Yeast Stress Responses

Topics in Current Genetics Series Editor: Stefan Hohmann

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Springer Berlin Heidelberg NewYork Hong Kong London Milan Paris Tokyo

Stefan Hohmann Willem H. Mager

(Editors)

Yeast Stress Responses With 61 Figures and 12 Tables

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Professor Dr. Stefan Hohmann Department of Cell and Molecular Biology/Microbiology G¨oteborg University Box 462, 405 30 G¨oteborg, Sweden Dr.Willem H. Mager Department of Biochemistry & Molecular Biology Division of Scheikunde, Vrije Universiteit De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

ISSN 1610-2096 ISBN 3-540-43926-9 Springer-Verlag Berlin Heidelberg New York Cataloging-in-Publication Data applied for Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at . This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under German Copyright Law. Springer-Verlag Berlin Heidelberg New York a member of BertelsmannSpringer Science+Business Media GmbH http://www.springer.de © Springer-Verlag Berlin Heidelberg 2003 Printed in Germany The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover-Design: design & production GmbH, Heidelberg Typesetting: Camera-ready by editors Data-conversion: PTP-Berlin, Stefan Sossna e.K. Printing: Saladruck, Berlin Bookbinding: St¨urtz AG, W¨urzburg Printed on acid-free paper SPIN 10849325 39/3150 ih - 5 4 3 2 1 0

Table of Contents

Introduction ............................................................................................................. 1 Stefan Hohmann and Willem H. Mager.............................................................. 1 What is stress? ............................................................................................ 1 Studies of stress responses ......................................................................... 2 Cell proliferation and stress ....................................................................... 3 Aim of the stress response.......................................................................... 4 Phases of the stress response...................................................................... 5 Sensing and signalling................................................................................ 6 Adaptation to stress .................................................................................... 8 Yeast as a model......................................................................................... 8 2 The environmental stress response: a common yeast response to diverse environmental stresses .......................................................................................... 11 Audrey P. Gasch ................................................................................................ 11 Abstract ......................................................................................................... 11 2.1 Introduction ............................................................................................. 11 2.2 The environmental stress response......................................................... 13 2.3 Responsiveness of ESR gene expression ............................................... 15 2.4 Transcript levels versus protein synthesis levels.................................... 18 2.5 Functions represented by genes repressed in the ESR........................... 19 2.5.1 Ribosome synthesis ......................................................................... 20 2.5.2 tRNA synthesis................................................................................ 21 2.5.3 General transcription ....................................................................... 22 2.5.4 RNA splicing and export................................................................. 22 2.5.5 Translation....................................................................................... 22 2.6 Functions represented by genes induced in the ESR ............................. 23 2.6.1 Carbohydrate metabolism ............................................................... 23 2.6.2 Fatty acid metabolism ..................................................................... 26 2.6.3 Respiration....................................................................................... 26 2.6.4 Oxidative stress defense.................................................................. 27 2.6.5 Autophagy and vacuolar functions ................................................. 28 2.6.6 Protein folding and degradation...................................................... 29 2.6.7 Cytoskeletal reorganization............................................................. 30 2.6.8 Signaling.......................................................................................... 31 2.7 Functional themes in the ESR ................................................................ 32 2.7.1 Differential expression of isozymes ............................................... 32 2.7.2 Coinduction of genes with counterproductive functions................ 32 2.7.3 Regulation of control steps of metabolic processes ....................... 34 2.8 The role of the ESR................................................................................. 34 2.9 Regulation of ESR gene expression ....................................................... 37 2.9.1 Rap1p............................................................................................... 37

