@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix dc: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Biochemistry and Molecular Biology, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Dambrowitz, Kirsten Amy"@en ; dcterms:issued "2009-10-07T00:00:00"@en, "2001"@en ; vivo:relatedDegree "Doctor of Philosophy - PhD"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """Ste12p is a transcriptional activator and an effector of two MAP kinase cascades in the yeast Saccharomyces cerevisiae. Ste12p activates transcription required for both pheromone response and filamentous growth. The goal of this work was to further define the domain structure of Ste12p and the mechanisms that govern Ste12p activity; In response to mating pheromone, Ste12p activates transcription of FAR1. Farlp interacts with the cell cycle machinery in order to cause G1 growth arrest. This work showed that overexpression of Ste12p in the absence of pheromone induced G1 growth arrest that was independent of FAR1 and of transcription from pheromone responsive elements. The growth arrest did not reflect cell death and could be eliminated by overexpression of Rst1p and Rst2p, two negative regulators of Ste12p. These data indicated a novel role for Ste12p in pheromone-responsive growth arrest. Rst1p and Rst2p, two negative regulators of Ste12p, are genetically redundant and, as such, are proposed to regulate Ste12p function by a common mechanism. This study demonstrated that overexpression of amino acids 262 to 594 of Ste12p activated transcription and that concurrent overexpression of either RST1 or RST2 abrogated that activation. The region 262 to 594 was able to interact with Rst1 p, but not with Rst2p, in yeast extracts. Rst2p, in contrast, interacted with the DNA binding domain of Ste12p. Further, Rst2p, but not Rst1p, was shown to inhibit interaction of the Ste12p DNA binding domain with DNA in vitro and to inhibit the function of a Ste12pDBD-VP16 fusion in vivo. In addition, recombinant Rst1p and Rst2p were shown to interact directly with these distinct domains of Ste12p in vitro. These results showed that Rst1p and Rst2p function by non-identical mechanisms, as they interact with separate domains of Ste12p in order to inhibit its function."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/13674?expand=metadata"@en ; dcterms:extent "9296449 bytes"@en ; dc:format "application/pdf"@en ; skos:note "DOMAIN FUNCTION AND REGULATION OF Ste12p by Kirsten Amy Dambrowitz B.Sc.(Hons), Queen's University, 1994 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE FACULTY OF G R A D U A T E STUDIES Department of Biochemistry and Molecular Biology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A June, 2001 © Kirsten Amy Dambrowitz, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Abstract Ste12p is a transcriptional activator and an effector of two MAP kinase cascades in the yeast Saccharomyces cerevisiae. Ste12p activates transcription required for both pheromone response and filamentous growth. The goal of this work was to further define the domain structure of Ste12p and the mechanisms that govern Ste12p activity; In response to mating pheromone, Ste12p activates transcription of FAR1. Far lp interacts with the cell cycle machinery in order to cause G1 growth arrest. This work showed that overexpression of Ste12p in the absence of pheromone induced G1 growth arrest that was independent of FAR1 and of transcription from pheromone responsive elements. The growth arrest did not reflect cell death and could be eliminated by overexpression of Rst1 p and Rst2p, two negative regulators of Ste12p. These data indicated a novel role for Ste12p in pheromone-responsive growth arrest. Rs t lp and Rst2p, two negative regulators of Ste12p, are genetically redundant and, as such, are proposed to regulate Ste12p function by a common mechanism. This study demonstrated that overexpression of amino acids 262 to 594 of Ste12p activated transcription and that concurrent overexpression of either RST1 or RST2 abrogated that activation. The region 262 to 594 was able to interact with Rst1 p, but not with Rst2p, in yeast extracts. Rst2p, in contrast, interacted with the DNA binding domain of Ste12p. Further, Rst2p, but not Rst lp , was shown to inhibit interaction of the Ste12p DNA binding domain with ii Abstract DNA in vitro and to inhibit the function of a Ste12pDBD-VP16 fusion in vivo. In addition, recombinant Rst lp and Rst2p were shown to interact directly with these distinct domains of Ste12p in vitro. These results showed that Rs t lp and Rst2p function by non-identical mechanisms, as they interact with separate domains of Ste12p in order to inhibit its function. iii Table of Contents Table of Contents Abstract ii Table of Contents iv List of Tables xi List of Figures xii List of Abbreviations xv Acknowledgements xix 1 Introduction 1 1.1 MAP kinase cascades affect gene expression in eukaryotes 1 1.1.1 Function and regulation of MAP kinase cascade components 1 1.1.2 MAP kinase cascades in Saccharomyces cerevisiae 3 1.1.3 The five MAP kinase cascades of yeast 3 1.2 The pheromone response pathway - A model MAP kinase cascade 6 1.2.1 Components of the P R P which function upstream of Ste12p 7 1.2.1.1 The cell surface receptors 7 1.2.1.2 The complex of Ste4p-associated P R P proteins 8 Ste5p interacts with Ste4p 8 Ste20p interacts with Ste4p 10 iv Table of Contents Ste50p associates with membrane-localized proteins 10 Ste4p-associated proteins localize to the shmoo tip 11 1.2.2 The MAP kinase cascade - S t e i l p , Ste7p and Fus3p 12 1.2.2.1 Fus3p'activates Ste12p and other effectors of the P R P 13 1.2.2.2 Fus3p and K s s l p function in different pathways. 14 1.2.3 Recovery from Pheromone Response 15 1.2.3.1 P R P signal attenuation at the cell surface 15 1.2.3.2 Attenuation of MAP kinase activity 17 1.2.3.3 P R P signal attenuation and transcription 18 1.2.4 Filamentous development: the pseudohyphal development and invasive growth pathways share components of the P R P 20 1.2.4.1 The phenotype of filamentous development 20 1.2.4.2 Signal transduction and filamentous development 21 1.2.4.3 The filamentation-invasion MAP kinase pathway 21 1.2.4.4 Maintaining MAP kinase cascade signaling specificity 22 1.2.5 The pheromone response and the cell cycle : 28 1.2.5.1 Cdc28p - the cell cycle kinase 28 1.2.5.2 The cyclins and CKIs of Start 29 1.2.5.3 Pheromone induced G1 arrest 31 Far lp is a pheromone-induced CKI 33 Additional mechanisms for growth arrest 36 1.2.5.4 Cdc28p activity impacts on the P R P 37 v Table of Contents 1.3 Ste12p - A transcription factor for MAP kinase-induced development.... 38 1.3.1 Important features of Ste12p function 38 1.3.2 DNA binding and protein-protein interactions of Ste12p 39 1.3.2.1 Pheromone responsive elements 39 1.3.2.2 Categories of Ste12p responsive promoters 42 1.3.3 The interaction of Ste12p with other transcriptional activators 43 1.3.4 MAP kinase-dependent phosphorylation of Ste12p 45 1.3.5 Regulatory domains of Ste12p 46 1.3.6 Overexpression of Ste12p... 47 1.3.7 Rst1 p (Dig1 p) and Rst2p (Dig2p) are MAP kinase substrates and negative regulators of Ste12p 48 1.3.7.1 RST1 and RST2 are redundant genes with non-identical functions 49 1.4 Transcriptional activators cause increased transcription by RNA polymerase II , 50 1.4.1 TFMD and TFIIA 53 1.4.2 The RNA polymerase II holoenzyme 56 1.4.2.1 The core components of RNA polymerase II 56 1.4.2.2 The Mediator complex 58 1.4.2.3 The general transcription factors 59 1.4.3 Accessory complexes alter chromatin structure 61 1.4.3.1 The SWI/SNF complex alters chromatin structure 61 vi Table of Contents 1.4.3.2 The S A G A complex contains a histone acetyltransferase.. 62 1.4.4 Activators recruit the complexes required for transcription to promoters 64 1.5 Project objectives 66 2 The phenotypes associated with Ste12p overexpression 68 2.1 Overexpression of Ste12p results in increased expression of FUS1 reporter genes 68 2.1.1 Induction of FUS1-lacZ 69 2.1.2 Induction of FUS1-HIS3 71 2.1.3 Contribution of the P R P to increased transcription 74 2.2 Overexpression of Ste12p results in growth arrest 76 2.2.1 Farlp-independent growth arrest 78 2.2.2 G1 growth arrest 78 2.2.3 Overexpression of nuclear localized Ste12pADBD causes growth arrest 80 2.2.4 Participation of the G1 cyclins 82 2.2.5 Ste12p overexpression does not reduce the viability of yeast.... 82 2.3 Overexpression of Ste12p increases mating in both STE12 and ste12 strains 87 vii Table of Contents 2.4 Nested deletions of STE12 show that amino acids 262 to 594 are sufficient to induce transcription and growth arrest 90 3 Rstlp and Rst2p interact with distinct regions of Ste12p 95 3.1 Overexpression of Rst1 p and Rst2p abrogates the effects of Ste12p overexpression 95 3.1.1 Rst lp and Rst2p inhibit growth arrest 95 3.1.2 Rst1 p and Rst2p reduce FUS1-lacZ expression 96 3.2 Rst lp and Rst2p interact with distinct regions of Ste12p 100 3.2.1 Rst lp interacts with amino acids 262 to 594 of Ste12p.. 105 3.2.2 Rst2p interacts with the DBD of Ste12p 105 3.2.3 Rst1 p interacts directly with Ste12p 107 3.3 Rst2p interacts with the Ste12p DNA binding domain in vivo 110 3.4 Rst2p disrupts the interaction of Ste12p with DNA in vitro 113 3.5 Accumulation of Ste12p is unnecessary for FUS1 induction in response to pheromone 115 4 Discussion 122 4.1 A mechanism for Farlp-independent G1 growth arrest 123 4.2 Interaction of Ste12p with negative regulators 125 viii Table of Contents 4.3 A mechanism for regulation of Ste12p by Rst2p 126 4.4 Mechanisms for regulation of Ste12p by Rst1 p 129 4.5 Rst1 p and Rst2p interact with different regions of Ste12p 130 4.6 Pheromone-responsive activation of transcription by Ste12p 132 4.7 A revised model for Ste12p regulation 133 4.8 Future work - Ste12p as a model transcription factor 136 4.9 Conclusion 138 5 Materials and Methods 140 5.1 Plasmids, strains and media 140 5.2 B-galactosidase assays 149 5.3 G1 Growth Arrest Assay 150 5.4 Quantitative Mating Assays 150 5.5 Expression and purification of recombinant proteins from E. coli 151 5.6 Expression of recombinant Ste12p in Spodoptera frugiperda (Sf9) cells152 5.7 Protein affinity precipitation of recombinant proteins 152 5.8 S D S - P A G E and western blot for detection of proteins 153 ix Table of Contents 5.9 Metabolic labeling and affinity precipitation of Ste12p 154 5.10 Northern and Southern blots 155 5.11 Electrophoretic Mobility Shift Assays 156 References................. ; >. 157 x List of Tables List of Tables Table 1. Promoters that respond to Ste12p 24 Table 2. G1 growth arrest by Ste12p overexpression does not reflect cell death.... 85 Table 3. G1 growth arrest by Ste12p overexpression does not reduce viability in farl yeast 86 Table 4. Ste12p overexpression increases mating efficiency 89 Table 5. Growth arrest by Ste12p deletion mutants 94 Table 6. Plasmids ..• 141 Table 7. Yeast Strains 146 Table 8. E. coli Strains 147 Table 9. Oligonucleotides for construction of STE12 deletions 148 xi List of Figures List of Figures Figure 1. The pheromone response pathway.... 2 Figure 2. MAP kinase cascades in yeast 5 Figure 3. Fus3p and K s s l p function in separate pathways 16 Figure 4. Promoters that respond to Ste12p 23 Figure 5. Pheromone response and the G1 to S transition 32 Figure 6. Features of Ste12p 40 Figure 7. A model for transcriptional activation 52 Figure 8. Overexpression of Ste12p induces FUS1-LacZ transcription 70 Figure 9. Overexpression of some domains of Ste12p induces FUS1-LacZ expression 72 Figure 10. Ste12p(215-473) induces FUS1-HIS3 transcription ....73 Figure 11. Induction of FUS1-LacZ expression by Ste12p overexpression is partially dependent upon the MAP kinase of the P R P 75 Figure 12. Ste12p overexpression induces growth arrest in both STE12 and ste12 yeast 77 Figure 13. Ste12p overexpression induces growth arrest in a F/\\R7-independent manner 79 Figure 14. Ste12p overexpression causes G1 growth arrest in a FAR1-independent manner ..81 Figure 15. Growth arrest by overexpression of Ste12pADBD/NLS 83 xii List of Figures Figure 16. Overexpression of CLN1 or CLN2 partially relieves growth arrest induced by Ste12p overexpression 84 Figure 17. Nested deletions of STE12 91 Figure 18. Amino acids 262 to 594 of Ste12p induce FUS1-LacZ transcription when overexpressed 92 Figure 19. Overexpression of Rst lp or Rst2p relieves growth arrest caused by Ste12p overexpression 97 Figure 20. Overexpression of Rst2p does not relieve growth arrest by overexpression of Ste12ADBD in ste12 yeast 98 Figure 21. Overexpression of Rst1 p or Rst2p inhibits induction of FUS1-LacZ by Ste12p overexpression 99 Figure 22. Overexpression of Rs t lp or Rst2p inhibits induction of FUS1-LacZ in response to pheromone 101 Figure 23. Recombinant Rst lp and Rst2p interact with Ste12p in yeast extracts 102 Figure 24. Rst lp and Rst2p interact with Ste12p in insect cell lysates 104 Figure 25. Rst lp interacts with amino acids 262 to 594 of Ste12p ....106 Figure 26. Rst2p interacts with the DNA binding domain of Ste12p 108 Figure 27. Rst2p interacts with amino acids 21 to 195 of the DNA binding domain of Ste12p... 109 Figure 28. Rs t lp interacts directly with Ste12pADBD 111 Figure 29. Rst2p inhibits the function of the Ste12p DNA Binding Domain 112 xiii List of Figures Figure 30. Rst2p inhibits the function of the Ste12p DNA Binding Domain 114 Figure 31. Rst2p disrupts the interaction of Ste12pDBD with DNA in E M S A ... 116 Figure 32. Disruption of DNA binding with increasing amounts of Rst2p 117 Figure 33. FUS1 induction in response to pheromone does not require novel protein synthesis 119 Figure 34. Ste12p does not accumulate in yeast cells in response to pheromone induction 121 Figure 35. A new model for Ste12p regulation 135 xiv List of Abbreviations List of Abbreviat ions %w/v % v/v a P A Y a-Gal4p a-histidine a-Ste12p A aa bp C CAK CDK CTD dATP DBD DNA DTT EDTA percent weight per volume percent volume per volume alpha beta delta gamma anti-Gal4p anti-histidine anti-Ste12p (deoxy)adenosine amino acid base pairs (deoxy)cytidine CDK activating kinase cyclin dependent kinase C-terminal domain deoxyadenosine triphosphate DNA binding domain deoxyribonucleic acid dithiothreitol disodium ethylenediamine tetraacetate xv List of Abbreviations E M S A ERK E. coli FIP F R E G G A P G D P GST GTP his\" HOG Inr K. lactis MAP kinase MAPK MAPKK M A P K K K MEK MEKK mRNA MW Ni electrophoretic mobility shift assay extracellular signal regulated kinase Escherichia coli filamentation-invasion pathway filamentous response element (deoxy)guanosine GTPase activating protein guanosine diphosphate glutathione-s-transferase guanosine triphosphate yeast media containing no histidine high osmolarity glycerol initiator Kluyveromyces lactis mitogen activated protein kinase mitogen activated protein kinase mitogen activated protein kinase kinase mitogen activated protein kinase kinase kinase M A P / E R K kinase M A P / E R K kinase kinase messenger ribonucleic acid molecular weight nickel xvi List of Abbreviations NR negative regulation O D 6 0 0 optical density at 600 nanometers PIC pre-initiation complex PID pheromone induction domain P M S F phenylmethylsulfonyl fluoride Pol II RNA polymerase II P /PRE P element and pheromone response element PQ P element and Q element P R E pheromone responsive element Pro proline P R P pheromone response pathway RNA ribonucleic acid S. cerevisiae Saccharomyces cerevisiae S D S sodium dodecyl sulphate Ser serine S R E stress responsive element S R F serum response factor T (deoxy)thymidine T C S TEF-1 , T e d and Aba DNA consensus sequence TEY threonine-glutamic acid-tyrosine Thr threonine T P C K N-tosyl-L-phenylalanine chloromethylketone TRIS tris(hydroxymethyl)aminomethane xvii • List of Abbreviations threonine-unspecified amino acid-tyrosine tyrosine upstream activating sequence upstream repressing sequence wild type xviii Acknowledgements Acknowledgements I thank Dr. Ivan Sadowski for his guidance, advice and support. I am grateful to the members of my supervisory committee, Dr. Jim Kronstad, Dr. Michel Roberge and Dr. Charlie Boone, for their combined encouragement, fair criticism and direction. Special thanks to Dr. Jim Kronstad and his lab members for sharing their office space. I am indebted to the many past and present members of the Sadowski lab for challenging, teaching, tolerating and championing me. I heartily thank them for exposing me to new concepts, including coffee breaks and brew pubs. I thank Dr. Norm Dovichi and the members of the NLLL and UBUBT for new challenges, technologies and friendships. I am grateful to my friends and family, near and far, whose humour, impatience, and encouragement have given me inspiration and energy. I am particularly grateful to Mom, Dad and Rob for their constant love, motivation and sense of perspective. Most importantly, I thank Chris Dambrowitz, for reminding me of the light at the end of the tunnel, even when I didn't want to see it. xix Introduction 1 Introduction 1.1 MAP kinase cascades affect gene expression in eukaryotes 1.1.1 Function and regulation of MAP kinase cascade components In order to survive and flourish in a constantly changing environment, all cells must be able to detect and respond to environmental signals. In many cases, the signal to which a given cell responds is detected at the cell surface and must be transmitted to intracellular or intranuclear effector molecules, which include transcription factors like Ste12p. One archetypal signal transduction pathway is the MAP kinase cascade, a three component signaling module that is conserved throughout eukarya (Gustin et al., 1998; Widmann et al., 1999). The three conserved components of a MAP kinase cascade are a MAP kinase (MAPK), a MAPK/extracellular signal-regulated kinase kinase (MAPKK or MEK) and a MAPK/extracellular regulated kinase kinase kinase (MAPKKK or MEKK). One example of a MAPK cascade is the pheromone response pathway, which is illustrated in Figure 1 (Bardwell et al., 1994; Gustin et al., 1998). The following sections will review MAP kinase cascades with particular focus on the Saccharomyces cerevisiae (yeast) pheromone response pathway and its effector, Ste12p. The activation of a MAP kinase cascade begins with an input signal, such as the activation of cell surface receptors by mating pheromone in yeast. The change in receptor activity activates an associated kinase (or kinases) or a GTP-1 Introduction p lasma membrane GTP polarized morphogenesis (|te12g) increase in transcription of pheromone-responsive genes PHEROMONE RESPONSE phosphorylation of Farlp Figure 1. The pheromone response pathway The pheromone response pathway in yeast is activated by pheromone binding to receptors at the cell surface. A signal transduction pathway, that includes a MAP kinase cascade, transmits the extracellular signal, culminating in the activation of Ste12p and Far lp , effectors of the pheromone response pathway. The tripartite pheromone response includes activation of transcription, G1 growth arrest and formation of a shmoo mating projection. Introduction associated protein, which, in turn, activates the MAPK cascade. The cascade of phosphorylations, from MEKK to MEK to MAPK, results in an active MAPK, which can phosphorylate its substrate proteins and stimulate changes in gene expression or protein activity within the cell. Most MAP kinase substrates are transcription factors, like Ste12p, although M A P kinases also phosphorylate kinases, phosphatases and other effector molecules. 1.1.2 M A P kinase cascades in Saccharomyces cerevisiae 1.1.3 The five M A P kinase cascades of yeast MAP kinase cascades have been studied in a wide range of eukaryotes, including Saccharomyces cerevisiae, Dictyostelium discoideum, Drosophila melanogaster and cultured mammalian cells (Widmann et al., 1999). Of these systems, the MAP kinases of yeast are the best characterized. With the advantages of genetic accessibility and a relatively small number of M A P kinases (6 yeast MAPKs, versus more than 12 mammalian MAPKs) , Saccharomyces cerevisiae is an excellent model for deciphering the mechanics of MAP kinase signaling (Madhani and Fink, 1998) (Garrington and Johnson, 1999; Widmann et al., 1999). The yeast Saccharomyces cerevisiae has five MAP kinase cascades that are required for pheromone response, invasive/filamentous growth, high osmolarity growth (HOG), cell integrity and spore wall assembly (Gustin et al., 1998; Madhani and Fink, 1998). The components of the pathways are described 3 Introduction in Figure 2, with predicted, but as yet undefined, components indicated by question marks. Each MAPK cascade in yeast has a unique MAP kinase, but other components of the signaling machinery are shared between the pathways. All of the pathways are believed to require an upstream kinase, such as Ste20p, which activates the MEKK of the pheromone response (PRP), filamentation-invasion (FIP) and high osmolarity glycerol (HOG) pathways (Leberer et al., 1992; Liu et al., 1993; Mosch et al., 1996; O'Rourke and Herskowitz, 1998; Ramer and Davis, 1993; Roberts and Fink, 1994). The MEK Ste7p is shared between invasive growth and pheromone response (Roberts and Fink, 1994) and Ste11p, a MEKK, is shared by the HOG, pheromone response and filamentation-invasion pathways (Posas and Saito, 1997; Roberts and Fink, 1994). Although the pathways share components, their input signals are distinct and their outputs are unique. For example, the P R P and FIP share an upstream kinase, MEKK and MEK, and activate a shared transcription factor, Ste12p, yet they control two independent developmental pathways in response to two different signals (Gustin et al., 1998). The different pathways may also have multiple functions under different growth conditions. The components of the FIP, for example, have been implicated in the maintenance of cell wall integrity during vegetative growth (Lee and Elion, 1999). In addition, the pathways may be activated sequentially. Global expression profiling shows that following P R P activation, the cell integrity 4 Introduction c o tio •o T— c (0 « \\ a z dep o o o o ° x: re .5>o E o c o > ro w v e o E o 1-(1) I-Q. HI LU Q. < H D Q. H co c CD c o Q . E o o CD E o co 0 i— CO x: CO O o o CO CO . °« CD ^ c E •- 2 co •S 0 \"O ffl-O CD co co -S 0 o Q. >> co co _ C0X3 .= O CO (0 0 0 ® m 3 T l CO zL 0 ^E'Q. g 5 o .= o ^ < Us •— **— CM j - 0 « * £ 2. CD 2 5Jb LL I— _ Q 5 Introduction pathway is activated, which may stabilize the cell during the formation of the shmoo mating projection (Roberts et al., 2000). There are six MAP kinases encoded by the yeast genome, five of which have been defined as members of the signaling cascades in Figure 2. The sixth kinase is Mlp lp, which is most similar to Mpklp, has been implicated as being a second MAP kinase in the cell integrity pathway signals (Gustin et al., 1998). Although the MAP kinases control several important life processes in Saccharomyces cerevisiae, they are not essential genes. A yeast strain with all six MAP kinase genes deleted remains viable, although it cannot mate, sporulate or regulate filamentation (Madhani et al., 1997). 1.2 The pheromone response pathway - A model MAP kinase cascade The yeast pheromone response pathway (PRP), shown in Figure 1, is an excellent model for studying MAP kinase regulation, and is one of the best characterized protein kinase cascades. When haploid yeast are exposed to pheromone from yeast of the opposite mating type, they rapidly (within one doubling time), differentiate into mating competent cells. The pheromone response includes G1 growth arrest, shmoo formation (a projection of the cell surface toward the nearest mating partner) and activation of transcription of pheromone responsive genes (Bardwell et al., 1994; Gustin et al., 1998). Pheromone responsive transcription is mediated through the transcription factor Ste12p, which is activated by the P R P . The pheromone response also includes activation of the genes and gene products that are 6 Introduction required for signal attenuation to allow reentry into the cell cycle in the absence of a mating partner (Dietzel and Kurjan, 1987a; Gustin et al., 1998; Kronstad et al., 1987). Regulation of Ste12p activity within the context of the pheromone response has been the major focus of this work. 1.2.1 Components of the PRP which function upstream of Ste12p 1.2.1.1 The cell surface receptors With the exception of cell-type specific pheromones and pheromone receptors, the P R P components are identical in a and a cells (Bardwell et al., 1994). The P R P is stimulated when pheromone from yeast of one mating type binds to a receptor on the cell surface of a yeast of the opposite mating type, a cells produce a factor, which is a 12-amino-acid farnesylated peptide exported from a cells by Ste6p, a transporter protein (Kuchler et al., 1989; McGrath and Varshavsky, 1989). a factor binds to Ste3p, the a factor receptor, at the cell surface of a cells and activates the P R P (Hagen et al., 1986). The P R P in a cells is stimulated by 13-amino-acid peptide a factor, produced and secreted from a cells, binding to Ste2p at the cell surface of a cells (Figure 1 )(Dmochowska et al., 1987; Jenness et al., 1983; Julius et al., 1983; Julius et al., 1984a; Julius et at, 1984b). Ste2p and Ste3p are members of the serpentine family of cell surface receptors, which have seven membrane-spanning hydrophobic a-helical domains (Dohlman et al., 1991). Intracellular domains of both Ste2p and Ste3p are 7 Introduction associated with a heterotrimeric G protein complex consisting of G p a l p (Ga), Ste4p (Gp) and Ste18p (Gy) (Boone et al., 1993; Clark et al., 1994; Stefan and Blumer, 1994). The association of Ste2p or Ste3p with pheromone results in a change in receptor conformation and a consequent dissociation of the G a from the G(3/y complex (Blinder et al., 1989; Dietzel and Kurjan, 1987b; Jahng et al., 1988; Kurjan and Dietzel, 1988; Miyajima et al., 1988; Whiteway et al., 1989). 1.2.1.2 The complex of Ste4p-associated PRP proteins The free G(3/y complex interacts with multiple proteins, including Ste5p, Ste20p, Ste50p, Far lp , Bemlp and Cdc42p (Cairns et al., 1992; Leberer et al., 1993; Stevenson et al., 1992; Whiteway et al., 1995). The protein complex is anchored to the plasma membrane through the prenylation and palmitoylation of Ste18p (Gy) (Hirschman et al., 1997; Hirschman and Jenness, 1999; Manahan et al., 2000; Pryciak and Huntress, 1998; Whiteway and Thomas, 1994). This complex of proteins at the cell surface propagates the pheromone signal, ultimately activating P R P effectors including Ste12p. Ste5p interacts with Ste4p Following pheromone stimulation, Ste5p is recruited to the plasma membrane by interaction with GB (Mahanty et al., 1999; Pryciak and Huntress, 1998; Whiteway et al., 1995). At the cell membrane, Ste5p acts as a scaffold for the kinases of the P R P MAPK cascade, associating with Ste11p (MEKK), Ste7p (MEK) and Fus3p (MAPK) (Choi etal . , 1999; Printen and Sprague, 1994). 