ring-type ubr1 and hect-typeufd4 ubiquitin ligases. Nat Cell Biol, 12(12):1177–85, 2010.[232] B. Szamecz, G. Boross, D. Kalapis, K. Kovacs, G. Fekete, Z. Farkas,V. Lazar, M. Hrtyan, P. Kemmeren, M. J. Groot Koerkamp, E. Rutkai, F. C.Holstege, B. Papp, and C. Pal. The genomic landscape of compensatoryevolution. PLoS Biol, 12(8):e1001935, 2014.[233] N. Mendl, A. Occhipinti, M. Muller, P. Wild, I. Dikic, and A. S. Reichert.Mitophagy in yeast is independent of mitochondrial fission and requires thestress response gene whi2. J Cell Sci, 124(Pt 8):1339–50, 2011.[234] J. van Leeuwen, C. Pons, J. C. Mellor, T. N. Yamaguchi, H. Friesen,J. Koschwanez, M. M. Usaj, M. Pechlaner, M. Takar, M. Usaj,B. VanderSluis, K. Andrusiak, P. Bansal, A. Baryshnikova, C. E. Boone,J. Cao, A. Cote, M. Gebbia, G. Horecka, I. Horecka, E. Kuzmin, N. Legro,W. Liang, N. van Lieshout, M. McNee, B. J. San Luis, F. Shaeri,139E. Shuteriqi, S. Sun, L. Yang, J. Y. Youn, M. Yuen, M. Costanzo, A. C.Gingras, P. Aloy, C. Oostenbrink, A. Murray, T. R. Graham, C. L. Myers,B. J. Andrews, F. P. Roth, and C. Boone. Exploring genetic suppressioninteractions on a global scale. Science, 354(6312), 2016.[235] D. Kaida, H. Yashiroda, A. Toh-e, and Y. Kikuchi. Yeast whi2 andpsr1-phosphatase form a complex and regulate stre-mediated geneexpression. Genes Cells, 7(6):543–52, 2002.[236] H. L. Fox, P. T. Pham, S. R. Kimball, L. S. Jefferson, and C. J. Lynch.Amino acid effects on translational repressor 4e-bp1 are mediatedprimarily by l-leucine in isolated adipocytes. Am J Physiol, 275(5 Pt 1):C1232–8, 1998.[237] T. Beck and M. N. Hall. The tor signalling pathway controls nuclearlocalization of nutrient-regulated transcription factors. Nature, 402(6762):689–92, 1999.[238] P. Lee, B. R. Cho, H. S. Joo, and J. S. Hahn. Yeast yak1 kinase, a bridgebetween pka and stress-responsive transcription factors, hsf1 andmsn2/msn4. Mol Microbiol, 70(4):882–95, 2008.[239] P. Lee, M. S. Kim, S. M. Paik, S. H. Choi, B. R. Cho, and J. S. Hahn.Rim15-dependent activation of hsf1 and msn2/4 transcription factors bydirect phosphorylation in saccharomyces cerevisiae. FEBS Lett, 587(22):3648–55, 2013.[240] D. H. Lee, M. Y. Sherman, and A. L. Goldberg. Involvement of themolecular chaperone ydj1 in the ubiquitin-dependent degradation ofshort-lived and abnormal proteins in saccharomyces cerevisiae. Mol CellBiol, 16(9):4773–81, 1996.[241] M. A. DePristo, D. M. Weinreich, and D. L. Hartl. Missense meanderingsin sequence space: a biophysical view of protein evolution. Nat Rev Genet,6(9):678–87, 2005.[242] G. V. Kryukov, L. A. Pennacchio, and S. R. Sunyaev. Most rare missensealleles are deleterious in humans: implications for complex disease andassociation studies. Am J Hum Genet, 80(4):727–39, 2007.[243] R. G. Gardner and R. Y. Hampton. A highly conserved signal controlsdegradation of 3-hydroxy-3-methylglutaryl-coenzyme a (hmg-coa)reductase in eukaryotes. J Biol Chem, 274(44):31671–8, 1999.140[244] A. Belle, A. Tanay, L. Bitincka, R. Shamir, and E. K. O’Shea.Quantification of protein half-lives in the budding yeast proteome. ProcNatl Acad Sci U S A, 103(35):13004–9, 2006.[245] T. Gidalevitz, A. Ben-Zvi, K. H. Ho, H. R. Brignull, and R. I. Morimoto.Progressive disruption of cellular protein folding in models ofpolyglutamine diseases. Science, 311(5766):1471–4, 2006.[246] J. H. Feder, J. M. Rossi, J. Solomon, N. Solomon, and S. Lindquist. Theconsequences of expressing hsp70 in drosophila cells at normaltemperatures. Genes Dev, 6(8):1402–13, 1992.[247] R. A. Krebs and M. E. Feder. Deleterious consequences of hsp70overexpression in drosophila melanogaster larvae. Cell Stress Chaperones,2(1):60–71, 1997.[248] H. Frauenfelder, S. G. Sligar, and P. G. Wolynes. The energy landscapesand motions of proteins. Science, 254(5038):1598–603, 1991.[249] L. Whitesell and S. L. Lindquist. Hsp90 and the chaperoning of cancer.Nat Rev Cancer, 5(10):761–72, 2005.[250] T. Hart, M. Chandrashekhar, M. Aregger, Z. Steinhart, K. R. Brown,G. MacLeod, M. Mis, M. Zimmermann, A. Fradet-Turcotte, S. Sun,P. Mero, P. Dirks, S. Sidhu, F. P. Roth, O. S. Rissland, D. Durocher,S. Angers, and J. Moffat. High-resolution crispr screens reveal fitnessgenes and genotype-specific cancer liabilities. Cell, 163(6):1515–26, 2015.141


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items