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2.9.2 Chromatin remodeling .................................................................... 39 2.9.3 Regulated mRNA turnover ............................................................. 41 2.9.4 Msn2p and Msn4p........................................................................... 43 2.9.5 Condition-specific transcriptional induction .................................. 48 2.9.6 Condition-specific cellular signaling.............................................. 50 2.9.7 Advantages of the complex regulation of ESR gene expression ... 53 2.10 Orchestration of cellular responses to stress ........................................ 54 2.11 Conclusions........................................................................................... 56 Acknowledgements....................................................................................... 57 References..................................................................................................... 57 3 The yeast response to heat shock...................................................................... 71 Amy Trott and Kevin A. Morano...................................................................... 71 Abstract ......................................................................................................... 71 3.1 Introduction............................................................................................. 71 3.2 The heat shock and environmental stress responses .............................. 72 3.2.1 Transcriptional regulators of heat shock gene induction ............... 72 3.2.2 Delineation of the Hsf1p and Msn2p/Msn4p heat shock regulons 73 3.2.3 The role of trehalose in thermotolerance........................................ 76 3.2.4 Thermal stress phenotypes in yeast ................................................ 77 3.3 Regulation of the heat shock factor Hsf1p............................................. 78 3.3.1 Regulation of Hsf1p transcriptional activation .............................. 79 3.3.2 The role of phosphorylation in Hsf1p regulation ........................... 81 3.3.3 Genetic and structural insights into DNA binding and regulation. 82 3.3.4 Sensing the proteome: regulation by protein chaperones .............. 84 3.3.5 Hsf1p-like proteins in yeast ............................................................ 86 3.3.6 Hsf1p and the cell cycle .................................................................. 88 3.4 New directions in protein chaperone biology ........................................ 91 3.4.1 Hsp90 chaperone complex subunits in yeast.................................. 91 3.4.2 Endogenous yeast Hsp90 substrates ............................................... 98 Hsf1 .................................................................................................................... 99 3.4.3 Protein chaperones and yeast prion propagation.......................... 101 3.5 Stress and aging .................................................................................... 105 3.6 Conclusions........................................................................................... 108 Acknowledgements..................................................................................... 108 References ................................................................................................... 109 4 The osmotic stress response of Saccharomyces cerevisiae ........................... 121 Markus J. Tamás and Stefan Hohmann .......................................................... 121 Abstract ....................................................................................................... 121 4.1 Introduction........................................................................................... 121 4.2 Structural and morphological effects caused by osmotic stress .......... 123 4.3 Glycerol and glycerol metabolism ....................................................... 124 4.3.1 Glycerol metabolic pathways........................................................ 125 4.3.2 Glycerol transport.......................................................................... 126

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4.3.3 Glycerol accumulation under osmotic stress: multiple levels of control ..................................................................................................... 127 4.4 Transport processes affected by osmotic stress.................................... 130 4.4.1 MIP channels: aquaporins and glycerol channels ........................ 130 4.4.2 Osmolyte uptake systems.............................................................. 132 4.4.3 Ion channels................................................................................... 133 4.5 Perception of and response to osmotic stress: the role of signalling pathways...................................................................................................... 133 4.5.1 S. cerevisiae MAPK pathways...................................................... 134 4.5.2 The HOG MAPK pathway in Saccharomyces cerevisiae............ 135 4.5.3 Control of gene expression ........................................................... 141 4.5.4 The cell integrity pathway............................................................. 150 4.5.5 Skn7p: a putative link between osmosensing pathways............... 156 4.5.6 Additional systems involved in osmotic stress signalling............ 162 4.5.7 Mechanisms of osmosensing ........................................................ 165 4.6 Metabolic adjustments .......................................................................... 167 4.7 Osmotic signalling in other yeasts: the S. pombe Sty1 pathway ......... 168 4.8 Conclusions ........................................................................................... 175 Acknowledgements..................................................................................... 177 References ................................................................................................... 177 5 Ion homeostasis in Saccharomyces cerevisiae under NaCl stress................ 201 Ingrid Wadskog and Lennart Adler................................................................. 201 Abstract ....................................................................................................... 201 5.1 Introduction ........................................................................................... 201 5.2 Yeast Na+ and K+ relations ................................................................... 202 5.2.1 Growth and intracellular ion levels............................................... 202 5.2.2 Why is K+ but not Na+ a preferred intracellular cation?............... 203 5.2.3 Na+ toxicity.................................................................................... 203 5.3 Adaptation to high concentrations of salt: role of ion transporters ..... 204 5.3.1 The plasma membrane H+-ATPase............................................... 205 5.3.2 K+ transport systems...................................................................... 207 5.3.3 The Pmr2Ap/Ena1p sodium transporter ....................................... 208 5.3.4 The Nha1p Na+/H+ antiporter........................................................ 209 5.3.5 Compartmentalization of Na+ ....................................................... 210 5.4 Regulation of ion homeostasis.............................................................. 212 5.4.1 Control at transcriptional level: ENA1.......................................... 212 5.4.2 Control on protein level ................................................................ 220 5.4.3 Regulation of the Trk1/2p system................................................. 221 5.5 Ion transporters and membrane targeting............................................. 221 5.5.1 Targeting of P-type ATPases to the plasma membrane ............... 222 5.5.2 Nhx1p is involved in membrane traffic out of the prevacuolar compartment ........................................................................................... 225 5.6 The genome-wide transcriptional response.......................................... 226 5.7 Conclusions ........................................................................................... 228 References ................................................................................................... 229