8 Introduction Disruption of these interactions interrupts P R P signaling (Choi et al., 1994; Inouye et al., 1997a; Kranz et al., 1994; Marcus et al., 1994; Printen and Sprague, 1994). The interaction of the P R P kinases with Ste5p has two results. First, interaction with Ste5p increases the efficiency of P R P signaling by co-localizing the kinases of MAPK cascade and Ste20p, the kinase that phosphorylates and activates Ste11p. (Leberer et al., 1992; Ramer and Davis, 1993; van Drogen et al., 2000). Second; by co-localizing and sequestering active P R P components, Ste5p prevents inappropriate crosstalk between MAP kinase cascades (Choi et al., 2000; Yashar et al., 1995). In addition to recruitment of Ste5p to the plasma membrane, the interaction of Ste5p with Ste4p is postulated to result in conformational changes to Ste5p. According to this model, the conformation changes rearrange the scaffolded. P R P kinases, making them more accessible to Ste20p and/or increasing their ability to cross-phosphorylate one another (Inouye et al., 1997b; Sette et al., 2000; Yablonski et al., 1996). The interaction of Ste5p and Ste4p, and subsequent activation of the P R P , requires the shuttling of Ste5p through the nucleus (Mahanty et al., 1999). Ste5p shuttles through the nucleus during vegetative growth, and pheromone treatment results in enhanced nuclear export of Ste5p (Mahanty et al., 1999). The requirement for nuclear shuttling may prevent cytoplasmic Ste5p from activating the downstream kinases in the absence of pheromone. 9 Introduction Ste20p interacts with Ste4p Another P R P component that interacts with Ste4p is Ste20p, the founding member of the p21-activated kinase (PAK) family. Ste20p activates Ste11 p (MEKK) through phosphorylation of Ser302 and/or Ser306 and Thr307 in yeast (Leberer et al., 1992; Ramer and Davis, 1993; van Drogen et al., 2000). Phosphorylation of Ste11p by Ste20p is required to transmit the P R P signal from the Ste4p/Ste18p complex to Ste12p (Leberer et al., 1992; van Drogen et al., 2000; Wu et al., 1995). Ste20p activation requires the interaction of Ste20p and Cdc42p, another membrane-associated member of the P R P (Moskow et al., 2000; Simon et al., 1995; Zhao et al., 1995). In addition, the Cdc42p-Ste20p interaction is required to localize Ste20 to regions of polarized growth, like the shmoo tip (Leberer et al., 1997a; Leberer et al., 1997b; Moskow et al., 2000; Peter et al., 1996; Ziman et al., 1993). Ste50p associates with membrane-localized proteins Another member of the complex of P R P proteins at the cell membrane is Ste50p (Rad et al., 1998; Rad et al., 1992). Ste50p acts in the P R P between Ste4p/Ste18p and Ste11p. Ste50p associates with the N-termjnal domain of Ste11p and disrupts the interaction between the regulatory (N-terminal) and catalytic domains (C-terminal) of Ste11p in vitro (Wu et al., 1999). The same study demonstrates that the interaction of Ste50p with Ste11p is important but not essential for Ste11p activation in pheromone response. Since Ste50p and 10 Introduction Ste5p interact with adjacent regions of Ste11 p, it may be that these two interactions, in concert with the activity of Ste20p, work to activate Ste11p during pheromone response. Ste4p-associated proteins localize to the shmoo tip Cdc42p, a Rho-like small GTP binding protein, in conjunction with its guanine nucleotide exchange factor, (Cdc24p, regulates morphological changes in yeast, including the development of the shmoo mating projections that yeast develop prior to conjugation (Adams et al., 1990; Drubin and Nelson, 1996; Li et al., 1995a; Zheng et al., 1994; Ziman et al., 1993). The mating projections form in the direction of the nearest mating partner in a manner that is dependent on Cdc42p (Dorer et al., 1995; Nern and Arkowitz, 1998; Nern and Arkowitz, 1999; Schrick et al., 1997; Segall, 1993). The activation of Cdc42p in response to pheromone requires the nuclear export of Cdc24p-Far1p complexes, mediated by the exportin Msn5p (Blondel et al., 1999; Nern and Arkowitz, 2000; Shimada etal. , 2000). Multiple protein-protein interactions coordinate shmoo formation. The Ste4p-Ste20p-Cdc24p-Ste5p complex is proposed to limit pheromone signaling and shmoo formation to the regions of the cell surface immediately adjacent to the activated pheromone receptors (Nern and Arkowitz, 1998; Shimada et al., 2000; Zhao et al., 1995). Simultaneously, the Bem1p-Ste20p-Ste5p-actin-Far1p complex is postulated to tether the signaling pathway to the cytoskeleton (Butty et al., 1998; Leeuw et al., 1995; Lyons et al., 1996) and polarize the cytoskeleton 11 Introduction along the pheromone gradient (Valtz et al., 1995). Although there is genetic evidence for the function of these complexes, evidence of their physical existence is still required. 1.2.2 The MAP kinase cascade - Ste11 p, Ste7p and Fus3p Following interaction of Ste5p with activated Ste4p, the P R P MAP kinase cascade is activated. The MEKK of the P R P , Ste11 p, is required for the activation of Ste7p (MEK) and consists of two domains, a kinase domain and a regulatory domain. Deletion of the N-terminal regulatory domain converts Ste11 p to a constitutively active form, which can activate the pheromone response in the absence of upstream components of the P R P (Cairns et al., 1992; Ramer et al., 1992; Stevenson et al., 1992). The mechanism for pheromone responsive activation of Ste11p has not been determined, but Ste20p can activate Ste11p as a kinase and Ste50 and/or Ste5p may interact with the regulatory domain of Ste11p, resulting in an active MEKK (Leberer et al., 1992; Ramer and Davis, 1993; van Drogen et al., 2000; Wu et al., 1999; Wu et al., 1995). Next, Ste11p (MEKK) phosphorylates Ste7p (MEK) and stimulates its ability to phosphorylate Fus3p (MAPK) in vitro and in vivo (Cairns et al., 1992; Zhou et al., 1993). In vitro, Ste11p has been demonstrated to phosphorylate Ste7p at residue Thr363, which is analogous to the activating phosphorylation of mammalian MEK1 (MEK) by MEKK (MEKK) (Neiman and Herskowitz, 1994; Zheng and Guan, 1994). Mutagenesis studies have indicated that phosphorylation of Ste7p at residue Ser359 is also required for pheromone 12 Introduction response, but serine phosphorylation of Ste7p by Ste11p has not been demonstrated in vitro (Neiman and Herskowitz, 1994; Pages et al., 1994; Zheng and Guan, 1994). Ste7p, like other MEKs, is a dual specificity kinase and phosphorylates Fus3p and K s s l p on both threonine and tyrosine in a -TEY-sequence in response to pheromone (Errede et al., 1993; Gartner et al., 1992; Ma et al., 1995), Ste7p is associated with Fus3p and K s s l p in tight complexes, which may contribute to signaling specificity (Bardwell et al., 1996). 1.2.2.1 Fus3p activates Ste12p and other effectors of the P R P Fus3p is the MAP kinase of the P R P . Upon activation by Ste7p in response to pheromone, Fus3p phosphorylates the effector proteins of the P R P . Fus3p substrates include Ste12p, a transcriptional activator that mediates pheromone responsive transcription, and Far lp , an effector of both cell cycle arrest and chemotropism (Chang and Herskowitz, 1990; Chang and Herskowitz, 1992; Elion etal . , 1993; Hung etal . , 1997; Song etal . , 1991). Fus3p also phosphorylates Rs t lp (Diglp) and Rst2p (Dig2p), negative regulators of Ste12p (Cook et al., 1996; Tedford et al., 1997). Activation of Ste12p and relief of repression leads to increased transcription of pheromone-responsive genes, which allows the yeast to proceed through the mating reaction. Characterization of a series of partial function mutants of Fus3p demonstrated that mutations in the residues of Fus3p that are conserved between MAP kinases can selectively affect multiple aspects of pheromone response including Ste12p activation, G1 arrest, shmoo formation and recovery 13 Introduction (Farley et al., 1999). Further,.the work showed that not all of these Fus3p functions are dependent upon activation of transcription by Ste12p (Farley et al., 1999). 1.2.2.2 Fus3p and Kss1 p function in different pathways Fus3p is closely related to the MAPK Kss lp , a gene whose overexpression promotes recovery from pheromone response (Courchesne et al., 1989; Elion et al., 1990). Early work indicated that the functions of K s s l p and Fus3p were redundant, as the mating competence of a fus3A kssIA strain is lower than that of a fus3A strain, but further work has revealed differences in both function and regulation that point to Fus3p as the P R P MAP kinase (Elion et al., 1991). First, Fus3p expression is limited to haploids and FUS3 gene expression is pheromone-induced, while K s s l p is expressed in all cell types and KSS1 gene expression is not induced by pheromone (Elion et al., 1990; Liu et al., 1993). Second, overexpression of KSS1 promotes recovery from pheromone induced arrest, whereas FUS3 overexpression increases pheromone sensitivity in yeast (Courchesne et al., 1989; Elion et al., 1990). Finally, Fus3p facilitates growth arrest in response to pheromone, through the phosphorylation of Far1 p. Kss1 p does not have an apparent growth arrest function (Elion et al., 1991; Elion et al., 1990; Farley et al., 1999; Tyers and Futcher, 1993). K s s l p can function in pheromone response, but does not normally function as a major positive regulator of pheromone response and is only able to do so when FUS3 is deleted from the genome. In other words, the absence of 14 Introduction FUS3 from the genome results in inappropriate crosstalk between M A P K pathways (Madhani et al., 1997). Other results show that, although K s s l p may function in the P R P , it is only in a manner that is much more limited than Fus3p and does not affect the same range of substrate proteins (Farley et al., 1999). These results demonstrate that, in cells with intact FUS3 and KSS1 genes, Fus3p is the principal MAP kinase of the P R P , while K s s l p functions primarily in filamentation (Figure 3). Global gene expression profiling confirms that Fus3p is the dominant MAP kinase in pheromone response. In kssIA yeast, Fus3p directs transcription in response to mating pheromone that is very similar to the response seen in wild type cells. In contrast, the transcription profiles in fus3A cells exposed to pheromone feature preferential induction of genes that have been implicated in filamentation (Roberts et al., 2000). 1.2.3 Recovery from Pheromone Response 1.2.3.1 PRP signal attenuation at the cell surface After a period of exposure to pheromone, yeast that have not found a mating partner will recover from pheromone-induced cell cycle arrest and reenter the cell cycle. Attenuation of the pheromone signal is mediated by several mechanisms which affect the P R P at multiple levels, ultimately reducing the activity of P R P effectors including Ste12p. 15 Introduction p h e r o m o n e s tarvat ion act ivat ion of mating r e p r e s s i o n of f i lamentat ion act ivat ion of f i lamentat ion Figure 3. Fus3p and K s s l p function in separate pathways Current models suggest that Fus3p and Ksslp function in separate developmental pathways in yeast. Fus3p is required to activate Ste12p for mating in response to pheromone. Ksslp, which can both repress and activate Ste12p function, controls the activity of Ste12p-Tec1p dimers at filamentous response elements. Figure adapted from (Madhani and Fink, 1998). 16 Introduction There are several proteins which down-regulate pheromone response upstream of the MAP kinase cascade. Bar1 p is a protease that is secreted by a cells that cleaves a factor, inhibiting propagation of the pheromone signal. When a cells are exposed to a factor, the expression of BAR1 is induced (Kronstad et al., 1987; MacKay et al., 1988). Ste2p and Ste3p undergo feedback regulation, as well. Upon activation of the P R P , the cytoplasmic tails of the pheromone receptors become hyperphosphorylated, and Ste2p ubiquitinated. These modifications target Ste2p for endocytosis, which eventually leads to downregulation of pheromone response (Feng and Davis, 2000; Hicke et al., 1998; Schandel and Jenness, 1994). Another mediator of pheromone response attenuation is Sst2p. SST2, a pheromone-inducible gene, encodes a putative GTPase activating protein (GAP) f o rGpa lp (Ga) (Apanovitch et al., 1998; Dohlman et al., 1995; Dohlman et al., 1996). In order for Ga to dissociate from Gp/y, it must exchange a bound G D P for a GTP. Hydrolysis of G T P to G D P results in the re-association of Ga with Gp/y and inhibition of pheromone signaling. A second negative regulator of G protein-mediated signaling is SIG1, a gene that suppresses dominant negative Gp by an unknown mechanism. Deletion of SIG1 results in pheromone hypersensitivity (Leberer et al., 1994). 1.2.3.2 Attenuation of M A P kinase activity Fus3p (MAPK), the terminal kinase of the P R P , is the target of at least three phosphatases, that down regulate Fus3p activity and promote recovery from pheromone response. MSG5, a gene whose expression is pheromone-17 Introduction induced, encodes a dual specificity phosphatase that is able to dephosphorylate both phosphothreonine and phosphotyrosine residues (Doi et al., 1994). Ptp2p and Ptp3p work in concert as protein tyrosine phosphatases (Zhan et al., 1997; Zhan and Guan, 1999). As well as phosphorylating effectors of the P R P , Fus3p also phosphorylates both,Ste7p and Ste5p, two upstream members of the P R P cascade (Elion et al., 1993; Errede et al., 1993; Kranz et al., 1994). The significance of these phosphorylations is unknown, but it has been postulated that they may act. as attenuation signals. Kss1 p, which was cloned as a gene that promotes recovery from pheromone, inhibits the activated P R P MAP kinase cascade at or below the level of Ste11p (Cherkasova et al., 1999). Fus3p may also promote pheromone recovery through modulation of Mcmlp activity. Overexpression of the transcription factor Mcm lp can promote proliferation in the presence of pheromone in a Fus3p-dependent manner (Cherkasova et al., 1999). Inactivation of Fus3p may also promote recovery. Catalytically inactive Fus3p has been shown to repress activation of FUS1 transcription by active Fus3p and catalytically inactive Fus3p accumulates in yeast after extended exposure to mating pheromone (Choi et.al., 1999; Farley et al., 1999). 1.2.3.3 PRP signal attenuation and transcription Signal attenuation in the pheromone response may also be achieved by altering the activity of transcription factors. Rs t lp (Diglp) and Rst2p (Dig2p) are 18 Introduction known to be negative regulators of Ste12p and substrates of Fus3p and Kss1 p (Bardwell et al., 1998b; Cook et al., 1996; Pi et al., 1997; Tedford et al., 1997) and RST2 expression is pheromone induced (Cook et al., 1996). Although the Rstps have not been linked to recovery, the genes were cloned in two-hybrid screens as proteins that interact with C ln lp , Cln2p and K s s l p , all three of which are implicated in recovery (Cherkasova et al., 1999; Cook et al., 1996; Courchesne et al., 1989; Oehlen and Cross, 1994; Oehlen and Cross, 1998b; Tedford et al., 1997; Wu et al., 1998) (see The pheromone response and the cell cycle, below). POG1 is a gene which, when overexpressed, inhibits both pheromone-induced growth arrest and repression of CLN1 and CLN2 expression (Leza and Elion, 1999). Deletion of POG1 results in enhanced pheromone sensitivity. Initial studies indicate that Pog lp may enhance pheromone recovery through an increase in CLN2 expression (Leza and Elion, 1999). As described below, Cln2p-Cdc28p complexes may inhibit the function of Ste20p in order to promote recovery from pheromone-induced arrest (Leza and Elion, 1999; Oehlen and Cross, 1994; Oehlen and Cross, 1998b; Wu et al., 1998). 19 Introduction 1.2.4 Filamentous development: the pseudohyphal development and invasive growth pathways share components of the PRP 1.2.4.1 The phenotype of filamentous development Under certain conditions, including nutrient limitation, Saccharomyces cerevisiae will exhibit filamentous growth (Gustin et al., 1998). When diploid yeast undergo the dimorphic transition from the yeast form to the filamentous pseudohyphal form, they switch from a bipolar to an apical budding pattern, their buds cease to detach and they grow as filaments of elongated cells that project outward from a colony in solid media (Gimeno et al., 1992; Mosch and Fink, 1997). Haploid yeast grow in a similar filamentous form, but their invasive growth is limited to projections beneath the colony, because their budding pattern is bipolar (Roberts and Fink, 1994). The environmental signals that control filamentous growth are nutritional (Cullen and Sprague, 2000; Dickinson, 1994; Gimeno et al., 1992; Lo et al., 1997). Nitrogen starvation in the presence of abundant carbon can induce pseudohyphal development, as can the presence of metabolic byproducts(Gimeno et al., 1992; Lorenz et al., 2000a). In contrast, haploid yeast grow invasively in response to glucose depletion and their morphology is unaffected by nitrogen depletion (Cullen and Sprague, 2000). The list of genes induced by the filamentation-invasion pathway remains incomplete, although it is known to include TEC1, FL011 and PHD1 (Gimeno and Fink, 1994; Lo and Dranginis, 1998; Madhani and Fink, 1997; Oehlen and 20 Introduction Cross, 1998a; Rupp et al., 1999). Recent analysis has also shown that different genes may be induced in the filamentation-invasion response in haploids and diploid, although FL011 expression is induced in both (Madhani et al., 1999). 1.2.4.2 Signal transduction and filamentous development At least two distinct signal transduction pathways stimulate filamentous development in yeast. Ras2p can stimulate both pathways, possibly in response to the different signals (Gimeno et al., 1992; Kubler et al., 1997; Lorenz and Heitman, 1997; Mosch et al., 1999; Mosch et al., 1996). One pathway is controlled by Gpr lp (G-protein coupled receptor) stimulation of c A M P - P K A and modulates the activity of the transcription factor Flo8p (Liu et al., 1996; Lorenz et al., 2000b; Pan and Heitman, 1999; Rupp et al., 1999; Tamaki et al., 2000). The second pathway that regulates filamentous development in yeast is the filamentation-invasion MAP kinase cascade (Figures 2 and 3), which shares several components with the P R P including Ste20p, Ste50p, Ste11p, Ste7p, Rst lp , Rst2p and Ste12p (Liu et al., 1993; Roberts and Fink, 1994). The filamentation-invasion pathway (FIP) does not respond to pheromone, however, and the FIP MAP kinase is Kss lp , and not Fus3p (Madhani et a l , 1997; Roberts etal . , 2000). 1.2.4.3 The filamentation-invasion MAP kinase pathway In the filamentation-invasion pathway, Ras2p signals through Bmhlp and Bmh2p, two 14-3-3 proteins, to activate Cdc42p (Gimeno et al., 1992; Mosch et 21 Introduction al., 1996; Roberts et al., 1997). Active Cdc42p stimulates Ste20p, which in turn activates Ste11p to initiate the MAPK cascade (Leberer et al., 1997b; Mosch et al., 1996; Peter et al., 1996). Ste50p has also been implicated in the activation of Ste11p for filamentation-invasion, possibly by a mechanism analogous to its role in pheromone response (see Ste50p associates with membrane-localized proteins, above) (Rad et al., 1998). Upon activation of K s s l p , negative regulation of Ste12p by Rst lp and Rst2p is relieved and transcription of filamentation genes is induced (Bardwell et al., 1998a; Bardwell et al., 1998b; Cook et al., 1996; Cook et al., 1997; Madhani et al., 1997; Tedford et al., 1997). Activation of transcription in the filamentation-invasion pathway requires two transcription factors, Ste12p and T e d p. Ste12p and T e d p bind to filamentous response elements (FREs) (Figures 3 and 4) and direct transcription of genes such as FL011 (Baur et al., 1997; Gavrias et al., 1996; Laloux et al., 1994; Lo and Dranginis, 1998; Madhani and Fink, 1997; Mosch and Fink, 1997). FR E elements consist of a T e d p binding site adjacent to a Ste12p binding site (Madhani and Fink, 1997). 1.2.4.4 Maintaining MAP kinase cascade signaling specificity The FIP and the P R P control two separate developmental pathways, yet they share most of their signaling components, including the transcription factor, Ste12p (Gustin et al., 1998; Madhani and Fink, 1998). Several mechanisms have been proposed as models for how the two signal pathways may be kept 22 Introduction PRE: Ste12p ( A ) T G A A A C A PRE PQ: Ste12p p^Mcr Mcml mlp C 2 ( N ) G G : alp C T G T C A T T G T F J. P box Q box P/PRE: McmlpJMcmlp^ ^ Ste12p C 2 ( N ) 6 G 2 - ( A ) T G A A A C A Pbox PRE FRE: Ste12p C A T T C T ( A ) T G A A A C A TCS PRE 1/2 repressor binding site Figure 4. Promoters that respond to Ste12p Several categories of promoters have been defined that respond to Ste12p. P R E sequences [(A)TGAAACA] are found in pheromone responsive genes. PQ elements, which consist of a P box [CC(NNNN)GG] adjacent to a Q box [CTGTCATTGT], are found in a-specific genes. P /PRE sites, which combine a P box with a P R E , are found in a-specific genes. F R E elements, composed of a T C S [CATTCT] and a P R E , are found upstream of genes induced during filamentation. See Table 1 for examples of genes containing these elements. All of these elements are recognition sites for transcription factors. As depicted, Ste12p binds to P R E elements, Mcmlp binds to P boxes, a l p binds to Q boxes and T e d p binds to T C S elements. 23 Introduction Table 1. Promoters that respond to Ste12p Gene Name Type of Promoter Gene Product Function Reference Ty1 LTR Filamentous Yeast transposon (Company et al., 1988; Laloux et al., 1994; Mosch and Fink, 1997; Mosch et al., 1996) FL011 Filamentous Cell surface flocculin (Lo and Dranginis, 1998; Rupp et al., 1999) TEC1 Filamentous Transcription factor, required for invasive growth (Madhani and Fink, 1997; Oehlen and Cross, 1998a) PHD1 Filamentous Transcription factor, enhances invasive growth (Gimeno and Fink, 1994) S0K2 Filamentous Transcription factor, reduces invasive growth (Ward etal . , 1995) RST2/DIG2 Haploid Negative regulator of (Cook et al., specific pheromone response 1996) FUS1 Haploid Required for fusion of mating (Hagen et al., specific haploids 1991; McCaffrey et al., 1987; Trueheart et al., 1987) FAR1 Haploid Inhibits CLN-CDC28 function, (Chang and specific causes G1 growth arrest in response to pheromone Herskowitz, 1990) SST2 Haploid Recovery from pheromone (Dietzel and specific arrest Kurjan, 1987a) MSG5 Haploid specific Recovery from pheromone arrest (Doi etal . , 1994) KAR5 Haploid specific Nuclear fusion (Beh etal . , 1997) STE2 a-specific a-factor receptor (Hartig et al., 19.86; Nakayama et al., 1985) BAR1 a-specific a-factor protease, degrades a-pheromone (Kronstad et al., 1987) 24 Introduction MFA1 a-specific a-factor (Michaelis and Herskowitz, 1988) STE3 a-specific a-factor receptor (Jarvis et al., 1988) MFa1 a-specific a-factor (Achstetter, 1989) MFa2 a-specific a-factor (Jarvis et al., 1988) STE13 a-specific a-factor processing (Achstetter, 1989) 25 Introduction distinct. First, combinatorial control of transcription distinguishes filamentation-invasion from pheromone response. The cAMP pathway and the FIP converge on the FL011 promoter, simultaneously modulating gene expression from independent promoter elements (Rupp et al., 1999). A second mechanism that likely maintains PRP/FIP specificity is the interaction of Ste12p with different transcription factor partners. Ste12p interacts with different partners at F R E s and P R E s , although it is unknown how the partners are selected (Baur et al., 1997; Hwang-Shum etal . , 1991; Laloux et al., 1994; Madhani and Fink, 1997; Oehlen etal . , 1996; Yuan et al., 1993). Cell-type-specific gene expression distinguishes the FIP from the P R P in diploids, as diploids do not express Ste5p, Fus3p, pheromone receptors, Gpa lp , Ste18p or Ste4p (Elion et al., 1990; Hartig et al., 1986; Lebereret al., 1993; Mukai et al., 1993; Perlman et al., 1993; Sprague et al., 1983; Whiteway et al., 1989). Scaffold proteins, such as Ste5p, may also contribute to specificity, although no scaffold has been identified for the FIP (Widmann etal . , 1999). The MAP kinases of the P R P and FIP are different, which may be an important source of signal specificity. Originally, K s s l p and Fus3p were postulated to have overlapping functions in pheromone response and invasive growth, as deletion of one gene was insufficient to abolish either response. The two kinases do have different substrate proteins and different activities (see The M A P kinase cascade, above) and recent observations have demonstrated that the two MAP kinase have independent functions (Cook et al., 1997; Madhani et al., 1997; Roberts et al., 2000). Fus3p is the major M A P kinase of the P R P and 26 Introduction may exclude K s s l p from the Ste5p complex, effectively eliminating K s s l p from the pheromone response (Madhani et al., 1997). K s s l p acts as both a negative and a positive regulator of filamentous development (Bardwell et al., 1998a; Bardwell et al., 1998b). Deletion of KSS1 from the genome does not eliminate invasive growth, but it is likely that the cAMP signal and the elimination of the negative regulation by K s s l p combine to permit invasion (Madhani et al., 1997; Rupp et al., 1999). Collectively, these results indicate that the FIP and the P R P employ different and specific MAP kinases, which may prevent unwanted crosstalk. The role of Kss1 p as a negative regulator may also be essential to signaling specificity. K s s l p binds to Ste12p and potentiates Rst lp - and Rst2p-dependent inhibition of Ste12p function at filamentous response elements (FREs) (Bardwell et al., 1998a; Bardwell et.al., 1998b). Inactive K s s l p is postulated to stabilize the interaction between Rst lp and Ste12p in a complex containing all three proteins (Bardwell et al., 1998a; Bardwell et al., 1998b). Activation of , K s s l p by Ste7p in the FIP results in dissociation of K s s l p from Ste12p, relieving repression at F R E s (Cook et a]., 1997). A similar effect is observed at pheromone responsive elements (PREs), however, it is much weaker. In a kssIA strain, under conditions of vegetative growth, F R E reporters show sixty percent of FlP-induced activity, whereas P R E reporters show seven percent of PRP-induced function (Cook et al., 1997). These results suggest that an inactivated MAP kinase may act as a pathway-specific inhibitor of activator function. 27 Introduction 1.2.5 The pheromone response and the cell cycle Saccharomyces cerevisiae proliferate by budding to produce two progeny cells with identical genetic material. Newly budded yeast, in the presence of sufficient nutrients, proceed through the four ordered phases of the cell cycle (G1, S, G2 and M) in order to replicate their DNA and distribute their genetic material to their progeny (Herskowitz, 1988). Pheromone response halts the ordered progression of the cell cycle in haploid yeast before the process of DNA replication begins, which ensures that yeast mate with a 1N complement of DNA. As described below, the interaction of the cell cycle machinery and the P R P is dynamic. The P R P can halt cell cycle progression by several mechanisms, while the activity of the cell cycle machinery limits the growth arrest function of the P R P to the G1 phase of the cell cycle. The following sections will review the events surrounding the G1 to S transition in yeast and the effects of pheromone and Ste12p-dependent transcription on those events. 