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6 Oxidative stress responses in yeast................................................................. 241 Michel B. Toledano1, Agnes Delaunay1, Benoit Biteau1, Daniel Spector1,2, Dulce Azevedo1,3 ............................................................................................. 241 Abstract ....................................................................................................... 241 6.1 Introduction........................................................................................... 241 6.2 Effects of oxygen free radicals on biological molecules ..................... 242 6.2.1 Some concepts of free radical chemistry...................................... 242 6.3 Biological effects of oxygen free radicals in yeast .............................. 245 6.3.1 Methods for measuring the cellular toxicity of ROS ................... 245 6.3.2 Cellular effects of ROS in S. cerevisiae ....................................... 247 6.4 Antioxidant defenses and thiol redox homeostasis.............................. 251 6.4.1 Metal containing antioxidants....................................................... 251 6.4.2 Thiol redox control pathways and peroxidase systems................ 254 6.5 Adaptive oxidative stress responses ..................................................... 262 6.5.1 S. cerevisiae adaptive responses to oxidative stress..................... 262 6.5.2 The genomic response underlying oxidative stress adapted states263 6.6 Control of S. cerevisiae oxidative stress responses ............................. 265 6.6.1 The Yap1 pathway ........................................................................ 266 6.6.2 Skn7 as a stress response coordinator........................................... 273 6.6.3 An H2O2-inducible Msn2/4 pathway ............................................ 274 6.5.4 Other regulators of the oxidative stress response in S. cerevisiae 275 6.7 Control of S. pombe oxidative stress responses ................................... 278 6.7.1 The stress-activated MAP kinase pathway................................... 279 6.7.2 Atf1, a bZip transcription factor substrate of Spc1/Sty1.............. 281 6.7.3 The S. pombe Yap1 homologue Pap1........................................... 282 6.7.4 The response regulator Prr1, a homologue of Skn7 ..................... 283 6.7.5 Two two-component phosphorelay systems contribute to the H2O2 response .................................................................................................. 284 6.8 Regulators of the oxidative stress response in other yeasts ................. 286 6.9 Conclusions........................................................................................... 287 Acknowledgements..................................................................................... 287 References ................................................................................................... 287 7 From feast to famine; adaptation to nutrient availability in yeast............. 305 Joris Winderickx1, Inge Holsbeeks1, Ole Lagatie1, Frank Giots1, Johan Thevelein1 and Han de Winde2,3 ...................................................................... 305 Abstract ....................................................................................................... 305 7.1 Introduction........................................................................................... 306 7.2................................................................................................................ 306 Setting the stage: limitation, starvation, and cell cycle checkpoints ......... 306 7.3 Specific responses to nutrient depletion............................................... 309 7.3.1 Carbon Source Signalling ............................................................. 309 7.3.2 Nitrogen Source Signalling........................................................... 326 7.3.3 Phosphor Limitation and Starvation ............................................. 333 7.3.4 Sulphur Limitation and Starvation................................................ 337 7.4 Common responses to nutrient depletion ............................................. 340

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7.4.1 General Concepts .......................................................................... 340 7.4.2 Nutrient signal integration and the control of metabolism and growth ..................................................................................................... 343 7.4.3 The FGM pathway; an integrator of responses to nutrient availability .............................................................................................. 345 7.4.4 Nutritional control by targets of rapamycin (Tor) proteins.......... 347 7.4.5 Glycogen and Trehalose metabolism............................................ 350 7.4.6 Morphological differentiation as a response to nutrient limitation353 7.5 Conclusions ........................................................................................... 357 References ................................................................................................... 358 Index...................................................................................................................... 387