1.2.5.1 Cdc28p - the cell cycle kinase Progression through the cell cycle in yeast is dependent upon the activity of Cdc28p, the cyclin dependent kinase (CDK) that coordinates the events of the Saccharomyces cerevisiae cell cycle. CDK activity is both positively and negatively regulated. Activation of CDKs requires cyclin binding and phosphorylation by a CAK (CDK activating kinase). Even in the presence of these activating stimuli, CKIs (cyclin dependent kinase inhibitors) can limit CDK 28 Introduction function by binding to CDK-cyclin complexes and directly inhibiting their activity (Mendenhall and Hodge, 1998). Cdc28p is a constitutively expressed protein that is regulated at the post-transcriptional level (Mendenhall and Hodge, 1998; Mendenhall et al., 1987). The activity of Cdc28p reflects the balance of cyclins and CKIs in a cell, which varies in response to proliferation and arrest signals. At several checkpoints in the cell cycle, influences such as nutrient availability, mating pheromone and DNA damage can halt or enhance cell cycle progression through an influence on Cdc28p activity (Mendenhall and Hodge, 1998). The first checkpoint of the cell cycle is Start, which occurs at the G1 to S transition. Cells that have passed the Start checkpoint begin to replicate their DNA, form buds and duplicate their spindle pole bodies. Passage through Start reflects a commitment to completing the cell cycle and once past Start, yeast are unable to respond to pheromone until they complete one cell cycle (Mendenhall and Hodge, 1998). Response to pheromone, however, arrests yeast at the Start checkpoint and inhibits further cell cycle progression (Gustin et al., 1998; Mendenhall and Hodge, 1998). 1.2.5.2 The cyc l ins and CKIs of Start There are five cyclins and two CKIs associated with Start in Saccharomyces cerevisiae. The three G1 cyclins are C ln lp , Cln2p and Cln3p (Andrews and Measday, 1998), the two B-type cyclins are Clb5p and Clb6p (Andrews and Measday, 1998), and S i d p and Far lp are the two CKIs (Mendenhall and Hodge, 1998). The relative amount of these cyclins and CKIs 29 Introduction in a cell is regulated by a combination of transcription and proteolysis and determines when the cells will progress through Start. C ln lp and Cln2p, which are 57% identical, are expressed maximally,at Start (Hadwiger et al., 1989; Tyers et al., 1993; Wittenberg et al., 1990). C ln lp -and Cln2p-Cdc28p complexes initiate bud formation, spindle pole body duplication and proteolysis of the S i d p CKI (Deshaies and Kirschner, 1995; Dirick et al., 1995; Lew and Reed, 1993; Schneider et al., 1996; Schwob et al., 1994; Tyers, 1996; Verma et al., 1997a; Verma et al., 1997b). CLN1 and CLN2 expression is activated in G1 by the S B F transcription factor in response to Cln3p-Cdc28p (Dirick et al., 1995; Nasmyth and Dirick, 1991; Ogas et al., 1991; Stuart and Wittenberg, 1995; Tyers et al., 1993; Wittenberg et al., 1990). C ln lp and Cln2p are ubiquitinated and degraded following Start (Blondel and Mann, 1996; Deshaies et al., 1995). Cln3p, the third G1 cyclin, is expressed throughout the cell cycle, with its levels increasing during the M/G1 phase under the control of the Mcmlp transcription factor (Kuo and Grayhack, 1994; Mclnerny et al., 1997). CLN1, CLN2 and CLN3 are genetically redundant and yeast require at least one G1 cyclin for viability (Cross, 1990; Richardson et al., 1989). A SIC1 deletion will restore viability to a cln1A cln2A cln3A strain (Tyers, 1996). S i d p is a CKI that inhibits the function of Clb5p- and Clb6p-Cdc28p complexes via exclusion of substrate (Mendenhall, 1993; Schwob et al., 1994). S i d p is only present in yeast during G1 (Donovan et al., 1994; Mendenhall et al., 1987; Schwob et al., 1994). Upon accumulation of C ln lp and Cln2p at Start, 30 Introduction S i d p is subject to ubiquitin-mediatedproteolysis (Dirick et al., 1995; Verma et al., 1997a; Verma et al., 1997b). The absence of S i d p enables Clb5p- and Clb6p-Cdc28p complexes to initiate DNA synthesis and spindle pole body separation (Schwob et al., 1994), two events which represent passage though Start. CLB5 and CLB6 are 50% identical and are maximally expressed at Start under the control of the MBF transcription factor (Epstein and Cross, 1992; Koch et al., 1993; Kuhne and Under, 1993; Schwob et al., 1994; Schwob and Nasmyth, 1993). Overall, the passage through the Start checkpoint from G1 to S phase requires the accumulation of sufficient Clnp-Cdc28p activity to initiate S i d p destruction and allow Clb5/6p-Cdc28p complexes to function (Dirick et al., 1995; Mendenhall and Hodge, 1998) (see Figure 5, NO pheromone). 1.2.5.3 Pheromone induced G1 arrest When haploid yeast respond to pheromone they arrest in G1 as unbudded cells with a 1N DNA content. Pheromone-induced G1 arrest is a result of inhibition of Clnp-Cdc28p activity. Several mechanisms have been proposed which may combine to inhibit Clnp-Cdc28p, including direct inhibition of kinase activity, degradation of G1 cyclin proteins and repression of the expression of G1 cyclin genes (Gustin et al., 1998; Mendenhall and Hodge, 1998). 31 Introduction c o » c re CO CD o C o 5 UIO the her Q . (Q X > o CO c o > O c o •4—» CO > < c o CO c CO 1_ •*—' CO o O CD •zz sz CO - t - : C « CO Q . £ CD CO 2 X 3 E H — O O CD CD O rt 0) x : > ? 8 CO CD - CL « C g o o Q . Q O 0) CO J C l i ^ w .9 CD W £ M — O T 3 o CO | S -CD O d \"° a> C 2 CO £ cn o c to T J O - D * * ZS O - Q _ Q C « CD CO £. ± i CO « CO CD •^i2 ® >, o - ° E °-b: CM ,o> . £ T J T J CD CD XT s 1 -CD CO 2 CD T J X 3 L L - C T J CO. C E o CD x : Q. T J C CO Q _ CM C m o 1- C L 3 1 -O) C i i O •go C T J O c l CD C L ^ -x) O C L — T 00 CD CM o o X J a) O £ o co £ = w ® 5 ^ 11 CD - \"5 CO O co • _ ' ZJ > o ' o > , CO > T J Q . to T J C •a % to CO X 5 \"co H 8 2 32 Introduction F a r l p is a pheromone-induced CKI Far1 p is a CKI and an effector of the pheromone response pathway that is required for pheromone-induced G1 arrest (Chang and Herskowitz, 1990). FAR1 is required for pheromone-induced repression of transcription. Analysis of global gene expression shows that pheromone exposure causes repression of multiple genes in a F>4R7-dependent manner. The repressed genes are generally cell cycle-regulated genes whose expression peaks outside of the G1 phase (Roberts et al., 2000). Repression is likely an indirect result of Far lp activation by Fus3p, since Far lp inhibits the function of Clnp-Cdc28p complexes in a pheromone-dependent manner, facilitating pheromone-induced G1 arrest (Gustin et al., 1998; Mendenhall and Hodge, 1998). The mechanism of Far lp action remains in dispute; for completeness, all proposed mechanisms of Far lp function are included in Figure 5. Far lp is known to inhibit all three Clnp-Cdc28p complexes in a PRP-dependent manner and has been detected in Clnp-Cdc28p immunoprecipitates from pheromone-treated yeast (Jeoung et al., 1998; Peter et al., 1993; Tyers and Futcher, 1993). Early evidence indicated that Far lp inhibits Clnp-Cdc28p by excluding other substrates. Clnp-Cdc28p complexes are able to phosphorylate Far lp , which implies that the kinase remains active (Tyers and Futcher, 1993). In some experiments, however, immunoprecipitated Cln2p-Cdc28p complexes from pheromone treated, FAR1 cells demonstrate reduced specific activity, compared to their counterparts from farl yeast (Peter et al., 1993). Importantly, when 33 Introduction Far lp is removed from Cln2p-Cdc28p-Far1p complexes by washing, Cln2p-Cdc28p kinase activity towards substrates other than Far lp is restored (Peter et al., 1993). In direct conflict with these observations, more recent work has demonstrated that Cln2p-Cdc28p complexes immunoprecipitated from pheromone-treated cells do not exhibit a Farlp-dependent reduction in specific activity (Gartner etal . , 1998). These authors agree that Far lp is present in Cln2p-Cdc28p complexes in pheromone treated cells, but they propose that Far1 p does not act through substrate exclusion (Gartner et al., 1998). The authors suggest that previous results were biased by CLN2 overexpression (Gartner et al., 1998). In contrast, all evidence to date supports direct inhibition of Cln3p-Cdc28p by Far lp in response to pheromone (Jeoung, et al., 1998). Further work will be required to determine the complete biochemical mechanism for Far lp action. Far lp CKI activity is modulated by regulation of its expression, protein degradation and pheromone-induced phosphorylation (Blondel et al., 1999; Blondel et al., 2000; Gustin et al., 1998; Mendenhall and Hodge, 1998). Maximal FAR1 gene expression is detected in G2/M and pre-Start G1 yeast (Oehlen et al., 1996). The G2/M expression is dependent upon Mcmlp and the G1 expression requires Ste12p (Oehlen et al., 1996). Far lp levels are highest during G1, not G2/M, due to increased protein stability as, outside of G1 , Far1 p is subject to ubiquitin-mediated proteolysis (McKinney et al., 1993; McKinney and Cross, 1995). At the G1-S transition, nuclear localized Far lp is ubiquitinated by 34 Introduction the S C F C d c 4 p complex and directed to the 26S proteasome (Blondel et al., 1999; Blondel etal . , 2000). The presence of pheromone increases FAR1 expression four-fold in a Ste12p-dependent manner (Chang and Herskowitz, 1990). While increased Far lp levels are required for pheromone-induced growth arrest, PRP-dependent Far lp phosphorylation is also required for Far lp to function as a CKI (Chang and Herskowitz, 1992; McKinney et al., 1993; Oehlen et al., 1996; Peter et al., 1993; Peter and Herskowitz, 1994; Tyers and Futcher, 1993). Pheromone-induced phosphorylation allows Far1 p to associate with Clnp-Cdc28p complexes in the nucleus (Chang and Herskowitz, 1992; Peter et al., 1993; Peter and Herskowitz, 1994; Tyers and Futcher, 1993). The association of Far lp with Clnp-Cdc28p complexes results in further phosphorylation and Far lp ubiquitin-mediated degradation, which may be a mechanism for recovery from pheromone response (Blondel et al., 1999; Blondel et al., 2000; Henchoz et al., 1997; Peter et al., 1993; Tyers and Futcher, 1993). Fus3p is postulated to be the PRP-dependent Far lp kinase (Chang and Herskowitz, 1992; Elion et al., 1993; Peter et al., 1993; Tyers and Futcher, 1993). Fus3, but not K s s l p , has been implicated in pheromone-induced arrest, functions upstream of Far lp in the P R P and is required for Far lp activity (Elion et al., 1991; Elion etal . , 1990; Elion etal . , 1993). Biochemically, it has been demonstrated that Far1 p is an in vitro substrate of Fus3p from pheromone 35 Introduction treated cells and Far lp from fus3A cells does not associate with Clnp-Cdc28p complexes (Elion et al., 1993; Peter et al., 1993; Tyers and Futcher, 1993). Together these results suggest a model for G1 growth arrest in which FAR1 expression and Far1 p phosphorylation increase in response to pheromone and allow Far lp to bind to and inhibit Clnp-Cdc28p complexes (Gustin et al., 1998; Mendenhall and Hodge, 1998). In this model, Far lp directly inhibits Clnp-Cdc28p complexes, which inhibits Glh3p-Cdc28p-mediated induction of CLN1 and CLN2 expression and prevents S i d p degradation (see Figure 5). Additional mechanisms for growth arrest Far lp inhibition of Clnp-Cdc28p complexes is the best understood mechanism for pheromone induced growth arrest in yeast, but other mediators have also been described. Hyperactivated Fus3p and K s s l p have been demonstrated to induce pheromone-responsive G1 arrest in farIA cells. In these yeast, Fus3p and K s s l p inhibit the transcription of CLN1, CLN2, and CLB5, which results in decreased Clnp-Cdc28p activity. Normally, Cln3p-Cdc28p activates transcription in G1 through the S B F transcription factor, but Fus3p and K s s l p do not appear to inhibit S B F , so other factors must be involved (Cherkasova et.al., 1999). FAR3 was isolated in a screen for yeast mutants which maintain Ste12p-responsive transcription but resist growth arrest in yeast that overexpress STE4, which encodes the Gp of the P R P (Horecka and Sprague, 1996). The screen also identified CLN3, FUS3 and FAR1 mutants. FAR3 is constitutively expressed 36 Introduction in all cell types and is not pheromone inducible. A far3A mutant increased the pheromone resistance of a farIA fus3A yeast strain, indicating that FAR3 inhibits growth by a separate pathway (Horecka and Sprague, 1996). In addition, FAR3 does not appear to alter Clnp-Cdc28p activity, CLN expression or Clnp proteolysis (Horecka and Sprague, 1996). A model for the action of the FAR3 gene product has not been proposed. G1 growth arrest can be induced by overexpression of Ste12p in the absence of pheromone (Dolan, 1996; Dolan and Fields, 1990). The arrest is independent of Far lp , so it may involve induction of other unidentified genes that mediate growth arrest (Dolan, 1996). 1.2.5.4 Cdc28p activity impacts on the P R P The basal activity of the P R P kinases Ste7p and Fus3p is increased in G1 but decreases, in a CLN1- and CLA/2-dependent manner, along with the expression of pheromone-responsive genes, as the Start checkpoint approaches (McKinney et al., 1993; Oehlen and Cross, 1994; Oehlen et al., 1996; Wassmann and Ammerer, 1997; Zanolari and Riezman, 1991). The decreased activity of the P R P correlates with peak expression of the G1 cyclins, indicating that the cell cycle may regulate P R P activity (Oehlen et al., 1996; Wassmann and Ammerer, 1997). Clnp-Cdc28p complexes may inhibit P R P function through the post-translational modification of Ste20p (Oehlen and Cross, 1998b; Wu et al., 1998). Overexpression of CLN2 represses P R P activation by pheromone, by deletion of 37 Introduction Gpa1 (Ga), and by Ste4p activation (Oehlen and Cross, 1998b). In contrast, CLN2 overexpression does not counteract P R P stimulation by activated Ste20p, Ste11p or Ste12p, indicating that Ste20p may be the target of Cln2p down-regulation (Oehlen and Cross, 1998b). In addition, Ste20p accumulates a post-translational modification (possibly phosphorylation) which correlates with both maximal CLN2 expression around Start and a decrease in P R P signaling (Oehlen and Cross, 1998b; Wu et al., 1998). The Ste20p modification correlates with a change in electrophoretic mobility that can be duplicated by Cln2p-Cdc28p complexes in vitro (Wu et al., 1998). The effect of G1 cyclins on the P R P is specific, as overexpression of CLB5 and CLB6 does not alter the mobility of Ste20p (Wu et al., 1998). Cln2p-dependent reduction of P R P signaling may limit pheromone response to G1 and may have a role in recovery from pheromone response (see PRP signal attenuation and transcription). 1.3 Ste12p - A transcription factor for MAP kinase-induced development 1.3.1 Important features of Ste12p function Ste12p is a transcription factor in the yeast Saccharomyces cerevisiae that responds to two separate MAP kinase signaling pathways; the pheromone response pathway (PRP) and the filamentation-invasion pathway (FIP) (Liu et al., 1993; Roberts and Fink, 1994) (see Filamentous development: the pseudohyphal development and invasive growth pathways share 38 Introduction components of the PRP, above). In response to both pathways, Ste12p binds to P R E s (pheromone responsive elements) and directs transcription (Dolan et al., 1989; Errede and Ammerer, 1989; Gavrias et al., 1996; Laloux et al., 1994; Liu et al., 1993; Madhani and Fink, 1997; Roberts and Fink, 1994). Since Ste12p responds to two separate signal transduction pathways and activates the transcription of separate cohorts of genes in response to those signals, any model of Ste12p function must consider how specificity of Ste12p function is maintained. A schematic of the known functional domains and protein-protein interactions of the 688 amino acid, 78 kDa Ste12p is shown in Figure 6 (Errede and Ammerer, 1989). The following sections will discuss the current model for Ste12p function and regulation, and address mechanisms for pathway-specific function of the transcription factor. 1.3.2 DNA binding and protein-protein interactions of Ste12p 1.3.2.1 Pheromone responsive elements Most Ste12p responsive promoters share a common sequence known as the pheromone responsive element (PRE), whose sequence is defined as 5'-(A)TGAAACA-3' (Kronstad et al., 1987; Van Arsdell and Thorner, 1987). The P R E was originally described as the UAS (upstream activating sequence) which mediates a-factor stimulation of a-specific genes (Kronstad et al., 1987; Van Arsdell and Thorner, 1987). Subsequently, it has been demonstrated that P R E s 39 Introduction 215 688 DBD T e d p H interaction 215 215 215 305 i ~ 301 335 PID 470 669 688 NR 688 a i p interaction Mcmlp interaction Figure 6. Features of Ste12p Three functional domains have been described for Ste12p. The DBD (amino acids 1 to 215) is the DNA binding domain, the NR (amino acids 305 to 669) is involved in negative regulation and the PID (amino acids 301 to 335) is the pheromone induction domain. Three transcription factor interaction domains have been identified in Ste12p. a l p interacts with amino acids 215 to 688 of Ste12p, Mcmlp interacts with amino acid 470 to 688 and T e d p interacts with amino acids 1 to 215. For details, see text. 40 Introduction direct both basal and pheromone-induced transcription of haploid-specific genes (Hagen et al., 1991). In addition to its function in pheromone response, a P R E element can form one half of the F RE (filamentous response element), which consists of a P R E adjacent to a T e d p binding site (Figure 4) (Laloux et al., 1994; Madhani and Fink, 1997). Several experiments have demonstrated that Ste12p binds directly to DNA at pheromone responsive elements. First, Ste12p was detected in E M S A complexes on the transcriptional control elements of a-specific genes and TY1 promoters, both of which contain P R E sequences (Dolan et al., 1989; Errede and Ammerer, 1989). Next, recombinant Ste12p was shown to bind directly to P R E s in both E M S A and footprint assays on both wild-type promoter fragments and oligonucleotides containing P R E s (Yuan and Fields, 1991). Recently, Ste12p was shown to bind to pheromone-responsive promoters by chromatin immunoprecipitation and microarray analysis (Ren et al., 2000). The study also demonstrated that Ste12p binds to selected promoters prior to pheromone response and that further Ste12p accumulates after pheromone treatment (Ren et al., 2000). The minimum Ste12p fragment required for binding to P R E s is amino acids 41 to 204, although amino acids 1 to 215 are generally referred to as the DNA binding domain (Figure 6) (Yuan and Fields, 1991). Amino acids 1-19, in fact, may reduce the ability of Ste12p to interact with DNA in vivo (Crosby et al., 2000). 41 Introduction 1.3.2.2 Categories of Ste12p responsive promoters Examples of Ste12p-inducible genes are listed in Table 1. Some genes, such as FL011, PHD1 and TEC1 are induced in yeast in the processes of filamentous development and invasive growth (Gimeno and Fink, 1994; Lo and Dranginis, 1998; Madhani and Fink, 1997). Other genes are induced by pheromone, and can be divided into three categories. STE2, the a-factor receptor, is expressed only in a cells and is a model a-specific gene (Hartig et al., 1986; Nakayama et al., 1985). Genes expressed only in a-cells, such as the a-factor receptor STE3, are called a-specific genes (Sprague et al., 1983). Some pheromone-inducible genes are expressed in both a and a yeast and are known as haploid-specific genes. FUS1, which is required for cell fusion, is a haploid-specific gene (McCaffrey et al., 1987; Trueheart et al., 1987). At each type of prompter, Ste12p activates transcription through a different protein-DNA complex, which may contribute to pathway-specific signaling (Figure 4). Haploid-specific genes, such as FUS1, have multiple P R E s in their promoters through which Ste12p directs both basal and pheromone-induced transcription, possibly as a homodimer (Hagen et al., 1991; McCaffrey et al., 1987; Trueheart et al., 1987; Yuan and Fields, 1991). Mcm1p-Ste12p complexes activate STE2 and other a-specific genes through adjacent Ste12p and Mcmlp binding sites, respectively P R E s and P boxes (Dolan et al., 1989). a-specific genes, like STE3, are regulated through PQ elements (Sengupta and Cochran, 1990). PQ elements consist of an Mcmlp binding site (P) adjacent to 42 Introduction an a l p binding site (Q), but do not have an associated P R E (Jarvis et al., 1988). Since Ste12p can interact with both a l p and Mcmlp , Ste12p activation of a-specific genes may be mediated by protein-protein interaction rather than protein-DNA interaction (Kirkman-Correia et al., 1993; Yuan et al., 1993). The fourth category of promoter to which Ste12p binds contains filamentous response elements (FREs), also known as S R E s (stress response elements) (Laloux et al., 1994; Madhani and Fink, 1,997). Genes involved in yeast filamentation, which include FL011 and TEC1, are regulated by T e d p and Ste12p bound to adjacent T C S and P R E elements in F R E s (Lo and Dranginis, 1998; Madhani and Fink, 1997). T C S (TEA (TEF-1, T e d , and AbaA motif) DNA consensus sequence) elements are T e d p binding sites (Baur et al., 1997). 1.3.3 The interaction of Ste12p with other transcriptional activators In order for the STE12 gene product to activate transcription of a- and a-specific pheromone-responsive genes, it must interact with M c m l p and a l p , two other transcription factors involved in pheromone response (Kirkman-Correia et al., 1993; Yuan et al., 1993). The carboxy-terminal 219 amino acids of Ste12p interact with Mcmlp to activate a-specific genes such as STE2 (Figure 6) (Errede and Ammerer, 1989; Kirkman-Correia etal . , 1993). The Ste12p (aa 469 to 688) interaction with Mcmlp was demonstrated by E M S A using myc-tagged deletions of Ste12p (Errede and Ammerer, 1989). The C-terminus of Ste12p also interacts with a l p (Figure 6). A ste12 phenotype in S. cerevisiae can be complimented by co-expression of a l p and Ste12p from K. lactis. Expression of a series of 43 Introduction chimeric Ste12p constructs, combining Ste12p domains from either species, in S. cerevisiae revealed that the C-terminus of Ste12p interacts with a l p . Recombinant Ste12p (aa 215 to 688) can also interact with a l p in the absence of DNA (Yuan et al., 1993). In addition, some Ste12p-a1p interactions may fall outside the carboxy terminus of Ste12p. The T-50 allele of STE12, which converts alanine 50 of Ste12p to a threonine residue, reduces the interaction of Ste12p and a l p , indicating that the Ste12p DNA binding domain may also be involved in the Ste12p-a1p interaction (La Roche et al., 1995). Another subset of pheromone-responsive genes that Ste12p controls is the karyogamy-specific genes, a set of genes required for nuclear fusion during mating. Both Ste12p and Kar4p modulate pheromone-responsive expression of these genes, which include KAR3, KAR5 and CIK1 (Gammie et al., 1999; Kurihara et al., 1996). Neither the DNA sequence elements nor the protein domains involved in the regulation of gene expression by Ste12p and Kar4p are known, although the KAR3 promoter does contain three potential P R E s (Kurihara etal . , 1996). Activated transcription from FREs is regulated by Ste12p and its transcription factor partner, T e d p (Baur et al., 1997; Gavrias et al., 1996; Laloux et al., 1994; Madhani and Fink, 1997). Although direct interaction of Ste12p and T e d p in the absence of DNA has not been demonstrated, their binding to the F R E is believed to be cooperative and only requires amino acids 1 to 215 of Ste12p (Baur et al., 1997; Madhani and Fink, 1997). In addition to regulation by 44 Introduction Ste12p and T e d p , genes required for filamentation-invasion may also require the input of other transcription factors, such as Flo8p, which regulates the expression of FL011, through independent sequence elements (Rupp et al., 1999). 1.3.4 MAP kinase-dependent phosphorylation of Ste12p Since Ste12p is known to function downstream of the MAP kinases Fus3p and K s s l p , one attractive model for Ste12p regulation is the accumulation of activating phosphorylations when the MAP kinases are stimulated. When Ste12p-Gal4p fusion proteins are expressed in yeast cells, it is possible to observe pheromone-dependent phosphorylation of the fusion proteins which correlates with transcriptional activation (Song et al., 1991). In addition, Ste12p has been demonstrated to be a substrate of pheromone-activated Fus3p (Elion etal . , 1993). Substantial effort has been expended to identify pheromone-induced phosphorylations of Ste12p. Ste12p has been shown by peptide mapping to have eight constitutive phosphorylated peptides, whose phosphorylation is independent of the protein kinases of the pheromone response pathway (Hung et al., 1997). Ste12p accumulates two transient minor phosphopeptides in the presence of pheromone which are dependent upon the pheromone response pathway and localization of Ste12p to the nucleus (Hung et al., 1997). The identity of the two phosphopeptides is not known. As a result, it is not possible to test their function or to demonstrate that the peptides are direct targets for Fus3p 45 Introduction or K s s l p phosphorylation. It is possible, therefore, that the phosphorylation of other target molecules, such as Rst lp (Diglp) and Rst2p (Dig2p) (described below) controls pheromone-responsive transcription (Cook et al., 1996; Tedford etal . , 1997). 1.3.5 Regulatory domains of Ste12p The regulatory domains of Ste12p were originally characterized by examining a series of Gal4p-Ste12p fusion proteins and by analysis of a set of linker and deletion mutants of Ste12p (Kirkman-Correia et al., 1993; Song et al., 1991). The analyses revealed an activation domain in amino acids 214 to 473 and demonstrated that amino acids 305 to 669 comprise a target of negative regulation in the absence of pheromone (Kirkman-Correia et al., 1993; Song et al., 1991). One study showed that amino acids 214 to 473 of Ste12p can activate transcription in the absence of pheromone, when fused to Gal4p. Activation by amino acids 214 to 473 was one hundred and eighty fold more efficient than activation by amino acids 214 to 688, demonstrating that the C-terminal amino acids reduce the activity of Ste12p (Song et al., 1991). Confirming these results, the second study showed that three separate deletions in the region of STE12 encoding amino acids 305 to 669 of Ste12p resulted in substantially (six- to twenty- fold) increased levels of basal transcription from a FUS1-LacZ reporter (Kirkman-Correia et al., 1993). 46 Introduction Subsequently, amino acids 301 to 335 of Ste12p have been identified as the pheromone inducible domain (PID) of Ste12p (Figure 6) (Pi et al., 1997). If this domain is absent from Ste12p, the activity of a FUS1-LacZ reporter is no longer pheromone inducible. In addition, this domain confers pheromone inducibility on Ste12p-Gal4p fusions. It has been proposed that PID function may be regulated by phosphorylation. However, alanine substitution of the serine and threonine residues in the pheromone inducible domain of Ste12p has no apparent effect on Ste12p function, indicating that phosphorylation of Ser or Thr residues does not control PID function. Interestingly, the mutation of two tyrosines (residues 310 and 317) in the Ste12p \"minimal pheromone induction domain\" to alanines resulted in increased basal transcription and loss of pheromone induction of Gal4p-Ste12p fusions (Pi et al., 1997). Tyrosine phosphorylation of Ste12p has not been demonstrated (Hung et al., 1997). Based on these data, the PID may contribute to both the activation and inhibition of Ste12p function. The PID is much smaller than either the activation domain (amino acid 214 to 473) or the regulatory domain (amino acids 305 to 669) defined by earlier studies. Future work will determine whether other amino acids within these domains have important regulatory functions. 1.3.6 Overexpression of Ste12p When Ste12p is overexpressed in yeast cells, it is possible to observe some of the phenotypes of pheromone response in the absence of pheromone 47 Introduction (Dolan, 1996; Dolan and Fields, 1990). STE12 overexpression leads to an increase in transcription of pheromone-inducible genes and the arrest of the cells in the G1 stage of the cell cycle, independent of the FAR1 gene product. As well, the overexpression of STE12 can suppress the mating defect of ste mutants. Since STE12 overexpression can increase the activity of Ste12p in the absence of pheromone and deletions of regions of Ste12p can eliminate negative regulation (Dolan, 1996; Dolan and Fields, 1990; Kirkman-Correia et al., 1993), it has been postulated that Ste12p has an inherent activation function which is masked in the absence of pheromone. One possible explanation of these results is that there is a direct negative regulator (or regulators) of Ste12p, similar to the Gal80p regulator of Gal4p. Two candidate proteins for the role of direct negative regulator of Ste12p are Rst lp (Diglp) and Rst2p (Dig2p) (Bardwell et al., 1998a; Bardwell et al., 1998b; Cook et al., 1996; Pi et al., 1997; Tedford et al., 1997). 1.3.7 Rstlp (Diglp) and Rst2p (Dig2p) are MAP kinase substrates and negative regulators of Ste12p Rst lp (Diglp) and Rst2p (Dig2p) are negative regulators of Ste12p and regulate both its pheromone-responsive and filamentous growth functions (Bardwell et al., 1998a; Bardwell et al., 1998b; Cook et al., 1996; Pi et al., 1997; Tedford et al., 1997). Rs t lp (Diglp) and Rst2p (Dig2p) were cloned simultaneously in two separate two-hybrid screens (Cook et al., 1996; Tedford et al., 1997). One screen identified Rst lp (Diglp) and Rst2p (Dig2p) as proteins that interact with Cln1 and Cln2p, two of the G1 cyclins (Tedford et al., 1997), 48 Introduction while the other screen cloned genes encoding proteins that interact with K s s l p (Cook et al., 1996). Further characterization has shown that Rs t lp (Diglp) and Rst2p (Dig2p) are nuclear proteins which co-immunoprecipitate with Kss lp , Fus3p and Ste12p, and that Rst lp (Diglp) and Rst2p (Dig2p) are substrates for MAP kinase phosphorylation by both K s s l p and Fus3p (Cook et al., 1996; Tedford etal . , 1997). In addition to being substrates for the MAP kinases, Rs t lp (Diglp) and Rst2p (Dig2p) are known to be essential to the negative regulation function of K s s l p (Bardwell et al., 1998a; Bardwell et al., 1998b). K s s l p , the M A P kinase that regulates the invasive growth pathway, functions as both an activator and an inhibitor of Ste12p-dependent transcription (Madhani et al., 1997). Recent data has demonstrated that the negative regulation function of K s s l p is dependent upon Rst lp (Diglp) and Rst2p (Dig2p) (Bardwell et al., 1998a; Bardwell et al., 1998b). Although K s s l p , Ste12p, Rst lp (Diglp) and Rst2p (Dig2p) coexist in a complex, the mechanism for the negative regulation has yet to be described (Cook et al., 1996; Tedford et al., 1997). 1.3.7.1 RST1 and RST2 are redundant genes with non-identical functions Rst lp (Diglp) and Rst2p (Dig2p) are novel genes with substantial sequence homology to one another (27% amino acid identity, 42% amino acid similarity) but no significant primary sequence similarity to other known proteins (Cook et al., 1996; Tedford et al., 1997). Disruption of either RST1 or RST2 has no detectable effect on S. cerevisiae; however, disruption of both genes results in 49 Introduction constitutive invasive growth and elevated transcription of pheromone-responsive genes (Cook et al., 1996; Tedford et al., 1997). In addition, rst1 rst2 cells grow slowly, but all three rst1 rsi2-associated phenotypes are commonly lost after the cells have been grown for several generations. The loss of the rst1 rst2 phenotype can be attributed to accumulation of loss of function mutations in the STE12 gene (M. Tyers, pers. comm.). Taken together, these results indicate that Rst lp (Diglp) and Rst2p (Dig2p) may be the direct negative regulators of Ste12p and that they are redundant. Two significant differences between Rs t lp (Diglp) and Rst2p (Dig2p) have been observed. First, Rs t lp (Diglp) is constitutively expressed, while Rst2p (Dig2p) is pheromone induced (Cook et al., 1996). Second, the two proteins are not the same size. Rs t lp (Diglp) is 452 amino acids, while Rst2p (Dig2p) is 323 amino acids, and the Rs t lp (Diglp) sequence contains several stretches of amino acid sequence that have no homology to Rst2p (Dig2p) (Cook et al., 1996; Tedford et al., 1997). So, although the two genes RST1 and RST2 have been assumed to be redundant, the genes and the mechanisms by which they are regulated are not identical. 1.4 Transcriptional activators cause increased transcription by RNA polymerase II Transcriptional activators, including Ste12p, regulate transcription in response to the physiological needs of the cell (Kornberg, 1999). In order to mate effectively, yeast cells that are exposed to mating pheromone must 50 Introduction differentiate into mating competent cells (Herskowitz, 1988). The pheromone response stimulates obligate transcriptional activation of gene expression, including a one-hundred fold increase in the cellular level of FUS1 transcripts (Hagen et al., 1991; McCaffrey et al., 1987; Trueheart et al., 1987). The increase in FUS1 expression is a result of activation by Ste12p, which, upon stimulation by the pheromone response pathway, causes increased RNA polymerase II (Pol II) transcription of FUS1 and other pheromone responsive genes (Dolan et al., 1989; Errede and Ammerer, 1989; Fields and Herskowitz, 1985; Fields and Herskowitz, 1987; Song et al., 1991). Activators are essential to regulated gene expression, since the amount of Pol II in a cell is limited and activators increase the amount of Pol II transcription from specific promoters (Hampsey and Reinberg, 1999; Kornberg, 1999). Activation may act through multiple protein-protein interactions, since Pol II is an enzyme which functions in conjunction with a large number of other protein complexes (Berk, 1999; Hampsey and Reinberg, 1999; Kornberg, 1999). A schematic of the protein complexes required for activated transcription of a eukaryotic promoter is presented in Figure 7. The following sections will outline the function of these Pol ll-associated factors, including TFIIA, the general transcription factors, the Pol II core components, the Mediator and the nucleosome remodeling complexes. The review will describe the steps which precede transcription initiation and the mechanisms by which transcriptional activators may affect the formation of the Pol II complex at promoters . 51 Introduction iTATAAAl -|TATAAA| TATA box Start UAS Upstream Activating Sequence Figure 7. A model for transcriptional activation An RNA polymerase II pre-initiation complex including TFIIA, TFIID, the general transcription factors and the Mediator, is assembled at a hypothetical eukaryotic promoter. In this model, the activator enhances complex formation by interaction with Mediator components. Prior to the formation of the PIC, nucleosomal rearrangement by either SWI/SNF or SAGA complexes may have exposed the DNA elements to which the transcription factors are bound. Adapted from (Malik and Roeder, 2000). 52 Introduction 1.4.1 TFIID and TFIIA In order to initiate transcription of a structural gene, a pre-injtiation complex (PIC), consisting of Pol II and the general transcription factors, must be assembled at the promoter (Hampsey, 1998). The first step in PIC assembly is promoter recognition by TFIID (Buratowski, 2000; Green, 2000; Kornberg, 1999). TFIID, a general transcription factor which contains TATA binding protein (TBP) and the TAFnS, associates with the promoter in the absence of other factors and nucleates the formation of the PIC. The binding of TBP to the promoter is the correlated with transcriptional efficiency (Kuras and Struhl, 1999; Li et al., 1999). The TBP subunit of TFIID binds directly to the TATA box, straddling the DNA and bending the DNA in order to form a context for the subsequent TFMB interaction (see The general transcript ion factors, below) (Kim et al., 1993; Nikolov et al., 1992). The TATA box is a core cis regulatory element found in typical class II eukaryotic promoters and is located 40 to 120 bp upstream of the transcription start site. Together with other core promoter elements, the TATA box defines the site where Pol II and the GTFs will bind and form the pre-initiation complex (PIC) (Hampsey, 1998; Leuther et al., 1996; Li et.al., 1994). Binding of TBP to the core promoter is stabilized by the factor TFIIA, which interacts with both TBP and the DNA flanking the TATA box (Buratowski et al., 1989; Imbalzano et al., 1994; Kang et al., 1995; Lee et al., 1992). TFIIA also inhibits the function of Mot lp, a protein that can dissociate TBP from the TATA element, and yTAF130, which inhibits TBP binding to the TATA sequence (Auble 53 Introduction et al., 1994; Kokubo et al., 1998). In addition to its core promoter functions, TFIIA has activator-dependent functions (Stargell et al., 2000). TFIIA can interact directly with both activation domains and coactivators and TFIIA is necessary for the activator-dependent stabilization of the TFIID-TATA complex (Clemens etal . , 1996; Damania etal . , 1998; Kobayashi etal . , 1995; Lieberman and Berk, 1994; Ozer et al., 1994; Ranish et al., 1999). The other components of TFIID are the TAFns. In Saccharomyces cerevisiae, twelve TAFns have been identified, all of which have homologues in higher eukaryotes (Green, 2000). Unlike TBP, which is generally required for Pol II transcription, and whose occupancy of promoters is correlated to transcriptional activity, TAFns are promoter-selective (Kuras et al., 2000; Lee et al., 2000; Li et al., 2000). Genome-wide analysis has demonstrated that each TAFn affects the expression of a characteristic subset of genes, ranging from three to sixty-seven percent of the genome, which indicates that TAFns may act as promoter selectivity factors (Lee et al., 2000). TAFns have multiple functions, including contacting the DNA in the core promoter region, and histone acetyltranferase function (Green, 2000). Several observations indicate that certain TAFns may interact with core promoter sequences that flank the TATA box and confer binding specificity on TFIID (Green, 2000; Verrijzer et al., 1995; Verrijzer and Tjian, 1996). For example, yTAF145 recognizes and selects core promoters in a UAS-independent manner (Shen and Green, 1997). TAF M s may also provide a catalytic activity that is essential for transcription; yTAF145 and its mammalian homologue, TAFII250, 54 Introduction have intrinsic histone acetyltransferase (HAT) activity, and could acetylate other proteins in the transcription complex (Mizzen et al., 1996). TAFII250 has also been demonstrated to act as a ubiquitin activating and conjugating enzyme for histone H1 (Pham and Sauer, 2000). Both of these modifications are postulated to affect nucleosomal architecture and transcription activity (Mizzen et al., 1996; Pham and Sauer, 2000). TAFnS may also interact transcriptional activators and increase TBP binding to promoters (Klebanow et al., 1997; Li et al., 1999; Moqtaderi et al., 1996; Poon et al., 1995; Reese et al., 1994). Activators have been shown to interact with isolated TAFnS, although TAFnS are found in complexes, so the protein-protein interactions of isolated TAFnS may not be physiologically relevant (Chen et al., 1994; Goodrich et al., 1993; Hoey et al., 1993; Reese et al., 1994; Thut et al., 1995). In addition, it has been shown that yTAF17-dependence of transcription is conferred by the UAS sequence, indicating a yTAF17-activator interaction (Michel et al., 1998; Moqtaderi et al., 1998). A recent study has also demonstrated that, at TAFn-dependent promoters, TAFnS are co-recruited with TBP in a manner consistent with direct interactions between TAFnS and activators (Li etal . , 2000). In addition to their role in TFIID, the TAF M s are found in S A G A , another multi-subunit complex that affects transcription (see The SAGA complex contains a histone acetyltransferase below) (Brown et al., 2000; Green, 2000). 55 Introduction 1.4.2 The RNA polymerase II holoenzyme Following the association of TFIID and TFIIA with the core promoter elements, the RNA polymerase II holoenzyme is recruited to the promoter (Ranish et al., 1999). The Pol II holoenzyme is a DNA-independent complex of proteins composed of the RNA polymerase II core enzyme, the Mediator complex and, possibly, the general transcription factors (Kim et al., 1994; Koleske and Young, 1994; Thompson et al., 1993). The scope of holoenzyme function in vivo was highlighted when DNA microarray analysis was used to monitor the effects of temperature sensitive holoenzyme components on transcription in yeast (Holstege et al., 1998). Srb4p, a member of the Mediator complex, was demonstrated to be required for transcription of most yeast genes, whereas mutation of SrblOp, a cyclin-dependent kinase from the holoenzyme, only affects selected genes (Holstege et al., 1998). While the limited effect of SrblOp indicates that some holoenzyme components are specific to particular genes or gene families, the global requirement of Srb4p for transcription indicates that the holoenzyme functions at most promoters. 1.4.2.1 The core components of RNA polymerase II The twelve subunit RNA polymerase II complex, which is conserved in all eukaryotic organisms, is the enzyme that transcribes protein coding genes (Hampsey, 1998; Kornberg, 1999). Recent crystallographic analysis of yeast Pol II shows that the enzyme forms a clam shaped molecule around the DNA, with 56 Introduction binding sites for both DNA and RNA and a pore beneath the active site for entry of the NTPs and the exit of transcripts (Cramer et al., 2000). The two dominant components of the \"clam\" are Rpb1 p ((3') and Rpb2p (B), the two largest components of Pol II, which are responsible for binding DNA and synthesizing RNA, respectively (Hampsey, 1998). The smaller Pol II subunits are arranged around the periphery of the structure and help to form a set of \"jaws\" to grip the DNA downstream of the active center, and a clamp which holds the DNA in place at the active center (Cramer et al., 2000). Multiple aspects of Pol II function are coordinated through the C-terminal domain (CTD) of Rpb lp (Dahmus, 1996; Kim et al., 1994; Koleske and Young, 1994; Lu etal . , 1991; O'Brien etal . , 1994). The phosphorylation state of the CTD reflects the shift of Pol II from initiation to elongation. The CTD has multiple repeats of the heptapeptide sequence Y S P T S P S that can be either phosphorylated or unphosphorylated (Conaway et al., 2000; Dahmus, 1996). The switch to the phosphorylated state accompanies the transition of the Pol II complex from initiation to elongation (Dahmus, 1996; Lu et al., 1991; O'Brien et al., 1994). The CTD also interacts with several Srbp components of the Mediator (Nonet and Young, 1989; Thompson et al., 1993), connecting the Pol II and Mediator components of the holoenzyme. 57 Introduction 1.4.2.2 The Mediator complex The Mediator was originally identified as a factor required by . transcriptional activators to stimulate transcription with reconstituted Pol II and GTFs in vitro (Flanagan et al., 1991). The Mediator complex of Saccharomyces cerevisiae consists of approximately twenty proteins, including ,Srb2p, Srb4p to Srb11p, Med lp to Med4p, Med6p to Med8p, GaM 1 p, Sin4p, Rgr lp and Rox3p (Gustafsson et al., 1998; Kim et al., 1994; Koleske and Young, 1994; Lee et al., 1997; Li et al., 1995b; Myers et al., 1998; Nonet and Young, 1989; Thompson et al., 1993). The exact composition of the Mediator, like the holoenzyme, depends upon the method of purification; for example, one Mediator preparation does not include Srb8p to Srb11p, although it does contain the other Srbps (Myers et al., 1998). Prior to the identification of the Mediator complex, nearly two thirds of the proteins in the Mediator were identified in disparate genetic screens for genes that affect transcription control in yeast. The SRB genes were isolated and characterized as extragenic supressors of RPB1 CTD truncation mutations (Nonet and Young, 1989; Thompson et al., 1993), while GAL11 was originally identified as a gene required for maximum expression of galactose-metabolizing enzymes (Suzuki et al., 1988). Although the Mediator has sometimes been classified as a co-activator of transcription, not all components of the Mediator increase transcription. In fact, Srb8p to Srb11p, Sin4p, Rgr lp and Rox3p have all been identified as repressors of transcription in genetic screens (Gustafsson 58 Introduction etal . , 1998; Hengartner et al., 1995; Kuchin e t a l , 1995; Li eta l . , 1995b; Liao et al., 1995; Song et al., 1996). The presence ofrepressors in the complex implies that the Mediator may have both positive and negative effects on transcription. As a result, it has been suggested that the Mediator complex integrates both positive and negative regulatory signals and acts as a \"control panel\" for transcription (Malik and Roeder, 2000). 1.4.2.3 The general transcription factors The general transcription factors (GTFs) are a group of proteins which are associated with some forms of the holoenzyme and are absolutely required for Pol II transcription (Hampsey, 1998; Koleske and Young, 1994). The GTFs include TFIIB, TFIIF, TFIIE and TFIIH. TFIIB links many components of the PIC, since discrete domains of TFIIB interact with TBP, TAFns, Pol II, and multiple subunits of TFIIF (Barberis et al., 1993; Buratowski et al., 1989; Buratowski and Zhou, 1993; Fang and Burton, 1996; Gonzalez-Couto et al., 1997; Goodrich et al., 1993; Ha et al., 1993). In addition to contacting multiple PIC components and stabilizing the TFIID-TATA complex, TFIIB influences both transcriptional start site (Leuther et al., 1996; Li et al., 1994) and the unidirectional assembly of the PIC (Lagrange et al., 1998; Littlefield etal . , 1999). In addition to its role in basal transcription, TFIIB is a target of gene-specific activators. The activator-TFIIB interaction influences transcription in two ways. First, activators increase TFIIB recruitment and thereby activate 59 Introduction transcription (Kim and Roeder, 1994; Lin et al., 1991; Roberts et al., 1995; Roberts et al., 1993). Second, the interaction of TFIIB with activators has been demonstrated to induce a conformational change in TFIIB, which may increase its ability to interact with other components of the transcription apparatus (Roberts and Green, 1994; Wu and Hampsey, 1999). The three remaining GTFs in the PIC are TFIIE, -F and -H . TFIIE interacts with TFIIF, TFIIH and Pol II in the PIC (Flores et al., 1988; Li et al., 1994; Maxon and Tjian, 1994). TFIIF, which is closely associated with TFIIE (Flores et al., 1988; Sawadogo and Roeder, 1985), stabilizes the interaction of Pol II with TFIIB and further stabilizes the PIC by altering DNA structure in the core promoter (Buratowski etal . , 1991; Forget etal . , 1997; Robert etal . , 1998). TFIIH has both helicase and kinase activity and, in addition to its role in initiation,, TFIIH is involved in nucleotide excision repair (Feaver et al., 1991; Lu et al., 1992; Schaeffer et al., 1993; Serizawa et al., 1992;'Serizawa et al., 1993; Svejstrup et al., 1996; Tirade etal . , 1999). TFIIE, -F and -H are also involved in the transition of the Pol II complex from pre-initiation to transcript elongation (Conaway et al., 2000). TFIIE stimulates TFIIH, a nine-subunit GTF complex, to begin the process of ATP-dependent promoter melting, which is required for promoter clearance (Kugel and Goodrich, 1998). In order to unwind the DNA at the transcription start site, TFIIH exerts torque on the DNA helix several nucleotides downstream of the start site and unwinds the helix (Kim et al., 2000). TFIIF and TFIIH, together, are necessary for the conversion of Pol II from the initiation state to the elongation 60 Introduction state, which requires phosphorylation of the Rpb lp (Dvir et al., 1997; Feaver et al., 1991; Lu etal . , 1992; O'Brien etal . , 1994; Serizawa etal . , 1992). The phosphorylation of the CTD by TFIIH is also stimulated by TFIIE, in conjunction with Gal11p, a component of the Mediator complex (Sakurai and Fukasawa, 1998). 1.4.3 Accessory complexes alter chromatin structure The coiling of eukaryotic DNA around histone octamers in nucleosomes represses transcription by occluding the binding sites for transcription factors. In addition, higher order chromatin structure and modification of histones can repress transcription in entire domains of chromatin (Kornberg, 1999; Kornberg and Lorch, 1999). Two families of accessory complexes associated with transcription counteract the effects of chromatin. One class of accessory complex is typified by the SWI/SNF complex, which remodels nucleosomes in an ATP-dependent manner (Peterson and Workman, 2000). The second class of remodeling complex, which includes the S A G A complex, chemically modifies the histone proteins (Brown et al., 2000). 1.4.3.1 The SWI/SNF complex alters chromatin structure The SWI/SNF complex is a multi-subunit, DNA-dependent ATPase which affects gene transcription by altering chromatin structure (Peterson and Workman, 2000). SWI/SNF reduces the total length of DNA per nucleosome and increases the accessibility of DNA to DNA binding proteins (Bazett-Jones et al., 61 Introduction 1999; Cote et al., 1994; Kwon et al., 1994; Logie and Peterson, 1997). The complex is conserved in all higher eukaryotes and is known to be associated with, but not an integral part of, the holoenzyme (Peterson and Workman, 2000). SWI/SNF complexes have been demonstrated to be recruited to promoters by activator proteins and, in vitro, SWI/SNF recruitment stimulates Pol II transcription of nucleosome arrays (Cosma et al., 1999; Krebs et al., 1999; Natarajan et al., 1999; Neely et al., 1999; Yudkovsky et al., 1999). A recent study shows that SWI/SNF can be recruited to a promoter by one activator and can subsequently participate in the chromatin remodeling that exposes the binding site for a second transcriptional activator, demonstrating that SWI/SNF can affect multiple aspects of activator function (Cosma etal . , 1999). SWI/SNF is a low abundance complex (100-500 copies/cell) and is required for transcription of only five percent of yeast genes (Cote et al., 1994; Holstege et al., 1998). Other chromatin remodeling may be facilitated by the R S C (remodels the structure of chromatin) complex, a higher abundance complex which has similar subunits and identical in vitro activity to the SWI/SNF complex (Cairns et al., 1996). 1.4.3.2 The SAGA complex contains a histone acetyltransferase Reversible acetylation of lysine residues in the N-terminal tails of histones is often associated with transcriptional activation of associated genes (Brown et al., 2000; Kornberg and Lorch, 1999). It is postulated that the histone acetylation weakens the affinity of histones for DNA, altering the nucleosome conformation 62 Introduction and increasing the accessibility of DNA to transcription factors (Kadonaga, 1998; Struhl, 1998; Wade and Wolffe, 1997). One complex which has histone acetyltransferase (HAT) activity and is associated with Pol II transcription is the S A G A complex. S A G A consists of 14 peptides, including the Gcn5p histone acetyltransferase (Brown et al., 2000). The other components of the complex include the Ada proteins, which interact with activators (Grant et al., 1997), the Spt proteins, which interact with TBP (Grant et al., 1997), several TAF M s , which are shared with TFIID (Grant et al., 1998a), and Tra lp , which interacts with activators (Grant et al., 1998b). The other components of S A G A increase the ability of Gcn5p to acetylate nucleosomal histones (Grant et al., 1997; Grant et al., 1999). The S A G A complex can be recruited to promoters by specific activators. In vitro, recruitment by these activators results in increased transcription in nucleosomal arrays (Ikeda et al., 1999; Utley et al., 1998). In vivo, transcriptional activation of the HIS3 gene is correlated with acetylation of local histone H3, and both activation and acetylation are dependent upon the acetylase function of Gcn5p (Kuo et al., 1998). Together, these results indicate that recruitment of S A G A to a promoter results in histone acetylation and transcriptional activation. S A G A shares several TAFn components with TFIID, and global transcription analysis has shown that the shared TAFn subunits collectively contribute to the transcription of seventy percent of yeast genes (Lee et al., 2000). Both S A G A and TFIID have subunits with HAT activity and, since the complexes have common subunits, questions have been raised as to whether 63 Introduction the complexes have overlapping functions (Green, 2000). Global transcription analysis revealed that, although the two HATs can compensate for the loss of one another, the two complexes are not identical in function. The complexes have overlapping functions at some genes, while other genes are specifically dependent on either TFIID or S A G A (Lee et al., 2000). Other HAT complexes have been identified in yeast (Brown et al., 2000). The other complexes have different histone specificities and one, Elongator, is involved in elongation and not initiation (Otero et al., 1999; Wittschieben et al., 2000; Wittschieben etaL, 1999). 1.4.4 Activators recruit the complexes required for transcription to promoters Activators are known to bind to DNA and to contact multiple components of the transcription machinery; however, a series of protein-protein contacts that leads to increased transcription has not been defined. Presently, activator interactions with the transcription machinery can be classified into two groups: interaction with proteins or complexes that relieve repression by nucleosomes, and interaction with Pol II and its associated factors (Kornberg, 1999). Through these interactions, activators may stimulate a two-part process of activation, first, by facilitating the local rearrangement of the nucleosome complexes that are inhibiting transcription and second, by recruiting the transcriptional machinery (Berk, 1999). 