List of contributors

Adler, Lennart Department of Cell and Molecular Biology, Göteborg University Box 462, S-405 30 Göteborg Sweden Azevedo, Dulce Laboratoire Stress Oxydants et Cancer, LSOC/SBGM/DBJC/DSV Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette France Biteau, Benoit Laboratoire Stress Oxydants et Cancer, LSOC/SBGM/DBJC/DSV Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette France Delaunay, Agnes Laboratoire Stress Oxydants et Cancer, LSOC/SBGM/DBJC/DSV Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette France De Winde, Johannes Division Bakery Ingredients, Beijerinck Laboratory, DSM Life Sciences Alexander Fleminglaan 1, NL-2613 AX Delft The Netherlands Gasch, Audrey Lawrence Berkeley National Lab 1 Cyclotron Road, Mailstop 84-355, Berkeley, CA 94720 USA Giots, Frank Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, B-3001 Heverlee Belgium

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List of contributors

Hohmann, Stefan Department of Cell and Molecular Biology, Göteborg University Box 462, S-405 30 Göteborg Sweden Holsbeeks, Inge Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, B-3001 Heverlee Belgium Lagatie, Ole Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, B-3001 Heverlee Belgium Mager, Willem Department of Biochemistry and Molecular Biology, Vrije Universiteit De Boelelaan 1083, NL-1081 HV Amsterdam The Netherlands Morano, Kevin Microbiology and Molecular Genetics, University of Texas Medical School 6431 Fannin St, JFB 1.765, Houston, TX 77030 USA Spector, Daniel Laboratoire Stress Oxydants et Cancer, LSOC/SBGM/DBJC/DSV Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette France Tamás, Markus Department of Cell and Molecular Biology, Göteborg University Box 462, S-405 30 Göteborg Sweden Thevelein, Johan Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, B-3001 Heverlee Belgium Toledano, Michel Laboratoire Stress Oxydants et Cancer, LSOC/SBGM/DBJC/DSV Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette France

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Trott, Amy Microbiology and Molecular Genetics, University of Texas Medical School 6431 Fannin St, JFB 1.765, Houston, TX 77030 Wadskog, Ingrid Department of Cell and Molecular Biology, Göteborg University Box 462, S-405 30 Göteborg Sweden Winderickx, Joris Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, B-3001 Heverlee Belgium

1 Introduction Stefan Hohmann and Willem H. Mager Department of Cell and Molecular Biology/Microbiology, Göteborg University, Sweden; Department of Biochemistry and Molecular Biology, Free University of Amsterdam, The Netherlands

What is stress? All cell types, even individual cells in multi-cellular organisms, have the ability to respond to changes in environmental conditions. Such responses require a complex network of sensing and signal transduction leading to adaptations of cell growth and proliferation as well as to adjustments of the gene expression programme, metabolic activities, and other features of the cell. Environmental conditions that threaten the survival of a cell, or at least prevent it from performing optimally, are commonly referred to as cell stress. The response and adaptation mechanisms to stress are highly complex. Hence, research on stress responses, specifically in times of global gene and protein expression analyses, can easily turn into a journey through almost all aspects of cell biology. In fact, many fundamental principles of cellular and molecular biology have been discovered while studying how cells respond to stressful conditions. A well-established example illustrating that many “stress proteins” fulfill homeostatic functions are heat-shock proteins that control protein folding as chaperones. Due to the complexity of stress responses and their link to fundamental cellular functions, it is problematical to focus a volume on stress responses. Instead, we have tried to concentrate on a number of specific, well-studied stress conditions that lead to overlapping though distinct responses. Each such type of stress is treated in a specific chapter. In addition, the overlap of the responses, i.e. the responses common to all stress conditions, is the subject of a separate chapter. Finally, we have focused on a specific organism, the budding yeast Saccharomyces cerevisiae, because this organism is particularly well studied and it is an important model system in cellular and molecular biology. Another important model is the fission yeast Schizosaccharomyces pombe, which is mentioned in many of the chapters. Single-celled organisms living freely in nature, such as yeasts, face large variations in their natural environment. Rapidly acting mechanisms are crucial for the survival of these cells to sudden environmental changes and powerful adaptation mechanisms are essential to maintain their capacity to proliferate. Environmental changes may be of a physical or chemical nature: temperature, pressure, radiation, Topics in Current Genetics, Vol. 1 S. Hohmann/P.W.H. Mager (Eds.) Yeast Stress Responses Springer-Verlag Berlin Heidelberg 2003