64 Introduction Several observations support the model that transcriptional activators activate transcription by localizing accessory complexes to promoters. Activators can interact with both the SWI/SNF and S A G A complexes, bringing chromatin remodeling complexes to promoters where they can increase the accessibility of DNA to proteins (see Accesso ry complexes alter chromatin structure, above). Furthermore, tethering Gcn5p to the DNA as a LexA-Gcn5p fusion can activate transcription of a reporter gene, demonstrating that localization of HAT activity to a promoter activates transcription (Candau et al., 1997). Multiple lines of evidence support the hypothesis that activators recruit the components of the transcriptional machinery. First, many components of the transcription machinery can interact with activation domains (Gill et al., 1994; Goodrich et al., 1993; Hoey et al., 1993; Klemm et al., 1995; Koh et al., 1998; Roberts et al., 1993; Stringer et al., 1990). Second, when components of the transcription complex are fused to DNA binding domains, thereby increasing their local concentration at selected promoters, the fusion proteins function as artificial activators (Chatterjee and Struhl, 1995; Farrell et al., 1996; Gaudreau et al., 1997; Gonzalez-Couto et al., 1997; Xiao et al., 1995). Third, it has been demonstrated that a high affinity protein-protein interaction mimics the function of an activation domain (Gaudreau et al., 1998). Gal11Pp (potentiator), an altered form of Ga l l 1 p, interacts with the dimerization domain of the activator Gal4p. In yeast strains expressing Ga l l 1Pp, the Gal4p dimerization domain, which has no inherent activation function, can substitute for an activation domain in a GaM 1Pp dependent manner (Gaudreau et al., 1998). 65 Introduction 1.5 Project objectives The unifying goal of this research was to further define both the domain structure of Ste12p and the mechanisms that regulate Ste12p activity. The first part of the work focussed on the effects of Ste12p overexpression. The results can be considered in two parts. First, the experiments showed that STE12 overexpression causes F/4/?7-independent G1 growth arrest. The work also demonstrated that the arrest was independent of transcription from P R E elements. Second, the experiments showed that overexpression of Ste12p can induce transcription of a FUS1 reporter gene. Based on these results, it was postulated that Ste12p overexpression might titrate a direct negative regulator or might activate transcription through Ste12p multimerization. The minimal domain of Ste12p that is required for transcriptional induction and growth arrest was determined to be amino acids 262 to 594. The second part of the work sought to determine whether amino acids 262 to 594 of Ste12p could interact with negative regulators of Ste12p. Overexpression of RST1 or RST2, two negative regulators of Ste12p, was found to inhibit the growth arrest and transcriptional induction that is caused by STE12 overexpression. Further work also demonstrated direct interactions between both Rs t lp and Rst2p and Ste12p. Rst lp interacts with amino acids 309 to 547 of Ste12p, while Rst2p interacts with the DNA binding domain. The interaction between the Ste12p DNA binding domain and Rst2p interferes with the Ste12p-DNA interaction. Taken together, these results demonstrate that Rs t lp and 66 Introduction Rst2p interact directly with distinct domains of Ste12p and may regulate Ste12p by different mechanisms. 67 Results 2 The phenotypes associated with Ste12p overexpression The goal of this work was to define both the functional domains of Ste12p and the mechanisms that regulate Ste12p activity. In order to characterize the domain structure of Ste12p, the following series of experiments examined the effects of STE12 overexpression on responses that are normally associated with pheromone treatment. STE12 overxpression was shown to induce transcription from a pheromone responsive promoter, induce growth arrest prior to the G1-S transition and increase mating efficiency in yeast. By examining the activity of a series of Ste12p deletions in these tests, I demonstrated that overexpression of amino acids 262 to 594 of Ste12p can relieve negative regulation of Ste12p in the absence of pheromone. 2.1 Overexpression of Ste12p results in increased expression of FUS1 reporter genes Deletion of amino acids from the carboxyl-terminal region (amino acids 309 to 669) of Ste12p increases the activity of both Ste12p and Ste12p-Gal4p fusions (Kirkman-Correia et al., 1993; Song et al., 1991). One possible explanation for the increased activity of Ste12p might be the elimination of an interaction with a negative regulator. If this model for Ste12p regulation is correct, it is possible that overexpression of Ste12p could also alleviate negative regulation. 68 Results In order to evaluate the effects of Ste12p overexpression on transcription, I examined the change in .transcription from a FUS1-LacZ reporter when Ste12p is overexpressed in yeast. Four Ste12p derivatives were used in these experiments; Wt Ste12p(1 to 688), Ste12pADBD(215 to 688), Ste12p(215-473) and Ste12p(473-688). All four proteins were expressed from a yeast episomal (high copy number) plasmid under the control of a GAL1 promoter. 2.1.1 Induction of FUS1-lacZ When Wt Ste12p and Ste12pADBD were expressed in STE12 yeast, activity from a FUS1-LacZ reporter increased more than 100-fold over a period of two hours following galactose induction (Figure 8A). The induction of FUS1-LacZ activity did not require the addition of pheromone. When the same constructs were expressed in ste12 yeast, only Wt Ste12p was able to activate the transcriptional response (Figure 8B), which was expected, since Ste12pADBD is not able to bind to P R E elements. An increase in Ste12p activity when Ste12p is overexpressed is consistent with either the titration of a negative regulator or the multimerization of Ste12p leading to transcriptional activation. Amino acids 473 to 688 of Ste12p, which correspond to the C-terminal one half of Ste12pADBD, have been identified as the focus of negative regulation of Ste12p in the absence of pheromone (Kirkman-Correia et al., 1993; Song et al., 1991). Since Ste12pADBD ovexpression can activate a FUS1-lacZ reporter, I sought to determine whether overexpression of Ste12p(473-688) or 69 Results O.. O o CM C •>- O o CJ T J C o , o CJ) 3 •a CO £$ o a CO r* o LO - r o o o LO (Zoei-LSru) CD CO o O 03 CO CD CO O Q . 'E. CD E o Q . C\\J CD Q CO Q < C L C\\J * ; C/D T -c n c T J O O . C CD CO •g 'E t o ro C L CD £ CD E CO c ro C CO O ~ J <<-.9-co CO c CO g t o t o 2 _ C L O X O LU CD > CD CD O i _ o J2 >, ro .tr\" CD > o ro CD to ro T J t o o CN T J CD _ T J JS c ro C. O Q . CD o CM N Cj CO CD , -O CD E E Q CD to CO 5 (A C L 3 t o ro CD E ro co TJ C cn a N CM O CO aJ i n 00 «^ a> w 3 « » S LL. >-CO _ Q ° CD O 3 4= c o E \" >, CD tr £ £ CO T J CD c: > 3 CD Q T J OQ CD 9 < t o C L tO CM ro S ro C/) C L CO CM - J S £ CO u. CD o C CD ZJ c r CD to CM T— CO o c to c ro -*—* c o o CM\" I CO CL a CD > C L 70 Results Ste12p(215-473) might also induce FUS1-LacZ activity. When either Ste12p(473-688) or Ste12p(215-473) was overexpressed in yeast, neither protein caused significant induction of a FUS1 reporter construct (Figure 9). 2.1.2 Induction of FUS1-HIS3 In contrast to their weak induction of expression from a FUS1-lacZ reporter, both Ste12p(473-688) and Ste12p(215-473) induced expression of a FUS1-HIS3 reporter (Figure 10). In Figure 10, yeast which are histidine auxotrophs, but which carry a FUS1-HIS3 reporter gene, were transformed with plasmids that express galactose-inducible Ste12p constructs. As expected, in the absence of histidine, the strain was unable to grow on galactose medium (Figure 10, top slide, vector). In contrast, the yeast which expressed Ste12p(473-688) or Ste12p(215-473) were able to grow on his\" galactose, with Ste12p(215-473) supporting more robust growth. This method is a more sensitive assay for FUS1 promoter activity, since the activation of FUS1-HIS3 is required for cell survival in medium lacking histidine (C. Boone, pers. comm.). This experiment demonstrated that both Ste12p(473-688) and Ste12p(215-473) retain an ability to relieve the negative regulation of Ste12p, however, that activity is not sufficient to be observed in a (3-galactosidase assay of FUS1-LacZ activity. Based on the results in Figures 8 and 9, Wt Ste12p and Ste12pADBD would also be expected to induce FUS1-HIS3 expression. This induction could 71 Results Expression Plasmid Figure 9. Overexpression of some domains of Ste12p induces FUS1-LacZ expression Yeast (•: SY2585: STE12, FUS1-LacZ ; • : W303a::SUL-1: ste12, FUS1-LacZ) transformed with plasmids encoding Wt Ste12p, Ste12pADBD, Ste12p(215-473) or Ste12p (473-688) under the control of a GAL1 promoter were induced with 2% galactose. After two hours, expression of FUS1-LacZ was assayed by measurement of (3-galactosidase activity. The vector, pYeDP8-1/2, contains no STE12 sequence. 72 Results STE12 on his- GALACTOSE STE12 on his- GLUCOSE Ste12p Xbal BamHI DNA BINDING | HttHHHI 215 473 68 Figure 10. Ste12p(215-473) induces FUS1-HIS3 transcript ion Yeast (SY2585: STE12, his3::FUS1-HIS3) transformed with plasmids encoding Wt Ste12p, Ste12pADBD, Ste12p(215-473) or Ste12p (473-688) under the control of a GAL1 promoter were grown for three days on glucose- or galactose-containing media lacking histidine. The vector, pYeDP8-1/2, contains no STE12 sequence. 73 Results not be observed by monitoring growth (as in Figure 10), however, because overexpression of Wt Ste12p or Ste12pADBD causes growth arrest in yeast (see Overexpression of Ste12p results in growth arrest, below). In contrast, overexpression of either Ste12p(215-473) or Ste12p(473-688), did not result in growth arrest, allowing us to observe the FUS1-HIS3 induction. On his\" glucose media (Figure 10, bottom slide, vector) the unmodified strain is able to grow slowly. This is due to the weak constitutive expression of His3p in the parent strain (SY2585; C. Boone, pers. comm.). 2.1.3 Contribution of the PRP to increased transcription The basal activity of Ste12p (activity in the absence of pheromone) at some promoters has been shown to be dependent upon the function of the P R P (Fields etal . , 1988; Hagen etal . , 1991). For example, expression of the FUS1 gene, whose UAS is composed solely of four P R E elements, is dependent upon the activity of multiple components of the P R P , even in the absence of pheromone (Hagen et al., 1991). To determine whether the function of overexpressed Ste12p is also dependent upon the function of the P R P , I examined whether deletion of the MAP kinases that activate Ste12p, and that are penultimate to Ste12p in the P R P , could reduce transcriptional induction when Ste12p is overexpressed. In Figure 11, FUS1-LacZ induction by Ste12p overexpression was quantified in a fus3 strain and in a fus3 kss1 strain. As expected, induction of transcription by Wt Ste12p and Ste12pADBD was reduced in both of these strains. Previous 74 Results 100-, i i i vector WtSte12p Ste12pADBD Expression Plasmid Figure 11. Induction of FUS1-LacZ expression by Ste12p overexpression is partially dependent upon the MAP kinases of the PRP Yeast ( • :SY2585: STE12, FUS1-LacZ; L J : W H Y 3 - 1 : STE12, fus3, FUS1-LacZ; • :WHY2-7: STE12, fus3, kssl, FUS1-LacZ) transformed with plasmids encoding Wt Ste12p or Ste12pADBD under the control of a GAL1 promoter were induced with 2% galactose. After two hours, expression of FUS1-LacZ was assayed by measurement of p-galactosidase activity. The vector, pYeDP8-1/2, contains no STE12 sequence. 75 Results work had demonstrated that deletion of KSS1 in a fus3 strain further reduced the activity of the P R P (Elion et al., 1991). The trend was repeated here, as deletion of both kinases had a more marked phenotype than deletion of FUS3 alone. Both of these observations showed that the activity of overexpressed Ste12p is dependent upon P R P activity. However, the deletion of FUS3 and KSS1 does not eliminate the induction of FUS1 expression altogether, indicating that some of the activity of overexpressed Ste12p is independent of Ste12p modification by the P R P . This observation supports the hypothesis that accumulation of excess Ste12p is sufficient to overcome a physical limitation to activation by Ste12p, such as binding of a negative regulator. 2.2 Overexpression of Ste12p results in growth arrest According to the current model, Ste12p contributes to G1 arrest by directing increased transcription of Far lp , the CDK inhibitor (see FaMp is a pheromone-induced CKI). In order to evaluate the effect of Ste12p overexpression on cell cycle progression, I examined the growth of yeast in which Ste12p derivatives were being overexpressed (Figure 12). Overexpression of Wt Ste12p or Ste12pADBD prevented growth of both STE12 and ste12 yeast (Figure 12, galactose). Neither Ste12p(215-473) nor Ste12p(473-688) elicited a growth arrest response (Figure 12, galactose), however, demonstrating that residues on either side of amino acid 473 of Ste12p are required for growth arrest. 76 Results Xbal Ste12p I DNA BINDING I 215 BamHI 4 7 3 Figure 12. Ste12p overexpression induces growth arrest in both STE12 and stel2 yeast Yeast (SY2585: STE12; W303a::SUL-1: ste12) transformed with plasmids encoding Wt Ste12p, Ste12pADBD, Ste12p(215-473) or Ste12p (473-688) under the control of a GAL1 promoter were grown for three days on glucose- or galactose-containing media. The vector, pYeDP8-1/2, contains no STE12 sequence. 77 Results 2.2.1 FaMp-independent growth arrest Although Ste12pADBD could not induce FUS1-LacZ transcription (Figure 8B) in a ste72 strain, Ste12pADBD was able to cause growth arrest in the same strain (Figure 12, lower left panel). If Ste12pADBD induced growth arrest without inducing FUS1-LacZ, then it is possible that Ste12p overexpression caused growth arrest by a mechanism that is also independent of the induction of FAR1 gene expression. To test this model, I examined the growth of farl yeast strains in which Ste12p was being overexpressed (Figure 13). Ste12p overexpression was sufficient to cause growth arrest in both FAR1 and farl strains. Both Wt Ste12p and Ste12pADBD induced this arrest, demonstrating that an intact Ste12p DBD is not required for the phenotype. Based on these data, I concluded that Ste12p overexpression causes growth arrest that is independent of Far l p and independent of the direct interaction of Ste12p with P R E s . 2.2.2 G1 growth arrest Ste12p overexpression results in growth arrest on solid medium, but arrest on solid media cannot be directly attributed to a stage in the cell cycle. Pheromone-induced arrest is limited to the G1 phase of the cell cycle, at the Start checkpoint. Cells in the G1 phase of the cell cycle, such as those arrested by 7 8 Results ste12 FAR1 ste12 farl f Vector iff W-t Ste^2p % ^ S t e - % A D B D f StejIj^ADBD /V4Stej;gp GLUCOSE ste12 FAR1 Vector/.*. 3 Stel 2pADBD ste72 far* Vector .EpADBD Figure 13. Ste12p overexpression induces growth arrest in a FAR1-independent manner Yeast (yA03: ste12, FAR1; yA02: ste12, farl) transformed with plasmids encoding vector pYeDP8-1/2, Wt Ste12p or Ste12pADBD under the control of a GAL1 promoter were grown for three days on glucose- or galactose-containing media. The vector, pYeDP8-1/2, contains no STE12 sequence. 79 Results pheromone, have not formed a bud and can be readily identified under the microscope based on this morphology (Guthrie and Fink, 1991). To determine whether Ste12p overexpression causes G1 (pheromone-like) arrest, I examined the change in yeast growing in liquid culture when Ste12p overexpression is induced (Figure 14). Cells growing in mid log phase were isolated and determined to be 25 to 30 percent unbudded, prior to induction of Ste12p expression. Upon induction of Ste12p expression, the cells began to accumulate in the G1 phase, reaching a peak of 75 to 80 percent unbudded (G1) cells after approximately two hours (Figure 14). This accumulation of cells in the G1 phase was independent of both FAR1 and genomic STE12, as both genes could be deleted without eliminating the arrest. G1 growth arrest in response to mating pheromone occurred within approximately one cell cycle, or ninety to one hundred and twenty minutes, and arrested cultures were generally greater than ninety percent unbudded (Bucking-Throm et al., 1973; Guthrie and Fink, 1991; Hartwell, 1973). The Far lp independent growth arrest caused by Ste12p overexpression was not as complete, but it did occur as rapidly. 2.2.3 Overexpression of nuclear localized Ste12pADBD causes growth arrest Ste12pADBD does not have a nuclear.localization signal and, when Ste12pADBD is overexpressed, it is observed in both the nucleus and the cytoplasm of yeast (Hung et al., 1997). Since SteT2p is a nuclear protein, it is 80 Results CL CM o S £ co Q 00 Q < CL CM •3 CO —I i CO ^ > 0 CO 5 CD CO peppnqun % c o peppnqun % T m o LO LO CM peppnqun % CD M - TJ O CD X o ~ 8 * co CD CD -C +^ +i i_ C CD CO F CO TJ E > £ ^ ro Q ® cD CL > o CM CD i_ r - C £ ^ CO CD ° fOTJ § P- CD £ CT CM > CD CD ^ - 5 > W jB 2 C2-CM '« — O \\» , CJ LU asz y c o T-j ro c o co ^ £ o 2 c 0) m c ^ w iP. O c S 9- V 8 •o 46 LO in C\\J peppnqun % o m !? co 81 Results possible that Ste12pADBD is only able to cause growth arrest as a consequence of inappropriate localization. When galactose-inducible Ste12pADBD with a nuclear localization signal was expressed in yeast, most of the protein was nuclear localized (Hung et al., 1997), and Ste12pADBD/NLS induced growth arrest in both STE12 and ste12 strains (Figure 15). This indicates that the growth arrest function of Ste12pADBD is not due to inappropriate localization. 2.2.4 Participation of the G1 cyclins G1 growth arrest in response to pheromone includes inhibition of Cdc28p-cyclin complexes. As shown in Figure 16, overexpression of either C ln lp or Cln2p, but not Cln3p, relieved some of the growth arrest that was observed when Ste12pADBD was overexpressed. A vector control showed growth on glucose and no growth on galactose (data not shown). This indicates that overexpression of Ste12p may affect the function of specific Cdc28p-cyclin complexes. The effect may be direct interference with complex function or may be a result of altered expression of the CLN genes. 2.2.5 Ste12p overexpression does not reduce the viability of yeast One possible explanation for the accumulation of yeast in the G1 phase of the cell cycle when Ste12p is overexpressed is that a high concentration of Ste12p in yeast is lethal during the G1 to S transition. To determine whether Ste12p overexpression resulted in cell death, I measured the viability of yeast in which Ste12p had been overexpressed (Tables 2 and 3). 82 Results GALACTOSE Figure 15. Growth arrest by overexpression of Ste12pADBD/NLS Yeast (SY2585: STE12; W303a::SUL-1: ste12) transformed with a plasmid encoding Ste12pADBD with a nuclear localization signal (Ste12pDBD/NLS) under the control of a GAL1 promoter were grown for three days on glucose- or galactose-containing media. The vector, YEplac112, contains no STE12 sequence. 83 Results Figure 16. Overexpression of CLN1 or CLN2 partially rel ieves growth arrest induced by Ste12p overexpression Yeast (W303a::SUL-1: ste12) transformed with plasmids encoding Ste12pADBD and C ln lp , Cln2p or Cln3p under the control of a galactose inducible promoter were grown for three days on either glucose- or galactose- containing media. 84 Results Table 2. G1 growth arrest by Ste12p overexpression does not reflect cell death Strain 1 Plasmid 2 Percent Colonies/ Viability Unbudded 3 ODeoo Unit4 Relative to Vector 5 (x 106) STE12 Vector 26 3.9 1.0 STE12 WT Ste12p 76 3.3 0.85 STE12 Ste12pADBD 74 3.7 0.95 ste12 Vector 17 3.8 1.0 ste12 WT Ste12p 70 2.9 0.76 ste12 Ste12pADBD 76 2.9 0.76 Cells were grown to OD6oo«0.6 and Ste12p expression was induced with 2% galactose. After cells were grown a further two hours, the optical density and the percent of unbudded cells was determined for each culture. Simultaneously, 100 uL of a 10\"4 dilution of each culture was plated in duplicate on glucose plates. After three days, colonies formed on each plate were counted. 1 ST£72 was SY2585, ste12 was W303a::SUL-1. 2AII plasmids had a GAL1 promoter. 3Percent Unbudded was determined as in Materials and Methods. 4Colonies/OD6oo Unit is the number of colonies counted on the plate relative to the ODeoo of the culture two hours after induction. Each value reflects an average of two plates. 5Relative viability is defined as Colonies/OD6oo Unit relative to the vector control. 85 Results Table 3. G1 growth arrest by Ste12p overexpression does not reduce viability in farl yeast Strain 1 Plasmid 2 Colonies/ Viability ODeoo Unit Relative to Vector 4 (x 10 6) 3 STE12, FAR1 Vector 3.3 1.0 STE12, FAR1 WTste12p 3.3 1.0 STE12, FAR1 Ste12ADBD 3.2 0.96 STE12, farl Vector 3.2 1.0 STE12, farl WT ste12p 3.4 1.1 STE12, farl Ste12ADBD 3.5 1.1 ste12,FAR1 Vector 3.2 1.0 ste12, FAR1 WT ste12p 2.8 0.88 ste12, FAR1 Ste12ADBD 2.8 0.88 ste12, farl Vector 2.4 1.0 ste12, farl WT ste12p 2.3 0.96 ste12, farl Ste12ADBD 2.5 1.1 Cells were grown to OD6oo*0.6 and Ste12p expression was induced with 2% galactose. After cells were grown a further two hours, the optical density of the culture was determined. Simultaneously, 100 u.L of a 10\"4 dilution of each culture was plated in duplicate on glucose plates. After three days, colonies formed on each plate were counted. ^STE12, FAR1 was W3031-B, STE12, farl was SY2587, ste12, FAR1 was yA03 , and ste12, farl was yA02. All plasmids had a GAL1 promoter. 3Colonies/OD6oo Unit is the number of colonies counted on the plate relative to the ODeoo of the culture two hours after induction. Each value reflects an average of two plates. 4Relative viability is defined as Colonies/OD6oo Unit relative to the vector control. 86 Results To measure viability, the yeast were grown to mid log phase in liquid culture and Ste12p overexpression was induced by the addition of galactose. The yeast were exposed to galactose for two hours, which has been demonstrated to be sufficient time for both G1 growth arrest (Figure 14) and FUS1-lacZ induction (Figure 8). Following galactose exposure, the cells were transferred to glucose media and following three days growth, the viability of yeast in the galactose treated cultures was determined. Similar to the results in Figure 14, Ste12p overexpression resulted in the accumulation of greater than seventy per cent unbudded cells in yeast cultures. The yeast in the G1 arrested cultures were found to be 76 to 110 per cent viable relative to the unarrested (vector) controls. If Ste12p overexpression caused cell death in G1 phase, one would predict that only the unarrested cells, which represented 25 to 30 % of the culture, would have been viable. Similar viability was observed after growth arrest of STE12, ste12, FAR1 and farl yeast. Taken together, these results indicate that overexpression of Ste12p and Ste12pADBD can induce G1 growth arrest in yeast that is independent of FAR1 and of STE12, which implies that the arrest is independent of direct interaction between Ste12p and P R E s . 2.3 Overexpression of Ste12p increases mating in both STE12 and ste12 strains Ste12p overexpression results in both transcriptional induction and G1 growth arrest, two components of the pheromone response. As shown in this work, the G1 growth arrest induced by Ste12p is independent of Far lp , the CKI 87 Results which has been implicated in pheromone-induced growth arrest, indicating a novel function for Ste12p. A third measure of P R P and Ste12p function is the mating efficiency of yeast. In order to further characterize the effects of Ste12p overexpression, and to compare them to a normal pheromone response, I examined the mating efficiency of yeast which overexpress Ste12p. When a cells expressing Ste12p were mated to an a strain (HLY334), overexpression of Ste12p increased mating efficiency (Table 4). In STE12 yeast, overexpression of Ste12pADBD increased mating efficiency by a factor of 12.9, compared to yeast expressing only genomic STE12. In a ste12 strain, which otherwise did not mate, Wt Ste12p overexpression allowed 77% mating efficiency. As was the case for FUS1-LacZ induction by Ste12p overexpression, a STE12 gene with an intact DBD was required for mating (Table 4, absolute mating efficiency of ste12 yeast). Collectively, these data indicate that Ste12p overexpression increases mating efficiency through increased transcriptional activation. Since the increased mating required an intact Ste12p DBD and Ste12p overexpression did not substitute for pheromone induction of the P R P (data not shown), it is unlikely that a novel function of Ste12p causes the increased mating efficiency. This is consistent with previous observations that Ste12p increases mating through transcriptional activation (Fields and Herskowitz, 1985; Fields and Herskowitz, 1987). 88 Results Table 4. Ste12p overexpression increases mating efficiency Plasmid 1 Relative Mating Absolute Mating Efficiency of STE12 Efficiency of ste12 Yeast 3 Yeast 2 (%mated) Vector 1.0 0 WT Ste12p 1.6 77 Ste12pADBD 12.9 0 STE12 (SY2585) or ste12 (W303a::SUL-1) yeast was mated to a ten-fold excess of HLY334(Mat a) cells. 1AII plasmids had a GAL1 promoter. 2Mating efficiency was calculated relative to the number of STE12 yeast added to the mating reactions. Relative mating efficiency was calculated relative to the vector-bearing yeast. 3Mating efficiency was calculated relative to the number of STE12 yeast added to the mating reactions. All numbers reflect an average of two plates. 89 Results 2.4 Nested deletions of STE12 show that amino acids 262 to 594 are sufficient to induce transcription and growth arrest To define the minimum fragments of Ste12p capable of inducing FUS1-LacZ transcription and/or growth arrest in yeast, I made a set of nested deletion mutants of STE12 expressed from the GAL1 promoter (Figure 17). I found that sequences C-terminal to amino acid residue 594 or N-terminal to residue 262 can be deleted from overexpressed Ste12p while maintaining elevated FUS1-LacZ expression in a STE12 strain (Figure 18). Overexpression of Ste12p mutants p1(215 to 641), p2(215 to 594), p5(262 to 688), p10(262 to 594) or p11 (262 to 641) induced FUS1-LacZ activity in the absence of pheromone (Figure 18). In contrast, Ste12p mutants p3(215 to 547), p4(215 to 500), p6(309 to 688), p7(356 to 688), p8(403 to 688), p9(450 to 688) or p12(309 to 547) did not elevate FUS1-LacZ transcription when overexpressed. The minimum active fragment, amino acids 262 to 594 (p10) was sufficient to elevate transcription of FUS1-LacZto levels comparable to those induced by much larger fragments of Ste12p (Figure 18). Fragments of Ste12p with amino acids deleted from either extremity of the p10 sequence were not able to activate FUS1-LacZ. 90 Results Minimum Active Fragment 262 594 Xbal BamHI I DNA BINDING | 215 473 688 Expression Plasmid Wt 262 309 356 403 450 262 2 6 2 3 0 9 ' ADBD P1 P2 - p i 5 0 0 P6 P7 _P8 *fi9 P10 P11 P12 641 594 547 594 641 547 Figure 17. Nested deletions of STE12 Nested deletions of STE12 expressed under the control of the GAL1 promoter. The minimum active fragment (encodes amino acids 262 to 594) is the minimum fragment required to induce FUS1-LacZ activity and growth arrest. 91 Results WtSte12p- | Ste12pADBD p1H Expression Plasmid P2- f p3-p4-p5-p6-B P7 -P p8-p9-a p10-p11 • p12-CEH Vector - L 0 T=3-3=1 25 50 - 1 75 Relative G-galactosidase Activity Figure 18. Amino acids 262 to 594 of Ste12p induce FUS1-LacZ transcription when overexpressed Yeast (SY2585: STE12, FUS1-LacZ) transformed with plasmids encoding Wt Ste12p, Ste12pADBD or constructs p1 through p12 (Figure 17) under the control of a GAL1 promoter were induced with 2% galactose. After two hours, expression of FUS1-LacZ was assayed by measurement of (3-galactosidase activity. The vector, pYeDP8-1/2, contains no STE12 sequence. 92 Results The deletion mutants (Figure 17) were also evaluated for their ability to induce growth arrest in STE12 and ste12 strains (Table 5). Constructs p1, p2, p5, p'1'0 and p11 all caused growth arrest in both STE12 and ste12 yeast. Parallel to the results for FUS1-LacZ induction, constructs p3, p4, p6, p7, p8, p9 and p12 were not able to prevent growth of yeast on galactose. The minimum fragment of Ste12p whose overexpression caused growth arrest is amino acids 262 to 594. This is the same fragment which, when overexpressed, induces the activity of FUSI-LacZ. The (+) and (-) signs in Table 5 indicate whether the observed growth was most similar to the vector (+) or WT Ste12p (-) control. Expression of all of the deletion mutants was confirmed by western blot (data not shown). Collectively, these results identified amino acids 262 to 594 as the minimum fragment of Ste12p that can induce transcription and cause growth arrest. If there is a negative regulator of Ste12p whose activity can be titrated by an increase in Ste12p expression, amino acids 262 to 594 are sufficient to titrate it. Alternately, Ste12p overexpression might cause activation by Ste12p aggregation. If so, then 262 to 594 must be able to aggregate with Wt Ste12p and cause activation, or must interact with and inhibit factors which prevent aggregation of individual Wt Ste12p molecules with one another. In either case, one can predict that there is a mechanism for negative regulation of Ste12p that acts through amino acids 262 to 594 of Ste12p 93 Results Table 5. Growth arrest by Ste12p deletion mutants Ste12p Construct Amino acids Expressed Growth on-Galactose' Vector None + WTSte12p 1 to 688 Ste12pADBD 215 to 688 p1 215 to 641 p2 215 to 594 p3 215 to 547 + p4 215 to 500 + p5 262 to 688 p6 309 to 688 + p7 356 to 688 + p8 403 to 688 + p9 450 to 688 + p10 262 to 594 p11 262 to 641 p12 309 to 547 + All Ste12p species are expressed under the control of a GAL1 promoter. 2Growth was evaluated after 3 days on galactose and compared to growth of the same transformant on glucose. The growth of all constructs was compared to Vector (+) and WT Ste12p (-) controls which had been grown in the same experiments. Identical results were obtained using the strains SY2585 (STE12) and W303a::SUL-1 (ste12). 94 Results 3 Rstlp and Rst2p interact with distinct regions of Ste12p The following experiments examine the roles of Rst l p and Rst2p as functionally redundant negative regulators of Ste12p at P R E s . The results in Chapter 2 demonstrated that Ste12p overexpression can activate transcription and/or growth arrest in the absence of pheromone. The following data show that Rst lp and Rst2p can interact with Ste12p in vitro and can decrease Ste12p function in vivo. Further, the data indicate that Rst lp and Rst2p regulate Ste12p by different mechanisms. 3.1 Overexpression of Rst1 p and Rst2p abrogates the effects of Ste12p overexpression Overexpression of Ste12p resulted in the induction of Ste12p-responsive transcription and growth arrest in a manner consistent with the titration of a direct negative regulator from endogenous Ste12p (Dolan, 1996; Dolan and Fields, 1990). If Rs t lp and Rst2p act as direct negative regulators of Ste12p, then simultaneous overexpression of RST1 or RST2 should reduce the effect of STE12 overexpression. To determine if this was true, I concurrently overexpressed STE12 and RST1 or RST2. 3.1.1 Rstlp and Rst2p inhibit growth arrest Both Wt Ste12p and Ste12pADBD caused growth arrest when overexpressed in yeast (Figures 12,13 and 14). Concurrent overexpression of 95 Results either RST1 or RST2 prevented growth arrest by Ste12p overexpression in STE12 yeast, as could be predicted for negative regulators of Ste12p function. Overexpressing Wt Ste12p or Ste12pADBD caused growth arrest, but the yeast grew vigorously when either RST1 or RST2 was overexpressed simultaneously (Figure 19, galactose). In ste12 yeast, however, overexpression of RST2 did not prevent the growth arrest induced by Ste12pADBD as efficiently as overexpression of RST1 did (Figure 20, ste12, galactose). One possible explanation for this phenotype is that Rs t ip and Rst2p interact with different parts of Ste12p, and that Rst2p cannot interact efficiently with Ste12p species that do not have an intact DNA binding domain. 3.1.2 Rst1 p and Rst2p reduce FUS1-lacZ express ion A second phenotype of overexpression of Ste12p is the induction of FUS1-LacZ transcription in the absence of pheromone (Figure 8). Galactose-induced transcription of Wt Ste12p and Ste12pADBD may increase FUS1 activity by competing with endogenous Ste12p for interaction with a negative regulator. If this is true, then overexpression of the negative regulator should counteract the effect of increased Ste12p levels in the cell. Expression of either Rs t lp or Rst2p under the control of a galactose-inducible promoter reduced the induction of FUS1-LacZ by Wt Ste12p and Ste12pADBD in a dose-dependent manner (Figure 21). If Rst1 p or Rst2p was overexpressed from an episomal (high copy number) plasmid, either protein reduced activation by Wt Ste12p or Ste12pADBD (Figure 21, panel A), while expression from a centromeric (low copy number) 96 Results GALACTOSE GLUCOSE Figure 19. Overexpression of R s t l p or Rst2p relieves growth arrest caused by Ste12p overexpression Yeast (SY2585: STE12) transformed with plasmids encoding Wt Ste12p DBD or Ste12pADBD and Rst lp (1) or Rst2p (2) under the control of a GAL1 promoter were grown for three days on galactose- or glucose-containing media. The vector, pYeDP8-1/2, contains no STE12 sequence. The control (c), YEplac181, contains no RSTsequence. pMT556 and pMT558 encode Rs t lp and Rst2p, respectively. 97 Results GALACTOSE GLUCOSE STE12 ste12 STE12 ste12 Ste12pADBD Ste12pADBD ^ Wmmv • GALACTOSE GLUCOSE Vector Figure 20. Overexpression of Rst2p does not relieve growth arrest by overexpression of Ste12ADBD in ste12 yeast Yeast (SY2585: STE12, W303a::SUL-1: ste12) transformed with plasmids encoding Ste12pADBD and Rst lp (1) or Rst2p (2) under the control of a GAL1 promoter were grown for three days on galactose- or glucose-containing media. The vector, pYeDP8-1/2, contains no STE12 sequence. The control (c), YEplac112, contains no RSTsequence. pG1T and pG2T encode Rst lp and Rst2p, respectively. 98 Results E CO -2 o Q . C L CM to CO CD O DH DH n r-aaavdsLais r LO ~ r ~ o LO ~r LO CM •g 'E to _ro CL c o to to CD i C L X LU C L CM 0 -4—• CO AJJAIPV esepjsopeieo-t) h aaavdzLejs j opeA L O T r o LO LO CM (Zoei-LStld) AijAjpv esepjsopeieQ-'a •g E to ro CL c g to to CD i — C L X LU C L CM a) » CO c o \"35 w a> Q . Q X OQ £ a CD Q > C L O CM co o N OQ Q O Q. CO CM CO -g 'E to ro C L co -= to _CO ro o E ~ 2 o >r CD I\" CO tlj CD \" W £ ro CD • a S 0 4^ to o to 2> I _ r o C L o CD ^ i5 ^ CM CO CO ™ B s f ,ro m co. CO C L O CD CD « - T J CM E to c ro to DH w O a . T 3 CD O ZJ T J C 0 1— 0 CD O E o to ZJ o 0 cz c 4= ° 10 cc: o co —i i CO ^ > LL. c o to 2 Q . U J X r-~ a> co *- . . to S? LO CH oo ^ s ° CM CO cz O o CD •!= 0 T J C ZJ C L CM > O 11 -2 O i _ o CD CM < ^ • oo O C L CD D E £ C i _ 0 o ° o 0 0 CQ .iz 0 s CO CO ° to 5= ro _ - 5 ro i _ E £ 8 I 0) <° d r -cr T -0 (T) L L - 0 O £ c o • i £ iS to 0 to . I S c? LL >- CO 0 N < -V ® CO ro ^> C L LL C L ^ < CM CD to 0 * C T J ro c C L ro T— -*-» T - CO o DH - S 0 C L -O LU O >\" cz 1\" ro oi < ~ CO 0 T J S I - i i ro T - C L 00 — T - ro ° E JS o C L co LU >- 0 0 > o 0 C L CO 0 L _ C L CM —^< to DH T J C ro C L -4—' to Q : 0 T J O O c 0 -I—' ro J Z 99 Results plasmid also reduced the induction of FUS1-LacZ, but to a lesser extent (Figure 21, panel B). RST1 or RST2 overexpression reduced induction of FUS1-LacZ expression by Ste12p in both the presence and absence of pheromone (Figures 21 and 22). After a two-hour induction with both pheromone and galactose, overexpression of either RST gene reduced FUS1-LacZ activity by nearly one half as compared to a vector control (Figure 22). The overexpression of single RST genes from these vectors was insufficient to eliminate activation by Ste12p. However, if negative regulation by Rst1 p and Rst2p is dependent upon the recruitment of a co-regulator, such as the M A P K kinase K s s l p , the amount of co-regulator in the cell may limit the effects of RstXp overexpression. Alternately, the expression vectors may express less than stoicheometric amounts of Rs t lp and Rst2p. 3.2 Rstlp and Rst2p interact with distinct regions of Ste12p Rst lp and Rst2p reside in complexes with Ste12p, K s s l p and/or Fus3p (Cook et al., 1996; Tedford et al., 1997). Since overexpression of Rs t lp or Rst2p inhibits the effects of Ste12p overexpression, I investigated whether Rst lp or Rst2p could interact directly with Ste12p. In Figure 23, recombinant GST-Rst1 p and GST-Rst2p were added to [35S]-methionine labeled yeast extracts expressing Ste12p constructs and recovered with glutathione agarose. GST-Rst1p interacted with both Wt Ste12p 100 Results 40 n i > 30 -\\ o < CO w CO T J CO O o CO Figure 22. Overexpression of Rstlp or Rst2p reduces induction of FUS1-LacZ in response to pheromone Yeast (SY2585: STE12, FUS1-LacZ) transformed with plasmids encoding Rst lp or Rst2p under the control of a GAL1 promoter were induced with 2% galactose and 2 |ng/ml a-factor. After two hours, p-galactosidase activity from a FUS1-LacZ reporter was assayed. The vector, pYeDP8-1/2, contains no STE12 sequence. The control (c), YEplac181, contains no RST sequence. pMT556 and pMT558 are episomal plasmids that encode Rst1 p and Rst2p, respectively. 1(H 0 Vector Rst lp Rst2p Expression Plasmid 101 Results Wt Ste12p Ste12pADBD Vector Q . Q . Q . Q- Q . Q . i - CM i - CM £ CN n to \"co - Q \"to u5 - Q \"to co MW — 205 rj »f- gam «m» — - 87 — 69 — 56 1 2 3 4 5 6 7 8 9 Figure 23. Recombinant R s t l p and Rst2p interact with Ste12p in yeast extracts Yeast (W303a::SUL-1: ste12) expressing Wt Ste12p (A) or Ste12pADBD (B) under the control of a GAL1 promoter were labeled with [3 5S]-methionine. Ste12p derivatives were recovered from yeast lysates by immunoprecipitation with a-Ste12p antibodies (lanes 1, 4 and 7) or by interaction with 5uig of either GST-Rst1p (lanes 2, 5 and 8) or GST-Rst2p (lanes 3, 6 and 9) and subsequent recovery with glutathione agarose. Recovered proteins were resolved by electrophoresis and visualized by autofluorography. The vector, pYeDP8-1/2, contains no STE12 sequence. 102 Results and Ste12pADBD proteins (Figure 23, lanes 2 and 5; labeled A and B) in yeast extracts. In contrast, GST-Rst2p was able to interact with Wt Ste12p (Figure 23, lane 3) but was unable to bind to Ste12pADBD (Figure 23, lane 6). These results show that recombinant GST-Rst1p and GST-Rst2p can interact with Ste12p in yeast extracts, but that the two inhibitors bind to different domains of Ste12p. Alternately, conformational changes to Ste12p caused by the DBD deletion may affect the structure of distal domains and thereby disrupt the interaction of Rst2p with Ste12pADBD. When GST-Rst1 p and GST-Rst2p were mixed with extracts of yeast containing no Ste12p (Figure 23, lanes 8 and 9) and recovered with glutathione agarose, Rs t lp bound a single protein of approximately 85 kDa, while Rst2p bound two proteins, one of approximately 85 kDa and another of approximately 92 kDa. These proteins have not been identified. Rst lp and Rst2p could also interact with recombinant Ste12p in baculovirus-infected insect cell extracts in the absence of any other yeast proteins (Figure 24, lanes 1 and 3). This indicates that Rs t lp and RSt2p can interact directly with Ste12p. This result, however, may be complicated by the fact that insect cells express MAP kinases. The inactive form of K s s l p regulates Ste12p and the K s s l p interaction with Ste12p is dependent upon the presence of Rst lp and Rst2p (Bardwell et al., 1998b). Thus it is possible that the interaction of GST-Rst1p and GST-Rst2p with Ste12p from insect cells is mediated by insect MAP kinases. 103 Results GST-Rst1p GST-Rst2p GST input Ste12p + - + - + - -want 1 2 3 4 5 6 7 8 MW 116 84 58 Figure 24. R s t l p and Rst2p interact with Ste12p in insect cel l lysates 5 jig of GST-Rst1 (lanes 1 and 2), GST-Rst2p (lanes 3 and 4) or G S T (lanes 5 and 6) were added to crude 100 no, of Sf9 (Spodoptera frugiperda) lysates containing Wt Ste12p (+) or no Ste12p (-) expressed from a baculovirus and protein complexes were recovered with glutathione agarose. After washing, the complexes were resolved by electrophoresis and Wt Ste12p (A) was detected by western blot with a-Ste12p antibodies. The input is crude Sf9 (Spodoptera frugiperda) lysate from cells infected with the Ste12p virus (+) (lane 8) or the wild-type virus (-) (lane 7). 104 Results 3.2.1 Rstlp interacts with amino acids 262 to 594 of Ste12p Overexpression of Ste12p(262 to 594) results in FUS1-lacZ induction which can be reduced by the overexpression of Rs t lp or Rst2p. In the experiment shown in Figure 25, I examined whether the same region of Ste12p can interact with Rst lp or Rst2p. I determined that recombinant GST-Rst1p could interact with Ste12p fragments including p1(215 to 641), p2(215 to 594) and p5(262 to 688) in yeast extracts (Figure 25, p1, p2, p5, lanes 2). GST-Rst1p did not recover fragments p4(215 to 500) or p7(356 to 688) and recovered p3(215 to 547) and p6(309 to 688) with reduced efficiency (Figure 25, p3, p4, p6, p7; lanes 2). These results show that amino acids 262 to 594 of Ste12p can interact with Rst lp , while a smaller fragment, Ste12p(309-547) can also interact, but with lower affinity. 3.2.2 Rst2p interacts with the DBD of Ste12p GST-Rst2p did not interact with any Ste12p fragment that did not include the Ste12p DNA binding domain (Figure 25, lanes 3). Since GST-Rst2p can interact efficiently with Wt Ste12p, I examined whether or not Rst2p interacts directly with amino acids 1 to 215 of Ste12p. In this experiment, both GST-Rst2p and GST-Rst1p interacted with purified recombinant 6-His-Ste12pDBD from E. coli (Figure 26, lanes 1 and 2), but the Rst2p-Ste12pDBD interaction was very 105 Results XJ < p1 CO DC P 2 P3 Rst2p < Rstlp Rst2p XJ < Rstlp a. CNJ +-> CO DC P 4 a. a r- CM -9 CO CO < CC DC MW — 193 — 112 — 87 — 69 — 56 X) < P 5 a o. T - CM * -CO CO CC CC P 6 a. a r- CM •Q CO CO < cc cc X! < P7 CO CC Q. CM •*-> CO CC MW — 193 — 112 — 87 — 69 — 56 Ste12p Xbal BamHI DNA BINDING 215 473 688 Figure 25. R s t l p interacts with amino acids 262 to 594 of Ste12p Yeast (W303a::SUL-1: ste12) expressing Ste12p constructs p1 through p7 (Figure 17) under the control of a GAL1 promoter were labeled with [ S]-methionine. Ste12p derivatives were recovered from yeast lysates by immunoprecipitation with a-Ste12p antibodies (lanes 1) or by interaction with 5 p.g of either GST-Rst1p (lanes 2) or GST-Rst2p (lanes 3) and subsequent recovery with glutathione agarose. Ste12p constructs encode the following amino acids: p1(215 to 641), p2(215 to 594), p3(215 to 547), p4(215 to 500), p5(262 to 688), p6(309 to 688), and p7(356 to 688). Recovered proteins were resolved by electrophoresis and visualized by autofluorography. Please see Figure 17 for a schematic of the Ste12p constructs. 106 Results strong whereas the Rst1 p interaction was weak. The interaction of GST-Rst1 p with Ste12pDBD was not consistently detected when this assay was repeated. 6-His-Gal4pDBD did not interact with either Rs t lp or Rst2p (Figure 26, lanes 5 and 6) and GST alone failed to interact with either Gal4pDBD or Ste12pDBD, confirming that the Rs t lp and Rst2p interactions with 6-His-Ste12pDBD are specific. This result also demonstrates that Rst2p and Rst1 p can interact directly with Ste12p in the absence of other proteins. Rst2p could interact with smaller fragments of the Ste12p DNA binding domain (Figure 27). The smallest fragment of the DNA binding domain that was found to interact with GST-Rst2p was amino acids 21 to 195 (Figure 27, lanes 13 to 16), the region of Ste12p implicated in DNA binding (Yuan and Fields, 1991). When the DNA binding domain was bisected, neither of the resulting halves was able to interact with Ste12p (Figure 27, lanes 17 to 24), indicating that Rst2p must contact amino acids at either end of the Ste12pDBD. When further deletions were made at either end of Ste12p, I found that Ste12pDBD fragments that contain amino acids 21 to 170 or amino acids 45 to 195 did not interact with GST-Rst2p (data not shown). This confirms that amino acids from both ends of the DBD are required for Rst2p interaction. Consistent with previous results, neither G S T nor GST-Rst1p interacted with the DNA binding domain of Ste12p. 3.2.3 Rstlp interacts directly with Ste12p GST-Rst1p was able to recover Ste12p from yeast extracts (Figure 23) and interacted efficiently with amino acids 262 to 594 (Figure 25). However, 107 Results Ste12p DBD Gal4p DBD Q. a C N i « CO CO CC CC 1 1-1 r— r-(/> (/) O O n 1 2 3 a Q. CM CO CO CC tr 3 1 1-a (/> c/> c O 5 6 CS) o 3 a c 8 Figure 26. Rst2p interacts with the DNA binding domain of Ste12p A two-fold molar excess of GST-Rst2p (lane 1), GST-Rst1 p (lane 2) or GST (lane 3) was mixed with 6-His-Ste12pDBD (labeled A). Proteins were recovered from solution by glutathione agarose, washed and resolved by electrophoresis. 6-His-Ste12pDBD was detected by western blot with polyclonal a-histidine antibodies. The 6-His-Ste12pDBD in lane 4 is equivalent to 1/12 of the 6-His-Ste12pDBD used in the interaction assays. In lanes 5 to 8, 6-His-Gal4pDBD was treated in the same manner. All proteins used in this assay were purified from E. coli. 108 Results amino 1 to 215 1 to 195 21 to 215 21 to 195 acids: Q . Q . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 amino ac ids: 1 to 108 109 to 215 Q . Q . Q _ Q . T - CN T - CM +-» 4 -1 * J CO CA CA CA DC CC DC CC • I 4-i I I H I- H 3 I— I— I— =J • > i u co co co a. co co co a. 57 — 39 — 34 — 17 18 19 20 21 22 23 24 Figure 27. Rst2p interacts with amino ac ids 21 to 195 of the DNA binding domain of Ste12p 3 ng of GST-Rst1 p (lanes 1, 5, 9, 13,17 and 21), GST-Rst2p (lanes 2, 6, 10, 14, 18 and 22) or G S T (lanes 3, 7, 11, 15, 19 and 23) was mixed with 1 mg of crude E. coli extracts expressing 6-His-Ste12pDBD constructs. Proteins were recovered from solution by glutathione agarose, washed and resolved by electrophoresis. 6-His-Ste12pDBD proteins were detected by western blot with polyclonal a-histidine antibodies. The 6-His-Ste12pDBD in lanes 4, 8, 12, 16, 20 and 24 is equivalent to 1/20 of the 6-His-Ste12pDBD used in the interaction assays. E. coli extracts in lanes 1 to 4 contain 6-His-Ste12pDBD(1 to 215); lanes 5 to 8 contain 6-His-Ste12pDBD(1 to 195); lanes 9 to 12 contain 6-His-Ste12pDBD(21 to 21); lanes 13 to 16 contain 6-His-Ste12pDBD(21 to 195); lanes 17 to 20 contain 6-His-Ste12pDBD(1 to 108), lanes 21 to 24 contain 6-His-Ste12pDBD(109 to 215). All G S T proteins used in this assay were purified from E. coli. 109 Results since Rst1 p simultaneously interacts with other proteins from the whole cell extract, it was not clear whether Rs t lp interacted directly with Ste12p(262 to 594). To determine whether Rst1 p could interact directly with the C-terminus of Ste12p, I expressed a trpE-Ste12pADBD fusion in E. coli. GST-Rst1p recovered trpE-Ste12ADBD from solution, but GST-Rst2p did not (Figure 28, lanes 2 and 3). From this result, I concluded that recombinant Rst1 p can interact with the C-terminus of Ste12p in the absence of other eukaryotic proteins. 3.3 Rst2p interacts with the Ste12p DNA binding domain in vivo Collectively, the results in Figures 21 to 28 demonstrate that Rs t lp and Rst2p can interact directly with separate regions of Ste12p. Rs t lp interacts with residues 262 to 594 the region of Ste12p which, when overexpressed, activates pheromone-like responses in yeast and Rst2p interacts with the Ste12p DBD. The data is insufficient, however, to establish that the Ste12p DNA binding domain is the domain through which Rst2p inhibits the activity of Ste12p. To examine the effect of Rst1 p and Rst2p on the DNA binding domain of Ste12p, I used a fusion of the Ste12p DNA binding domain (Ste12pDBD, amino acids 1 to 215) to the potent transcriptional activation domain of HSV-1 VP16 (Sadowski et al., 1988). The fusion, DBD-VP16, was expressed from a galactose-inducible promoter. Expression of DBD-VP16 in yeast activated transcription of FUS1-LacZ approximately 25-fold more than expression of the Ste12p DBD alone (Figure 29). When Rst2p was co-expressed with DBD-VP16, 110 \\ Results a a T— CM (/> OC DC 1 1 -1 1 -(/) O o O a c MW 116 84 Figure 28. R s t l p interacts directly with Ste12pADBD 5 ng of GST (lane 1), GST-Rst1p (lane 2) or GST-Rst2p (lane 3) were added to 100 Lig of E. coli lysates containing a trpE-Ste12pADBD fusion (Ste12p amino acids 216 to 688) and protein complexes were recovered with glutathione agarose. After washing, the complexes were resolved by electrophoresis and trpE-Ste12pADBD was detected by western blot with a-Ste12p antibodies. The input is crude E. coli lysate containing the trpE-Ste12pADBD fusion. 111 Results RstXp Expression Vector • Control • Rs t lp • Rst2p DBD DBD-VP16 Vector Expression Plasmid Figure 29. Rst2p inhibits the function of the Ste12p DNA Binding Domain Yeast (W303A::SUL-1: ste12, FUS1-LacZ) transformed with plasmids expressing Ste12pDBD or DBD-VP16 and Rst lp or Rst2p under the control of a GAL1 promoter were induced with 2% galactose. After two hours, (3-galactosidase activity from a FUS1-LacZ reporter was assayed. The vector, pYeDP8-1/2, contains no STE12 sequence. The control, Y E p l a d 112, contains no RST sequence. pG1T and pG2T are centromeric plasmids that encode Rst lp and Rst2p, respectively. 112 ' Results the activation of FUS1-LacZ was reduced (Figure 29). The overexpression of RST1, however, had no effect on the induction of FUS1-LacZ by DBD-VP16 (Figure 29). The expression of DBD-VP16 also activated transcription of endogenous FUS1. Consistent with the experiments using the FUS1-LacZ reporter gene (Figure 29), overexpression of Rst2p reduced FUS1 activation by DBD-VP16, while overexpression of Rs t lp did not (Figure 30 and data not shown). When DBD-VP16 was expressed in either RST2 or rst2 yeast, FUS1 expression was increased (Figure 30, lanes 2 and 4). When RST2 was expressed under the control of a galactose promoter in the same yeast, FUS1 activity was reduced (Figure 30, compare lanes 1 to 4 to lanes 5 to 8). These results demonstrate that Rst2p is able to inhibit Ste12p function through the specific interaction of Rst2p with the DNA binding domain of Ste12p. Consistent with the finding that Rst1 p interacts with amino acids 262 to 594 of Ste12p, Rs t lp was unable to regulate Ste12p through its DNA binding domain alone. 3.4 Rst2p disrupts the interaction of Ste12p with DNA in vitro Rst2p interacts specifically with the DNA binding domain of Ste12p and inhibits Ste12p function. Based on these observations, it is possible that is that Rst2p inhibits Ste12p DNA binding. I assayed this by observing the effects of Rst2p in an electrophoretic mobility shift assay (EMSA) of Ste12p. 1'13 Results Vector Rst2p Strain: RST2 RST2 rst2A rst2A co co Ste12p: Q CO Q Q_ > • Q CD Q Q CD Q Q_ > I Q CD Q RST2 RST2 rst2A rst2A CO CO Q CQ Q D -> I Q CQ Q O CQ Q Q_ > Q CQ Q FUS1 ACT1 8 Figure 30. Rst2p inhibits the function of the Ste12p DNA Binding Domain Yeast (W3031a: RST2; MTy1147:rst2) transformed with plasmids expressing Ste12pDBD or DBD-VP16 and a vector (lanes 1 to 4) or Rst2p (lanes 5 to 8) under the control of a GAL1 promoter were induced with 2% galactose. After two hours, RNA was prepared from the cells and the expression of both FUS1 (top) and ACT1 (bottom) was detected by northern blot. 114 Results The DNA binding domain of Ste12p, when expressed and purified from E. coli, bound specifically to a pheromone responsive element in an E M S A (Figure 31 and 32, lanes 1 to 5). When Rst2p was added to, the E M S A reaction, however, the interaction of Ste12pDBD with the DNA was reduced (Figure 31, lane 9). The efficiency of the Ste12pDBD interaction with DNA was inversely proportional to the amount of Rst2p present in the E M S A reaction (Figure 32, lanes 6 to 9). In contrast, the presence of Rs t lp in the E M S A did not affect the binding of the Ste12pDBD to DNA (Figure 31 lanes 6 and 7). It should be noted that, in Figure 32, equal masses of GST-Rst2p and G S T were used to titrate Ste12p. As G S T is a much smaller protein than Rst2p (approximately one fifth the size), the molar quantities of GST were higher than the molar quantities of GST-Rst2p! It should also be noted that the upper band in lane 13 is Ste12p-specific (data not shown), indicating that the G S T is not disrupting Ste12p binding, but stabilizing a larger Ste12p-DBD complex. These results demonstrate that Rst2p can prevent the binding of Ste12pDBD to DNA in vitro, indicating that Rst2p may inhibit Ste12p function by impeding its ability to bind to P R E s . 