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concentration of solutes and water, presence of certain ions, toxic chemical agents, pH and nutrient availability. In nature, yeast cells often have to cope with fluctuations in more than one such growth parameter simultaneously. Studies of stress responses Stress responses are often studied by rapidly shifting yeast cells from one condition to another, for instance, from 25°C to 42°C or from a medium without salt to 1M NaCl, and then following certain aspects of cellular behaviour such as gene expression. Dramatic shifts, commonly referred to as “sub-lethal”, may not always be relevant to the conditions which cells experience in nature. We also have to keep in mind that the term sub-lethal commonly refers to the population while large proportions of individual cells may in fact die. However, such “unphysiological”, dramatic shock treatments lead to clear and strong cellular responses that are far easier to study than gradual and slow adaptive changes. We certainly should not forget that sub-lethal shock treatments may, initially at least, cause substantial damage to the cell (and kill a large proportion) and hence probably lead to systemic responses that may not be characteristic for the adaptation to a given condition. For instance, shifting a cell rapidly to a very high NaCl concentration leads to dramatic water loss, thereby, certainly damaging many cellular components; the corresponding repair mechanisms will then be stimulated. Those are similar to the mechanisms evoked by heat shock and hence not characteristic for the way cells cope with NaCl. The specific response to NaCl stimulates transport systems to adjust ion homeostasis and to counteract the toxic effects of sodium ions. The call for caution in interpreting results from sub-lethal treatments may sound trivial, but there are many instances in literature where observations of cellular behaviour are based on analyses of heavily damaged or even dead cells. Along the same argument, it is noteworthy to mention that the severity of a stress affects the profile of a response dramatically. As discussed in some detail in chapters 2 and 4 and below, the more severe a stress the longer it takes the cell to respond, at least at the level of signal transduction and gene expression. For this reason it is highly important to study stress responses over time to capture actual responses and to monitor changes, for instance when comparing mutant and wild type strains. There are numerous examples in the literature where certain mutants were reported unable to mount a certain response. Closer inspection by time courses then revealed that the response had only shifted to a later time point. The reason for the delay in response in more heavily stressed cells is not well understood. It appears that certain rescue processes in the cell have to operate before signalling and gene expression responses are mounted. Obviously a virtue of studying yeast is the possibility to perform genetic analyses. Indeed, especially signalling pathways in stress responses are excellent examples where genetic analyses have been most instrumental not only to identify pathway components but also to order by epistasis analysis those components within a pathway and even provide information on the operation of the pathway. Throughout this volume, it will become clear that genetic analyses are a driving

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force in identifying genes/proteins important for the acquisition of tolerance to stress and for the elucidation of mechanisms controlling stress responses. Additional genes/proteins that potentially play a role in stress responses have now been identified by global gene and protein expression analysis. However, it often turns out to be difficult to demonstrate a role of a protein in a given stress response if the only known link is enhanced expression of the gene encoding that protein. The criteria in genetic analysis are that mutation and/or overexpression of the gene of interest cause a relevant stress phenotype such as diminished or enhanced growth or survival under elevated temperature or osmolarity. In fact, genetic alteration of the majority of genes identified by global expression analysis does not lead to such a scorable stress phenotype. There may be several reasons for that: functional redundancy; minor effects not detected by present methods; stimulation of compensatory pathways; indirect gene expression effects due to stimulation of a pathway not directly involved in the response to the particular stress; a gene product may be involved in adaptation but not required for growth under a certain condition; and so on. These examples illustrate that we probably have to reconsider our criteria and that we also need to perform more detailed experiments to identify phenotypes, such as cocultivation for many generations of wild type and mutant. Another approach is to return to global expression analysis also for phenotypic analyses and search for compensatory mechanisms that might be stimulated in the mutant and hence prevent a visible growth phenotype. Much work is ahead to link global expression data with cellular processes and phenotypes. An important aspect in the analysis of not only stress responses concerns cells and populations. Generally, we perform our studies on a cell culture consisting of billions of cells and for instance, the mRNA levels detected by Northern blot analysis represent the average of all those cells. Perhaps it is trivial to repeat that many of those cells may actually be dead after a sub-lethal stress treatment but apart from that, there are large variations in how individual cells behave. Analysis of stress responses in individual cells, for instance by using flow cytometry, has only recently attracted some attention. Averaging cells obviously has some statistical virtue, even though we cannot calculate the standard deviation. Especially theoretical approaches by modelling will however require some thought (or rather data) of how individual cells behave. Cell proliferation and stress Control of cell proliferation on the one hand and cellular stress responses on the other are very much interrelated and in some respect seem to be two sides of the same coin. For instance, stress treatments cause a transient arrest of the cell cycle, commonly in G1 or, under osmotic stress, also at the G2-M boundary. For instance, stress treatments cause a transient arrest of the cell cycle. Arrest commonly occurs in G1, under osmotic stress also at the G2-M boundary. Such cell cycle arrest may be needed to prevent damage during cell cycle phases in which the cell is specifically vulnerable (S and M) and allow adaptation while cells are in G1 (or