3.5 Accumulation of Ste12p is unnecessary for FUS1 induction in response to pheromone Ste12p bound to P R E s mediates pheromone responsive transcription. Many of the genes encoding pheromone response pathway components and P R P regulatory proteins, including STE12 and RST2, have multiple upstream 115 Results 1 2 3 4 5 6 7 8 9 10 11 ^^^^^^^^^^^^^^^^^^^^^^^^ Figure 31. Rst2p disrupts the interaction of Ste12pDBD with DNA in E M S A 3 ng of 6-His-Ste12pDBD was incubated in the presence of competitors (oligonucleotides or protein) on ice. After one hour, an oligonucleotide probe containing two P R E elements arranged tail to tail was added and the reactions were incubated for a further 30 minutes on ice. Reactions were resolved as described in Materials and Methods. A indicates the 6-His-Ste12pDBD-oligonucleotide complex. Lane 1 contains no inhibitors. Lanes 2 and 3 contain 10- and 100-fold excesses of unlabelled P R E oligonucleotide. Lanes 4 and 5 contain 10- and 100-fold excesses of unlabeled Gal4p UAS oligonucleotide (see Materials and Methods), respectively. Lanes 6 and 7 contain 1 and 5 Lig of GST-Rst lp . Lanes 8 and 9 contain 1^g and 5 ug of GST-Rst2p. Lanes 10 and 11 contain 4 tig and 16 jag of GST. 116 Results 1 2 3 4 5 6 7 8 9 10 11 12 13 Figure 32. Disruption of DNA binding with increasing amounts of Rst2p Reactions were performed as described in Figure 31. A indicates the 6-His-Ste12pDBD-oligonucleotide complex. Lane 1 contains no inhibitors. Lanes 2 and 3 contain 10- and 100-fold excesses of unlabeled P R E oligonucleotide. Lanes 4 and 5 contain 10- and 100-fold excesses of unlabeled Gal4p UAS oligonucleotide (see Materials and Methods), respectively. Lanes 6 to 9 contain 1 Lig, 2 ng, 4 ng and 10 Lig of GST-Rst2p and lanes 10 to 13 contain 1 fig, 2 Lig, 4 Lig and 10 ng of GST. 117 Results P R E s . Pheromone-stimulated transcription of genes encoding P R P components results in an amplification of signaling sensitivity in response to pheromone. Since STE12 has five consensus P R E s in its promoter, I examined whether Ste12p accumulation in response to pheromone relieves inhibition of Ste12p by Rs t lp and Rst2p. To determine whether Ste12p accumulation is required for activation of transcription in response to pheromone, I measured induction of FUS1 transcription in cells treated with cycloheximide. As measured by northern blot, inhibition of protein synthesis by cycloheximide did not prevent FUS1 induction Instead, cycloheximide treatment caused an approximately 100-fold superinduction of FUS1 transcription following a 30-minute treatment with a mating pheromone (Figure 33, FUS1). This result demonstrates that nascent Ste12p synthesis is not required for induction of transcription in response to pheromone. In the absence of cycloheximide, STE12 transcription increased two-fold in response to pheromone (Figure 33, STE12, lanes 3 and 4, or 7 and 8). In contrast, when yeast are treated with pheromone over a 30-minute period, Ste12p protein levels remained approximately constant (Figure 34, compare lanes 5 and 6). These results are consistent with the conclusion that FUS1 induction is not dependent upon novel synthesis of Ste12p. The increased FUS1 induction in response to pheromone when cycloheximide is present indicates that one or more proteins, whose synthesis is 118 Results RST2 rst2A CYC: + + - - + + - -0C: + - + - +- + -1 2 3 4 5 6 7 8 FUS1 RST2 WW * RST1 STE12 Figure 33. FUS1 induction in response to pheromone does not require new protein synthesis Yeast (W3031a: RST2, MTy1147: rst2) were treated for ten minutes with 0.1 mg/ml cycloheximide (CYC: lanes 1, 2, 5 and 6) followed by a thirty minute treatment with a-factor (a: lanes 1, 3, 5, and 7). Yeast in lanes marked (-) were grown for the same period but were not treated with either cycloheximide or a-factor. Gene expression of FUS1, RST2, RST1, STE12 and ACT1 was evaluated by northern blot. 119 Results stimulated in pheromone-treated yeast, inhibit pheromone induction. Rs t lp is not a likely candidate for this protein, as RST1 expression is not affected by a-factor treatment (Figure 33, RST1). Consistent with previous observations, however, the transcription of RST2 is slightly elevated in response to pheromone (Figure 33, RST2). A failure to accumulate Rst2p cannot be the sole mechanism for increased FUS1 expression in cycloheximide treated yeast, since superinduction of FUS1 is observed in both RST2 and rst2 strains. Other negative regulators of the P R P , which do not interact directly with Ste12p (see Recovery from pheromone response), are also known to be induced by pheromone treatment. Cycloheximide treatment may inhibit synthesis of multiple proteins required for attenuation of pheromone-responsive transcription. Alternatively, cycloheximide treatment may result in the loss of unstable regulatory proteins. Two other mechanisms may be involved in FUS1 superinduction. First, cycloheximide may enhance the stability of FUS1 mRNA, perhaps by trapping the RNA molecules on polysomes and thereby shielding them from cytoplasmic ribonucleases (Cochran et al., 1983; Edwards and Mahadevan, 1992; Oleinick, 1977). Alternately, the superinduction may be the result of transcriptional induction in response to cycloheximide, as has been observed for other promoters (Hu and Hoffman, 1993; Koshiba et al., 1995; Li et al., 2001). For example, cycloheximide may alter the chromatin structure adjacent to the FUS1 gene (Cesari et al., 1998). 120 Results t=0 t=30 pheromone: + STE12I + 193— I 112 — 87 — 69 — 56 — MW 4 5 6 7 8 t=0 t=30 + - + + + -pheromone: + - + STE12: + + - • 193 — 112 — * v ^ ^ J 1 - _ j j t a < u ^ _ 87 69 — \" ~ - - - . . -MW 9 10 11 12 13 14 15 16 Figure 34. Ste12p does not accumulate in yeast cel ls in response to pheromone induction Yeast (W3031a: STE12; yA06: ste12) were treated with a-factor (pheromone: lanes 1 ,3 ,5 and 7). Yeast in lanes marked (-) were grown for the same period but were not treated with a-factor. Immediately after pheromone treatment (t=0) or thirty minutes later (t=30), protein extracts were prepared from the cells and the amount of Ste12p (A) in 1 mg of each extract was determined by western blot with a-Ste12p polyclonal antibodies (TOP). a-Ste12p antibodies were preabsorbed onto extracts from ste12 yeast (see Materials and Methods). Antibody complexes were subsequently removed from the blot and the amount of Gal4p (B) present in the extracts was determined using a-Gal4p antibodies (BOTTOM). 121 Discussion 4 Discussion The unifying goal of this work was to further define both the domain structure of Ste12p and the regulatory mechanisms that control Ste12p activity. The results of this study revealed that overexpression of Ste12p(262 to 594) induces transcription, growth arrest and increased mating in yeast that is independent of Far lp . The results also showed that overexpression of STE12 can cause growth arrest that is independent of transcription from pheromone responsive elements. The stimulation of Ste12p-dependent responses by overexpression of amino acids 262 to 594 indicated that there may be a direct negative regulator of Ste12p that can be titrated by excess Ste12p. Alternately, the overexpression of Ste12p may result in the formation of Ste12p multimers which can activate transcription. If activation of transcription in response to pheromone by Ste12p is dependent upon the formation of Ste12p multimers, the presence of excess Ste12p might be sufficient to cause multimerization and activation in the absence of the pheromone stimulus. Overexpression of RST1 or RST2, two negative regulators of Ste12p, was shown to counteract the effects of STE12 overexpression. Additionally, Rst lp and Rst2p, which reside in complexes with Ste12p in vivo, were demonstrated to interact directly with distinct domains of Ste12p. Ste12p is a transcription factor which functions in a complex network of kinases, negative regulators and other transcription factors. This work 122 Discussion demonstrated a new role for Ste12p in growth arrest and showed that the two negative regulators, Rs t lp and Rst2p, must regulate Ste12p by separate mechanisms. 4.1 A mechanism for FaMp-independent G1 growth arrest Previously published work has shown that overexpression of Ste12p can induce G1 growth arrest that is F/\\f?7-independent (Dolan, 1996; Dolan and Fields, 1990). This work demonstrates that overexpression of Ste12p amino acids 262 to 594 is sufficient to induce G1 growth arrest that is independent of Far lp . Further, the growth arrest occurs in both STE12 and ste12 yeast, indicating that the arrest is independent of transcription from pheromone responsive elements. Like pheromone-induced arrest, the arrest occurs within one cell cycle. Also consistent with a PRP-like response, this arrest can be counteracted by the overexpression of CLN1 and CLN2, two G1 cyclins whose expression and activity is inhibited in pheromone response (see The pheromone response and the cell cycle). It is possible that Ste12p overexpression inhibits growth by activating transcription in a PRE-independent manner. For example, Ste12p might interact with DNA indirectly by interacting with another transcription factor, such as T e d p or Mcmlp (see DNA binding and protein-protein interactions of Ste12p). The FAR1 promoter, for example, contains two putative P R E s , but it also includes binding sites for Mcmlp , which may facilitate activation by overexpressed Ste12p (SCPD; http://cgsigma.cshl.org/jian/). 123 Discussion Some observations from this work support the model that Ste12p induces growth arrest by activating transcription. When deletions were made to define the smallest domains of Ste12p that could induce either G1 arrest or FUS1-LacZ induction, both phenotypes were linked to the same domain, amino acids 262 to 594. Amino acids 262 to 594 overlap with amino acids 216 to 356, which are known to function as an activation region of Ste12p and with the pheromone induction domain, amino acids 301 to 335 (Olson et al., 2000; Pi et al., 1997; Song et al., 1991). Since the residues that induce G1 arrest have not been differentiated from the residues that activate transcription, it is possible that the G1 arrest function requires that Ste12p act as an activator. In addition, overexpression of RST1 and RST2, two negative regulators of Ste12p, can counteract the overexpression of STE12 and restore growth. Rs t lp and Rst2p may counteract activation of transcription by Ste12p. Alternatively, Ste12p may be inhibiting growth by a transcription-independent mechanism. It is noteworthy that Rst lp and Rst2p, two cognate negative regulators of Ste12p, were cloned in a two hybrid screen as proteins that interact with C ln lp and Cln2p (Tedford et al., 1997). No explanation or function for this interaction has ever been demonstrated. Perhaps Ste12p interferes with cell cycle progression by either promoting or disrupting the interaction of Rstps and G1 cyclins. Regardless of whether or not Ste12p must direct transcription in order to facilitate growth arrest, it must inhibit cell cycle progression through a novel mechanism, since the arrest is independent of FAR1, the pheromone inducible 124 Discussion CKI. In addition, the mechanism is less potent than Fai ip- induced arrest, since only 75 to 80 percent of cells appear to arrest when Ste12p is overexpressed, while almost all cells arrest during pheromone response in response to Far lp (Guthrie and Fink, 1991). Future work must identify which gene or protein targets are affected by Ste12p in the process of causing growth arrest. Global gene expression analysis could compare Ste12p overexpression with pheromone induction (Roberts et al., 2000) to determine which genes are up-regulated when Ste12p is overexpressed in farl cells. Global protein-protein interaction by Repressed Trans-Activator (RTA™) analysis (US Patent No. 5,885,779) or \"protein chip\" analysis (MacBeath and Schreiber, 2000) could identify proteins that interact with Ste12p. Collectively, these data could identify the gene products required for FAR1-independent growth arrest. 4.2 Interaction of Ste12p with negative regulators In this work, I have demonstrated that overexpression of STE12 increases transcription from a FUS1 reporter in both STE12 and ste12 yeast, that Ste12pADBD induces transcription in STE12 yeast and that amino acids 262 to 594 correspond to the minimal domain of Ste12p that can induce FUS1-lacZ transcription. Previous results have shown that deletion of regions of the STE12 gene encompassed by amino acids 262 to 594 results in increased activity of both Ste12p and Ste12p-Gal4p fusions in yeast (Kirkman-Correia et al., 1993; 125 Discussion Song et al., 1991). All of these results are consistent with the model that a titratable, direct negative regulator inhibits Ste12p. Two known negative regulators of Ste12p activity are Rs t lp and Rst2p. Both of the Rstps are found in complexes that interact with Ste12p, K s s l p and Fus3p, and deletion of the RST1 and RST2 genes promotes both invasive growth and pheromone-response phenotypes (Cook et al., 1996; Tedford et al., 1997). Overexpression of the RST genes counteracts the effects of STE12 overexpression in a dose-dependent manner, indicating that these regulators may be the factors that are titrated when excess Ste12p is present in cells. Also consistent with the function of direct negative regulators, both Rs t lp and Rst2p bind directly to recombinant Ste12p and interact with Ste12p in yeast extracts. Rst1 p and Rst2p cannot function as titratable negative regulators, however. Deletion of RST1 and RST2 from the genome does not eliminate the activation of transcription that is observed when Ste12p is overexpressed (Olson et al., 2000). Therefore, excess Ste12p cannot simply be competing with endogenous Ste12p for Rst1 p and Rst2p. Although these results show that Rs t lp and Rst2p bind directly to separate domains of Ste12p and inhibit its function, they do not define a mechanism for inhibition of Ste12p by Rs t lp and Rst2p. 4.3 A mechanism for regulation of Ste12p by Rst2p Rst2p interferes with the interaction between Ste12p and the P R E in E M S A assays, indicating that Rst2p may inhibit Ste12p by preventing DNA 126 Discussion binding. Rst2p can also inhibit the function of the Ste12p DBD in vivo, since Rst2p inhibits the function of Ste12pDBD-VP16 fusions. These results show that Rst2p interacts with the DNA binding domain of Ste12p, but data presented in Figure 21 and in two-hybrid analysis (Pi et al., 1997) show that RST2 can inhibit the function of Ste12pADBD. The results in Figure 21 do not imply that Rst2p can inhibit Ste12pADBD directly, however. In this experiment, FUS1-lacZ transcription is measured in a STE12 strain, as Ste12pADBD alone cannot activate transcription. If, as demonstrated in recent work, Ste12p overexpression results in multimerization that activates transcription (Olson et al., 2000), the Rst2p may inhibit Ste12p by interacting with the DNA binding domain of the endogenous Ste12p, not the overexpressed Ste12pADBD. It is also possible that Rst2p interacts with Ste12pADBD as a member of a complex. Rst2p is required for the regulatory functions of Kss1 p, indicating that a Ste12p-Kss1 p-Rst2p complex may exist (Bardwell et al., 1998b). If this is true, a direct interaction between Rst2p and Ste12pADBD may not be required for inhibition. A further result that argues that Rst2p does not interact with the Ste12pADBD is shown in Figure 20. Rst2p has limited ability to counteract the growth arrest induced by Ste12pADBD in a ste12 strain, although it does prevent arrest in a STE12 strain. Independent of an interaction of Rst2p with the C-terminus of Ste12p, Rst2p inhibits the function of the Ste12p DBD at P R E s and disrupts DNA binding 127 Discussion in vitro. Based on these results, it is possible to postulate that Rst2p inhibits Ste12p simply by masking its DNA binding residues. However, alternate mechanisms may also be considered. Ste12p is postulated to bind to DNA as a dimer, although the dimerization domain has not been identified in amino acids 1 to 215. It is possible that Rst2p eliminates Ste12p dimerization, like the PIAS inhibitors of Stat transactivation (Chung et al., 1997; Liu et al., 1998). Another important function of amino acids 1 to 215 of Ste12p is interaction with T e d p , which is required for cooperative binding to F R E elements (Madhani and Fink, 1997). Since Rst2p is also a negative regulator of invasive growth, it is possible that interaction with the Ste12p DBD may disrupt Ste12p-Tec1p interactions. In this case, Rst2p may function to maintain MAP kinase cascade specificity. The increased expression of RST2 in the presence of pheromone could result in reduced Ste12p-Tec1p interaction or increased Ste12p-Kss1p interaction, resulting in reduced activation from P R E s . Incomplete understanding of the Ste12p DNA binding domain complicates the process of defining the function of Rst2p in the regulation of Ste12p. Although the DNA binding domain of Ste12p is generally defined as amino acids 1 to 215, the minimum fragment required for DNA binding is actually amino acids 40 to 204 (Yuan and Fields, 1991). As a result, the nominal DBD of Ste12p includes at least 51 amino acids that may have other functions, including dimerization and protein-protein interaction. The Rst2p interaction with amino acids 21 to 195 of Ste12p overlaps with 20 of the amino acids (aa 21 to 40) not implicated in DNA binding, indicating that it could be regulating other Ste12p 128 Discussion functions. The interaction does not overlap, however with amino acids 1 to 19 of Ste12p, which were recently identified as a negative regulatory domain of Ste12p (Crosby etal . , 2000). 4.4 Mechanisms for regulation of Ste12p by Rstlp Rst1 p interacts with the C-terminus of Ste12p, amino acids 309 to 547, a region that includes the pheromone induction domain (amino acids 301 to 335) and overlaps with amino acids 214 to 473 of Ste12p, which are known to activate transcription (Kirkman-Correia et al., 1993; Pi et al., 1997; Song et.al., 1991). Rs t lp may inhibit Ste12p by masking the activation domain in a pheromone-reversible manner. Recent observations have shown that Rst1 p, and not Rst2p, inhibits the function of Ste12pADBD in the absence of pheromone (Olson et al., 2000). When a LexAp-Ste12p(216 to 688) fusion is expressed in RST1 or rst2A cells in the absence of pheromone, only a low level of activity is observed from a LexA-LacZ reporter. When the same fusion is expressed In an rst1 strain, however, the LexA-LacZ reporter has a very high level of activity (Olson et al., 2000). This indicates that, in the absence of pheromone, Rst lp does, indeed, regulate the activity of Ste12pADBD in vivo. Based on the observed functions of other negative regulators, Rs t lp could inhibit Ste12p by several mechanisms. Rst lp could function like Gal80p, the inhibitor of Gal4p, and mask the activation domain of Ste12p in the absence of pheromone (Ma and Ptashne, 1987; Yano and Fukasawa, 1997). Rs t lp might 129 Discussion also interfere with interaction of Ste12p and other DNA binding proteins, such as Mcmlp and a l p . One other strong possibility is that Rs t lp stabilizes the interaction between Ste12p and a Ste12p-specific inhibitor, such as an inactive MAP kinase. This possibility is consistent with the observation that K s s l p inhibition of Ste12p is dependent upon Rst lp (Bardwell et al., 1998a). Rs t lp interacts in yeast extracts with Ste12p amino acids 309 to 547, a smaller fragment of Ste12p than is required for activation by overexpression. Recent work has demonstrated that this discrepancy may be observed because Rst lp preferentially interacts with Ste12p multimers (Olson et al., 2000). Several regions in the C-terminus spanning amino acids 216 to 688 are required for multimerization of Ste12p and the region 262-356 is required for activation. So, although amino acids 309 to 547 can support multimerization and Rs t lp interaction, they may lack the activation domain that would be required to increase FUS1-lacZ expression when Ste12p is overexpressed. 4.5 Rstlp and Rst2p interact with different regions of Ste12p Previous observations about Rs t lp and Rst2p have suggested that these two proteins function in the same manner. First, when RST1 and RST2 were cloned, it was suggested that the two genes were redundant, as both genes had to be deleted from the yeast genome before the phenotypes of invasive growth, slow growth and increased expression of pheromone responsive genes could be detected (Cook et al., 1996; Tedford et al., 1997). Second, sequence comparison showed that the proteins are 22% identical over their entire 130 Discussion sequence, sharing a 60 amino acid segment with 64% similarity (Tedford et al., 1997). Third, immunoprecipitation and kinase assays showed that both proteins are located in complexes with Ste12p, K s s l p and Fus3p and that the proteins are MAP kinase substrates. Collectively, these results led to the conclusion that Rst lp and Rst2p inhibit Ste12p by the same mechanism. One difference that was observed in the original studies of RST1 and RST2 may account for their apparent redundancy. RST2 expression is pheromone-induced, while RST1 is constitutively expressed (Tedford et al., 1997). As a result, the two genes might appear to have a single function when, in fact, they do not. Since RST2 is expressed only at low levels in the absence of pheromone, deleting RST2 will have little effect in vegetatively growing cells, where Rst lp is required for regulation of Ste12p activity. The effects of deletion of RST1, on the other hand, might be masked by increased activity of Ste12p resulting in the increased expression of RST2. This work shows that Rst lp and Rst2p, in fact, do not work by identical mechanisms. Rs t lp and Rst2p interact with different parts of the Ste12p protein and only Rst2p can inhibit the function of the Ste12p DNA binding domain. Rst lp , in contrast, interacts with and may inhibit the function of the Ste12p activation domain. These observations support the hypothesis that the apparent redundancy of Rs t lp and Rst2p may be due to their complementary, but non-identical, functions. 131 Discussion 4.6 Pheromone-responsive activation of transcription by Ste12p In these experiments, I have demonstrated that overexpression of STE12 is sufficient to overcome Ste12p inhibition. It is unlikely, however, that the sole mechanism of Ste12p activation in pheromone response is the accumulation of excess Ste12p. When yeast are treated with both cycloheximide and pheromone, FUS1 transcription increases one hundred fold in 30 minutes, demonstrating that Ste12p accumulation is not required for activation. In addition, when yeast are treated with pheromone, Ste12p levels remain approximately the same and STE12 transcription increases only two-fold. FUS1 transcription is known to increase more than 10-fold under the same conditions (McCaffrey et al., 1987; Trueheart et al., 1987). This demonstrates that the level of expression of Ste12p is not reflected in the level of induction of expression of pheromone responsive genes. Ste12p activation, therefore, must reflect a change in the state of Ste12p or of its associated negative regulators. Ste12p is activated by M A P kinases and Ste12p, Rst lp and Rst2p are all known targets of Fus3p and K s s l p (Cook et al., 1996; Tedford et al., 1997). Initially, activation of Ste12p could be mediated by changing the nature of the Ste12p-Rst1p interaction. For example, phosphorylation may dissociate the Ste12p-Rst1p-kinase interaction or alter it to expose the Ste12p activation domain. This model is supported by the observation that Rst1 p interacts reversibly with the pheromone induction domain (Pi etal . , 1997). 132 Discussion After a period of pheromone induction, Rst2p may accumulate in the cell. Since Rst2p is a pheromone-induced negative regulator of transcription, it is possible that Rst2p is required for recovery from pheromone and that, upon induction, Rst2p binds to Ste12p and inhibits transcription of pheromone responsive genes. Alternatively, Rst2p may function to maintain MAP kinase cascade specificity, by ensuring that Ste12p does not interact with T e d p and activate transcription from FREs . If either of these models proves to be true, phosphorylation of Ste12p and Rst2p may, in fact, facilitate interaction and inhibition. 4.7 A revised model for Ste12p regulation Recent work in the field of MAP kinase regulation has revealed new aspects of Ste12p function. It is now clear that Ste12p is an effector of two separate MAP kinase cascades and that it has DNA binding partners that are specific to both pheromone response and filamentation (Gustin et al., 1998; Madhani and Fink, 1998). In addition, Ste12p has also been demonstrated to be the target of negative regulation, in complexes with Rst lp , Rst2p and the MAPK K s s l p (Bardwell et al., 1998a; Bardwell et al., 1998b; Cook et al., 1997; Tedford etal . , 1997). Ste12p can no longer be simply visualized as a transcription factor that binds to P R E s and activates transcription upon MAP kinase phosphorylation. Any model of Ste12p function must now include the aspects of negative 133 Discussion regulation, relief of inhibition in response to MAP kinase stimulation and mechanisms for maintaining specificity of signaling. Based on my observations of Ste12p and its regulation by Rs t lp and Rst2p, I propose the model in Figure 35 for Ste12p regulation in response to pheromone. In the absence of pheromone, a complex of K s s l p and Rs t lp inhibits Ste12p bound to P R E s by binding to the activation domain of Ste12p. Upon pheromone induction, Ste12p and Rs t lp become phosphorylated, disrupting the complex and revealing the Ste12p activation domain. After a period of pheromone responsive transcription, Rst2p binds to Ste12p, disrupts DNA binding and stimulates recovery. Rst2p bound to the Ste12p DBD also disrupts the F R E complex, ensuring that there is no crosstalk between the FIP and the P R P . Several approaches could be used to address this model for Rst2p function. To determine whether pheromone induction of RST2 is required for Rst2p function, gene expression could be monitorred in yeast with a non-pheromone inducible RST2. If pheromone induction of RST2 is required for recovery, yeast with non-pheromone inducible RST2 should show increased expression of pheromone responsive genes after extended exposure to pheromone. If pheromone-induced RST2 prevents inappropriate activation from FREs , FIP gene expression should be induced by pheromone in yeast with non-pheromone inducible RST2. To explore whether or not Rst2p prevents the interaction of Ste12p with P R E elements in vivo, Ste12p interaction with DNA could be compared by chromatin immunoprecipitation or in vivo footprinting 134 Discussion NO PHEROMONE P H E R O M O N E INDUCTION Figure 35. A new model for Ste12p regulation Based on the results in this thesis, I propose a new model for Ste12p regulation. For details, see text. 135 Discussion in both RST2 and rst2 strains. It is also possible that Rst2p inhibits the function of the Ste12pDBD by preventing its oligomerization. Experiments similar to those published for Rst lp (Olson et al., 2000), could determine whether there is an oligomerization domain in the Ste12pDBD and whether it is the domain of Ste12p that interacts with Rst2p. This model does not include a function for Rst1 p at F R E s , due to a lack of data, but it may block the activation domain of Ste12p in the same manner. In filamentation-invasion, one might predict that Rst lp inhibition would be relieved by Ksslp-dependent phosphorylation. 4.8 Future work - Ste12p as a model transcript ion factor Ste12p is a transcription factor and an effector in multiple signal transduction pathways, and Ste12p activity is modulated by a web of protein-protein interactions. Because of its genetic accessibility and readily modulated activity, Ste12p has been and should continue to be an excellent model transcriptional activator. To date, the characterization of Ste12p has focused on the interaction of Ste12p with the pheromone and filamentation MAP kinase cascades. The work has resulted in a significant number of observations about how signal transduction impacts upon the activity of a transcriptional activator. What has not been directly addressed, however, is how the activator, Ste12p, transmits an extracellular signal to the RNA polymerase II transcriptional machinery. If the component(s) of the transcriptional machinery with which 136 Discussion Ste12p interacts were identified, it might be possible to define more precisely how Ste12p activates transcription. Consequently, it might also be possible to understand how specific protein-protein interactions modulate Ste12p activity. Such an approach to understanding Ste12p regulation might provide insight into the functions of multiple transcriptional activators. Several mechanisms that regulate activity are also observed for other transcription factors. Like the Ets-1 family of activators, Ste12p is directed to specific promoters by interactions with other sequence-specific transcription factors (Wasylyk et al., 1998). Like the Myc proto-oncogene, Ste12p directs transcription in response to multiple signal transduction pathways that control diverse developmental programs (Gustin et al., 1998; Sakamuro and Prendergast, 1999). If we understood how Ste12p interacts with the transcriptional machinery, we might then be able to understand how these modes of regulation affect transcription factor activity, both collectively and individually. Future study of Ste12p will also explore the regulation of Ste12p by Rst lp and Rst2p. Rst lp and Rst2p, which bind directly to Ste12p, interact with different regions of Ste12p and appear to function by different mechanisms. If we understood how Ste12p interacts with the Pol II transcriptional machinery, it might help us to decipher precisely how these negative regulators function and why two separate mechanisms are required. Ste12p could also be used as a model to develop novel methods to study transcription factors. Protein microarray technology may provide a practical method to determine the targets of Ste12p within the transcription machinery 137 Discussion (MacBeath and Schreiber, 2000; Zhu et al., 2000). Protein microarrays of the components of the transcription machinery could be probed with Ste12p to identify Ste12p targets. Interaction could then be confirmed by more traditional assays of protein-protein interaction, such as co-immunoprecipitation. If such a method were successful, it would be applicable to many transcription factors. 4.9 Conc lus ion This work has demonstrated that overexpression of STE12 can induce growth arrest and activate transcription. The growth arrest induced by Ste12p is Farlp-independent but otherwise reminiscent of pheromone-induced arrest, which suggests that Ste12p may have a second role in pheromone-induced growth arrest. This work has also demonstrated that the induction of transcription by Ste12p overexpression can be inhibited by overexpression of Rst lp and Rst2p and that Rst lp and Rst2p can bind directly to Ste12p. The observation that Rst1 p and Rst2p interact with different domains of Ste12p suggests that, although the two proteins are genetically redundant, they do not have identical functions. Rst2p is likely an inhibitor of Ste12p DNA binding and Rs t lp may inhibit the function of the Ste12p activation domain. Further work will be required to fully characterize the mechanisms of Ste12p inhibition by Rs t lp and Rst2p in combination with the P R P and FIP M A P kinases. Future work will also be required to determine how the inhibition is relieved to allow Ste12p to activate transcription. 138 Discussion The overriding goal for future study of Ste12p will be to understand how specificity of signaling is maintained when a single activator stimulates the genes required for two different signal transduction cascades. New technologies that explore global gene expression and global protein-protein interactions should facilitate reaching that goal. 139 Materials and Methods 5 Materials and Methods 5.1 Plasmids, strains and media Plasmids and strains used in these experiments are listed in Tables 6, 7 and 8. Yeast media was made as described (Ausubel et al., 1998; Guthrie and Fink, 1991; Kaiser et al., 1994). a-Factor (Sigma Chemical Company) was added at 2 ng/ml to liquid cultures. Cycloheximide was used at a concentration of 0.1 mg/mL. Plasmid transformations into yeast used the method of (Kaiser et al., 1994). Unless otherwise stated, E. coli were grown in LB media, supplemented with 100 ng/mL ampicillin when appropriate. DNA manipulation techniques including amplification by polymerase chain reaction (PCR), restriction enzyme digestion, DNA purification and ligation followed standard procedures (Ausubel et al., 1998; Sambrook et al., 1989). The plasmid pSC4 (Table 6) was used as the template for P C R amplification of STE12 sequences. The E. coli strain D H 5 a was used for the propagation of plasmids (Table 8). Plasmids used for this study are described in Table 6. Oligonucleotides used for P C R amplification of STE12 are described in Table 9. The sequences of plasmids containing PCR-amplified sequences were confirmed by DNA sequencing. Manual DNA sequencing with Sequenase T7 DNA polymerase (United States Biochemical Corporation) and automated sequencing using an 140 Materials and Methods Table 6. P lasmids Plasmid Name Plasmid Description Plasmid Source PYeDP8-1/2 pJL1 Ye/STE12AXbal pSC4 pA012 pA034 pUNK YEpNLS YEplac112 YEplac181 Ylplac204 Ylplac211 p1 Episomal plasmid with galactose-inducible promoter. Episomal plasmid, expresses WT Ste12 from a galactose-inducible promoter. Episomal plasmid, expresses Ste12ADBD, amino acids 216 to 688, from a galactose-inducible promoter. Plasmid containing STE12 genomic DNA; used as P C R template for STE12 cloning. Episomal plasmid, expresses Ste12, amino acids 215 to 473, from a galactose-inducible promoter. Episomal plasmid, expresses Ste12, amino acids 473 to 688, from a galactose-inducible promoter. Integrating plasmid, removes STE12 promoter and DBD when transformed into yeast. Episomal plasmid, expresses. Ste12ADBD, amino acids 216 to 688, with a nuclear localization signal from a galactose-inducible promoter. Episomal plasmid, TRP1 marker. Episomal plasmid, LEU2 marker. Integrating plasmid, TRP1 marker. Integrating plasmid, URA3 marker. Episomal plasmid, expresses Ste12, amino acids 215 to 641, from a galactose-inducible promoter. (Cullin and Pompon, 1988) W. Hung W. Hung (Fields and Herskowitz, 1987) P C R amplified STE12 with oligonucleotides A01 and A 0 2 , cloned into pYeDP8-1/2. P C R amplified STE12 with oligonucleotides A01 and A 0 2 , cloned into pYeDP8-1/2. Deleted Xbal fragment of pSC4, then cloned the Sphl/Sacl fragment into Ylplac211. W. Hung D. Gietz D. Gietz D. Gietz D. Gietz P C R amplified STE12 with oligonucleotides A01 and A 0 1 5 , cloned into pYeDP8-1/2. 141 Materials and Methods P2 Episomal plasmid, expresses P C R amplified STE12 with Ste12, amino acids 215 to 594, oligonucleotides A01 and from a galactose-inducible A014 , cloned into promoter. pYeDP8-1/2: P3 Episomal plasmid, expresses P C R amplified STE12with Ste12, amino acids 215 to 547, oligonucleotides A01 and from a galactose-inducible A 0 1 3 , cloned into promoter. pYeDP8-1/2. p4 Episomal plasmid, expresses P C R amplified STE12with Ste12, amino acids 215 to 500, oligonucleotides A01 and from a galactose-inducible A012 , cloned into promoter. pYeDP8-1/2. p5 Episomal plasmid, expresses P C R amplified STE12 with Ste12, amino acids 262 to 688, oligonucleotides A 0 7 and from a galactose-inducible A 0 4 , cloned into pYeDP8-promoter. 1/2. p6 Episomal plasmid, expresses P C R amplified STE12 with Ste12, amino acids 309 to 688, oligonucleotides A 0 8 and from a galactose-inducible A 0 4 , cloned into pYeDP8-promoter. 1/2. P7 Episomal plasmid, expresses P C R amplified STE12 with Ste12, amino acids 356 to 688, oligonucleotides A 0 9 and from a galactose-inducible A 0 4 , cloned into pYeDP8-promoter. 1/2. p8 Episomal plasmid, expresses P C R amplified STE12with Ste12, amino acids 403 to 688, oligonucleotides AO10 from a galactose-inducible and A 0 4 , cloned into promoter. pYeDP8-1/2. p9 Episomal plasmid, expresses P C R amplified STE12.with Ste12, amino acids 450 to 688, oligonucleotides A011 from a galactose-inducible and A 0 4 , cloned into promoter. pYeDP8-1/2. p10 Episomal plasmid, expresses BamHIinsert of p2, cloned Ste12, amino acids 262 to 594, into the BamHI-digested from a galactose-inducible . backbone of p5. promoter. p11 Episomal plasmid, expresses Bam HI insert of p1, cloned Ste12, amino acids 262 to 641, into the BamHI-digested from a galactose-inducible backbone of p5. promoter. p12 Episomal plasmid, expresses BamHI insert of p3, cloned Ste12, amino acids 309 to 547, into the BamHI-digested from a galactose-inducible backbone of p6. promoter. 142 Materials and Methods pMT556 Episomal plasmid, expresses RST1 under the control of a galactose-inducible promoter. M. Tyers pMT558 Episomal plasmid, expresses RST2 under the control of a galactose-inducible promoter. M. Tyers pG1T Centromeric plasmid, expresses RST1 under the control of a galactose-inducible promoter. M. Tyers pG2T Centromeric plasmid, expresses RST2 under the control of a galactose-inducible promoter. M. Tyers pJL1/trp Episomal plasmid, expresses WT Ste12 from a galactose-inducible promoter. TRP1 marker. W. Hung pAXba/trp Episomal plasmid, expresses Ste12ADBD (amino acids 216 to 688) from a galactose-inducible promoter. TRP1 marker. W. Hung pMT485 2 u plasmid, URA3 marker, expresses CLN1 under the control of a galactose-inducible promoter. M. Tyers pMT979 2 LI plasmid, URA3 marker, expresses CLN2 under the control of a galactose-inducible promoter. M. Tyers pMT42 2 LI plasmid, URA3 marker, expresses CLN3 under the control of a galactose-inducible promoter. M. Tyers pMT580 Expresses GST-Rst1 p in E. coli. M. Tyers pMT581 Expresses GST-Rst2p in E. coli. M. Tyers pGEX-4T3 GST expression vector for E. coli Pharmacia Biotechnology pSTVP/235 Episomal plasmid, expresses Ste12p DBD (amino acids 1 to 215) fused to an HSV1 VP16 activation domain from a galactose-inducible promoter. W. Hung pAO001 -50 to +820 of the FUS1 open P C R amplified FUS1 with reading frame in pGEM3Z(f+). oligonucleotides A 0 2 3 and A024 , cloned into pGEM3Z(f+)at the BamHI site. 143 Materials and Methods pAO002 pAO003 pV1.4 pV2 pV3 pV4 pAO006 pAOOOJ pAO008 pAO009 pAO010 Same as pAOOOt, opposite orientation. Episomal plasmid, expresses Ste12, amino acids 1 to 215, from a galactose-inducible promoter. Expresses 6-His-Ste12pDBD, amino acids 1 to 215, in E. coli. Expresses 6-His-Ste12pDBD, amino acids 1 to 195, in E. coli. Expresses 6-His-Ste12pDBD, amino acids 21 to 215, in E. coli. Expresses 6-His-Ste12pDBD, amino acids 21 to 195, in E. coli. Expresses 6-His-Ste12pDBD, amino acids 1 to 108, in E. coli. Expresses 6-His-Ste12pDBD, amino acids 109 to 215, in E. coli. Expresses 6-His-Ste12pDBD, amino acids 21 to 170, in E. coli. Expresses 6-His-Ste12pDBD, amino acids 21 to 155, in E. coli. Expresses 6-His-Ste12pDBD, amino acids 70 to 195, in E. coli. As for AO001. EcoRI/BamHI insert of pV1.4 cloned into pYeDP8-1/2. P C R amplified STE12 with oligonucleotides VT1 and VT2, cloned into pRSETA (Invitrogen). P C R amplified STE12 with oligonucleotides VT1 and SDB2, cloned into pRSETA (Invitrogen). P C R amplified STE12 with oligonucleotides SDB1 and VT2, cloned into pRSETA (Invitrogen). P C R amplified STE12 with oligonucleotides SDB1 and SDB2, cloned into pRSETA (Invitrogen). P C R amplified STE12 with oligonucleotides VT1 and A026 , cloned into pRSETA (Invitrogen). P C R amplified STE12 with oligonucleotides VT1 and A 0 2 5 , cloned into pRSETA (Invitrogen). P C R amplified STE12 with oligonucleotides SDB1 and A027 , cloned into pRSETA (Invitrogen). P C R amplified STE12 with oligonucleotides SDB1 and A028 , cloned into pRSETA (Invitrogen). P C R amplified STE12 with oligonucleotides AO30 and SDB2, cloned into pRSETA (Invitrogen). 144 Materials and Methods pAO011 Expresses 6-His-Ste12pDBD, P C R amplified STE12 with amino acids 45 to 195, in E. coli. oligonucleotides A029 and SDB2, cloned into pRSETA (Invitrogen). pMR8 PHD1 clone, use EcoRI/Mlul fragment as probe for Northern and Southern blot analysis. W. Hung pTES314 Expresses TRPE-Ste12p, amino acids 220 to 688, fusion in £. coli. W. Hung pDF33 850 nucleotide fragment of ACT1 in pUC18. D. McMaster 145 Materials and Methods Table 7. Yeast Strains Strain Genotype Source SY2585 W303a::SUL-1 W303'1a W3031b SY2587 y A O i yA02 yA03 MTy1147 MTy1154 WHY2-7 WHY3-1 HLY334 SY991 SY2625 Mat a, Ieu2, trp1, ura3, ade2, mfa2A::FUS1- C. Boone LacZ, his3::FUS1-HIS3 Mat a, Ieu2, trp1, ura3, ade2, mfa2A::FUS1- W. Hung LacZ, his3::FUS1-HIS3, ste12::LEU2 Mat a, Ieu2, trp1, ura3, ade2, his3, can1 E. Leberer Mat a, Ieu2, trp1, ura3, ade2, his3, can1 E. Leberer Mat a , farl, Ieu2, trp1, ura3, ade2, his3, can1 C. Boone Mat a, ste12, Ieu2, trp1, ura3, ade2, This study mfa2A::FUS1-LacZ, his3::FUS1-HIS3 Mat a, ste12, farl, Ieu2, trp1, ura3, ade2, , This study his3, can1 Mat a , ste12, Ieu2, trp1, ura3, ade2, his3, This study can1 Mat a, rst2::HIS3, Ieu2, trp1, ura3, ade2, M. Tyers h7s3, can1 Mat a, rst1::TRP1, Ieu2, trp1, ura3, ade2, M. Tyers his3, can1 Mat a, fus3::LEU2, kss1::URA3, Ieu2, trp1, W. Hung ura3, ade2, mfa2A::FUS1-LacZ, his3::FUS1-HIS3 Mat a, fus3::LEU2, Ieu2, trp1, ura3, ade2, W. Hung mfa2A::FUS 1 -LacZ, his3::FUS 1 -HIS3 Mat a , ura3-52 G. Fink Mat a, ade2, his3, Ieu2, trpt, ura3, can1, C. Boone mfa1::LEU2, mfa2::LEU2 \\ > Mat a, barl, ade2, Ieu2, trp1, ura3, can1, C. Boone his3::FUS1-HIS3 146 Materials and Methods Table 8. E. coli Strains Strain Genotype D H 5 a supE44, endA1, hsdR17 (rk-, mk+), th i l , recA1, gyrA96, relA1(F80lacZdM15) JM101 supE, thi, d(lac-proAB), [F\", traD36, proAB, laclqZdM15], [rk+, mk+], mcrA(+) NM522* F'[(proAB+laclqZdeltaM15), supEthi1delta(lacproAB)deltahsd(r-m-)lambda-deoR+] RR1 SupE44, hsdS20(r-Bm-B), ara14, proA2, lacY1, galK2, rpsL20, xy15, mtl1 * Gift from D. Kilburn 147 Materials and Methods Table 9. Oligonucleotides for construction of STE12 deletions Oligo- Ste12p Oligonucleotide sequence nucleotide amino name acid residue 0 A01 215F 5 ' - G G T A C C A T G T C T A G A A G A C C A T C T A G T A C A A C A A 0 7 262F 5 ' -GGTACCATGCCCTCTCAAATTAATGATTTTATT A 0 8 309F 5 ' - G G T A C C A T G G A C T A I I I ICTTGTATCTGTTGAA A 0 9 356F 5 ' -GGTACCATGTCTCTTCTTAATAGATACCCCTAT AO10 403F 5 ' - G G T A C C A T G G A C C C T A C C A G C T A C A T G A A G T A T A011 450F 5 ' - G G T A C C A T G C A A T C T T A C C C A A A C G G A A T G G T T A012 500R 5 ' -GAATTCTCATTGTGGATACAGCATATTGTTATC A 0 1 3 547R 5 ' -GAATTCTCACTGCATGGAATTTGAAC I I IGCAT A014 594R 5 ' -GAATTCTCAATTTCCTTGTGAAGACTTCATTCC A 0 1 5 641R 5 ' - G A A T T C T C A A G A A T C T T C G T C A C C A G C A C T T G G A 0 4 688R 5 ' -GAATTCTCAGGTTGCATCTGGAAGGTTTTTATC VT1 1F 5 ' - C T G G A T C C A T G A A A G T C C A A A T A A C C A A T A G T SDB1 21F 5 ' - C T G G A T C C A T G G A A A A C G A T G A A G T C A G T A A A G C T A029 45F 5 ' - C T G G A T C C A T G T T C T T T T T A G C C A C A G C G AO30 70F 5 ' - C T G G A T C C A T G G G C I I IGTCTCTTGTGTATTT A 0 2 5 109F 5 ' -CTGGATCCATGTTTGAAGAGGGTATTTTTTCA A026 108R 5 ' -TCGAATTCTCATTTTTTCTTTTGAACTACTTCTCT A028 155R 5 ' -TCGAATTCTCAAAAAAATACTTTCTGCTTTTTTTG A027 170R 5 ' - T C G A A T T C T C A T T C C A A C G C A T C C G SBD2 195R 5'-TCG AATTCTC ATG A A A A A G A T A A G G C G G G CTC ATT VT2 215R 5 ' -TCGAATTCTCATCTAGAATCTAAATGTTGAAGTAA °The number indicates the terminal amino acid encoded by the P C R amplification product. F oligonucleotides prime in the forward orientation, R oligonucleotides in reverse. 148 Materials and Methods ABI Prism Genetic Analyzer (Applied Biosystems (Canada) Inc.) were performed according to the manufacturer's instructions. Saccharomyces cerevisiae strains yA01 , yA02 and y A 0 3 (Table 7) were made by deletion of STE12 from the genome of SY2585, SY2587 and W3031-B using the plasmid pUNK (Table 6). To confirm the identity of the strains, potential ste12 strains were tested for their ability to respond to pheromone in a halo assay (a strains) or for their ability to secrete pheromone ( a strains (Guthrie and Fink, 1991)). The genotypes of new strains were confirmed by Southern blot (Ausubel et al., 1998; Sambrook et al., 1989). 5.2 p-galactosidase assays For p-galactosidase assays, yeast were grown to ODeoo* 0.8 in selective minimal media, induced with 2 % (% w/v) galactose and/or 2 ng/ml a-factor and then grown for two hours, unless otherwise stated, p-galactosidase activity was determined as described (Ruby et al., 1983). First, ODeooof the culture was determined. Then, 500 uL of the culture were harvested and resuspended in 250 LiL of Z buffer (100 mM sodium phosphate (pH 7.0), 10 mM potassium chloride, 1 mM magnesium sulphate and 0.27% p-mercaptoethanol), 50 uL 0.1% S D S (sodium dodecyl sulphate) and 50 LIL chloroform. Samples were vortexed briefly and incubated for five minutes at 30 °C. The assay was started by addition of 200 LAL O N P G (o-nitrophenylgalactoside, 0.004 ng/mL in Z-buffer) and stopped 149 Materials and Methods by addition of 500 uiL of 1M sodium carbonate (pH 11). Absorbance of the final sample at 420 nm was determined. The formula for calculation of (3-galactosidase activity is as follows: p-galactosidase activity = (1000 x A42o)/(OD 6oo x v x t ) Where: v = sample volume in ml_ (0.5 ml_) t = assay time in minutes. B-galactosidase activity values are reported as an average of three trials and error bars represent standard deviation of those three trials. For those values for which no error is reported, the standard deviation value is not large enough to distinguish with respect to the scale of the graph. 5.3 G1 Growth Arrest Assay ' The method of (Guthrie and Fink, 1991) was used to determine the percentage of yeast cells in a culture that were in the G1 phase of the cell cycle. Briefly, a 1 ml_ aliquot of yeast from a mid-log suspension culture was fixed in 10% formaldehyde and sonicated for 3 seconds on low power. Fixed cells were washed in 100 LIL of P B S and stored in P B S at 4°C until they could be counted. The ratio of unbudded to total cells was determined and expressed as a percentage for a minimum of 200 cells per sample. 5.4 Quantitative Mating Assays Mating assays were performed by standard techniques (Guthrie and Fink, 1991). The two strains to be mated were grown to O D 6 o o « 0.8 and filtered onto a 150 Materials and Methods common support. The yeast were transferred to galactose-containing plates and incubated at 30 °C for 4 to 5 hours. Yeast were then transferred to diploid-selective media and grown for two or three days, at which time the number of colonies formed was counted. One haploid strain was added to the mating reaction at a lower (10-fold diluted) frequency than the other. The number of yeast in the mating reaction from the limiting strain is defined as the \"number of haploids\" in the determination of mating efficiency. Mating efficiency is defined as the ratio of diploids formed to the number of haploids added to the mating reaction. 5.5 Expression and purification of recombinant proteins from E. coli 6-His-Ste12pDBD, GST-Rst1 p, GST-Rst2p and G S T were expressed in NM522 and trpE-Ste12pADBD was expressed in RR1 as previously described ((Ausubel et al., 1998), strains in Table 8). RR1 E. coli were propagated in M9 minimal media (Ausubel et al., 1998) NM522 cells were infected with T7 RNA poll phage (gift from D. Kilburn) for 6-His-Ste12pDBD expression. Following induction of protein expression, E. coli were washed twice in lysis buffer (1mM dithiothreitol, 0.1% Nonidet P40, 250 mM NaCI, 50 mM NaF, 5 mM EDTA, 50 mM Tris (pH 7.5), 1 mM P M S F , 1 |ag/ml leupeptin, 1 ng/ml pepstatin, 10 jag/ml soybean trypsin inhibitor, 10 ng/ml T P C K and 0.6 mM dimethylaminopurine) and lysed by sonication. Crude lysates were clarified by centrifugation for five minutes at 10 OOOxg. 151 Materials and Methods The GST-recombinant proteins were batch purified with glutathione agarose as described by the manufacturer (Sigma Chemical Company). Similarly, 6-His-Ste12pDBD was batch purified with Ni-agarose (Invitrogen). Lysates with trpE-Ste12pADBD were used without further treatment. C. Perelli-Hentschel used the method described for 6-His-Ste12pDBD to prepare 6-His-Gal4pDBD (Ausubel et al., 1998). 5.6 Expression of recombinant Ste12p in Spodoptera frugiperda (Sf9) cells Ste12p was expressed in Sf9 cells from a recombinant A c M N P V virus as described (Olson et al., 2000). At 72 hours post-infection, the cells were washed twice in SF9 extract buffer (20 mM Tris (pH 8.0), 150 mM NaCI, 1 mM EDTA, 1% Nonidet P40, 0.5% sodium desoxycholate, 1 mM P M S F , 3 mM dithiothreitol, 0.7 mM leupeptin, 2 L I M pepstatin, 2 mM benzamidine, 2 Lig/mL chymostatin, 100 iug/ml TPCK) and lysed in the same buffer by Dounce homogenization. The lysate was clarified by centrifugation at 12 OOOxg for 20 minutes and used without further purification. 5.7 Protein affinity precipitation of recombinant proteins Interaction of recombinant 6-His-Ste12pDBD, trpE-Ste12pADBD and Wt Ste12p from insect cells with GST-Rst1 p and GST-Rst2p was detected by incubation of selected proteins in lysis buffer at 4°C for one hour, followed by addition of glutathione agarose and agitation for ninety minutes. The glutathione 152 Materials and Methods agarose and associated proteins were recovered and washed, and the proteins were eluted in 1 X S D S sample buffer (Sambrook et al., 1989). Proteins were detected by western blot (see SDS-PAGE and western blot for detection of proteins). For these assays, 5 ng each of GST-Rst1 p, GST-Rst2p, G S T or 6-His-Ste12pDBD were used. One hundred micrograms each of the crude trpE-Ste12pADBD and Wt Ste12p (Sf9) extracts were used, and the Wt Ste12p (Sf9) extract was supplemented with 1 mg/mL bovine serum albumen. 5.8 SDS-PAGE and western blot for detection of proteins Proteins were resolved by S D S - P A G E as previously described (Ausubel et al., 1998) and detected by Coomassie blue staining or by western blot. ECL reagents were used as described by the manufacturer (Amersham Pharmacia Biotech) for luminescent detection. 2 % (%w/v) powdered skim milk was added to both primary and secondary antibody incubations. Three a-Ste12p antibodies were used in this work. Two polyclonal a-Ste12p antibodies, which interact with the C-terminal 472 and 215 amino acid residues of Ste12p, have been described previously (Hung et al., 1997). The third a-Ste12p antibody was generated against 6-His-Ste12pDBD in a New Zealand white rabbit by standard techniques (Olson et al., 2000). The antibody is specific to the N-terminal 215 residues of Ste12p. a-Ste12p antibodies were used at a 1:20 000 dilution. 6-His proteins were detected with polyclonal a-His antibodies from Santa Cruz Biotechnology. G S T proteins were detected with polyclonal a -GST 153 Materials and Methods antibodies from Santa Cruz Biotechnology. Primary antiboides from Santa Cruz were used at a 1:2000 dilution. Secondary antibody was horseradish-peroxidase-labeled goat- or donkey- anti-rabbit IgG serum from Gibco-BRL (Life Technologies); it was used at a 1:10 000 dilution. Denatured yeast protein extracts were prepared using the method of (Chang and Herskowitz, 1992). Cultures were grown to OD 6 o o « 0.8 and cells were harvested by centrifugation. Cells were lysed in S D S Sample Buffer (100 mM Tris pH 6.8, 4 % SDS, 10 % glycerol, 20 % p-mercaptoethanol) with acid-washed glass beads by heating the samples at 95 to 100 °C for five minutes. Samples were chilled on ice and, following centrifugation, analyzed by western blot. Approximately 20 jug of extract were used per lane. For the detection of endogenous Ste12p levels following a-factor treatment, polyclonal a-Ste12p antibodies were pre-absorbed onto extracts from ste72 yeast prior to incubation with the STE12 blot. Following Ste12p detection, antibodies were removed from the blot by washing in distilled water, then 0.2 M sodium hydroxide, followed by a final wash in distilled water, for five minutes each! The same blot was subsequently probed with a-Gal4p polyclonal antibodies (Sadowski et al., 1991). 5.9 Metabolic labeling and affinity precipitation of Ste12p Yeast transformed with galactose-inducible STE12 constructs were grown to OD6oo« 0.8 and starved for methionine for 20 minutes. Following starvation, 154 Materials and Methods 1.2 mCi of [35S]-methionine and 2% (% w/v) galactose were added and cultures were labeled for two hours at 30 °C. [3 5S]-methionine-labeled extracts were made as described (Tedford et al., 1997). Yeast were lysed with acid-washed glass beads by vigorous vortexing in lysis buffer (see Expression and purification of recombinant proteins from E. coli) followed by centrifugation at 17 000 x g for 30 minutes at 4 °C. Each extract was pre-cleared by incubation with 20 ng of purified GST protein (above) and 50 jaL of glutathione agarose (Sigma Chemical Company) for one hour at 4°C followed by brief centrifugation at 2000 rpm. Supernatants were incubated for 1 hour on ice with 5 u,g of GST-Rst1 p or GST-Rst2p followed by 1 hour of agitation at 4°C with 25 [iL of glutathione-agarose. Glutathione-agarose beads and associated proteins were recovered and washed three times in lysis buffer. Proteins were eluted for 30 minutes at 37°C in lysis buffer supplemented with 5 mM glutathione. Eluted proteins were resolved by S D S - P A G E (above) and detected by autofluorography. Immunoprecipitations with polyclonal a-Ste12p antibodies were performed as previously described (Hung et al., 1997; Olson et al., 2000). 5.10 Northern and Southern blots Genomic DNA or total RNA was isolated from yeast as described (DNA (Kaiser et al., 1994); RNA (Schmitt et al., 1990)). 10 ugof DNA or 20 ng of RNA was resolved by electrophoresis and transferred to nitrocellulose or nylon as 155 Materials and Methods described (Sambrook et al., 1989). Probes for northern and Southern analysis were random prime labeled using the Oligolabeling Kit (Promega) as described by the manufacturer. 5.11 Electrophoretic Mobility Shift Assays Electrophoretic mobility shift assays were performed as described in (Ausubel et al., 1998). The probe, which contains two P R E s arranged tail to tail consisted of two oligonucleotides (WH65A: 5'-T C G A C A T G T T T C A T T T G A A A C A A A G C - 3 ' and WH66A: 5'-TCGAGCTTTGTTTCAAATGAAACATG-3 ' ) which were annealed and labeled by Klenow end fill-in in the presence of [ 3 2P]-dATP. Approximately 20 pmol of probe was used per lane. Non-specific competitor oligonucleotide consisted of a Gal4p DNA binding site formed by annealing GS3 : 5 ' - T C G A C G G A G T A C T G T C C T C C G -3' and GS4: 5 ' - T C G A C G G A G G A C A G T A C T C C G - 3 ' . 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Subcellular localization of Cdc42p, a Saccharomyces cerevisiae 185 References GTP- binding protein involved in the control of cell polarity, Mol Biol Cell 4, 1307-16. 186 "@en ; edm:hasType "Thesis/Dissertation"@en ; vivo:dateIssued "2001-05"@en ; edm:isShownAt "10.14288/1.0090710"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Biochemistry and Molecular Biology"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Domain function and regulation of Ste12p"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/13674"@en .