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G2). Cell cycle arrest in G1 is especially well studied for nutrient starvation (chapter 7). Glucose is the favourite carbon source of Saccharomyces and glucose depletion stimulates wide cellular responses. One aspect of the adaptation to glucose depletion is the acquisition of tolerance to a range of different stress conditions. In fact, the same is true for cells depleted or starved for any other nutrient. For this reason, nutrient responses are treated in this volume, although a future volume of Topics in Current Genetics is planned to address nutrient-stimulated cellular responses in yeast and other model systems in more detail. The mechanisms underlying starvation-induced acquisition of stress tolerance are probably linked to cell cycle-controlled stress tolerance and to the activity of protein kinase A. It is well known that G1 cells, i.e. non-proliferating yeast cells, are more stress tolerant than actively dividing cells. Moreover, there are numerous indications in the literature that nutrient availability, control of residence in the G1-phase of the cell cycle, activity of protein kinase A (cAMP-dependent protein kinase), and stress tolerance are related (in fact correlated), although the detailed mechanisms are less well understood. However, a gene expression programme controlled by the transcription factors Msn2p and Msn4p as well as accumulation of the stress protectant trehalose are central to the acquisition of stress tolerance of G1 and nutrient-starved cells. Aim of the stress response The cellular response to stress is obviously aimed at protecting cells from the detrimental effects of stress and at repairing possible damage. Protective responses of living cells have initially been identified in studies of the heat shock response (chapter 3). Cells exposed to elevated temperature increase the synthesis of heat shock proteins many of which serve as molecular chaperones. They control the conformation of other proteins or keep protein complexes in a functionally competent state. The classical heat shock response studies revealed two fundamental features: first, the response leads to acquisition of stress tolerance. Once cells have been challenged with a mild stress, they become more resistant to severe stress. For instance, the fraction of yeast cells surviving a shift to 45°C is much lower among cells that have been grown at 24°C than for cells pre-conditioned at 37°C. Acquisition of stress tolerance holds for almost all stress conditions and is considered to be one of the main purposes of the cellular stress response. Interestingly, in several cases, exposure to one type of stress has been demonstrated to lead to tolerance to other types of stress as well. This phenomenon of cross-protection suggests that different stress conditions require common cellular responses, such as adjustment of energy metabolism and production of protective proteins (such as heat shock proteins) or small protective molecules (compatible solutes such as glycerol or trehalose). The second fundamental aspect of the stress response is that underlying molecular mechanisms also play an important role in normal unstressed cells such as the heat shock proteins mentioned previously. Under heat stress conditions, at which the risk of protein unfolding or complex dissociation

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may occur, the cell will simply need more of those molecular chaperones than under ambient conditions. In addition, for other stress responses, enhancement of basal cellular activities plays an important role. The response of yeast to hyperosmolarity conditions (osmostress) is characterised, amongst others, by adjustment of glycerol metabolism and initiation of accumulation of glycerol. Glycerol is normally produced by cells as a by-product of glycolytic metabolism (and serves here as a redox-valve) and excreted to the growth medium or re-metabolised (chapter 4). Ionic stress displays features in common with hyperosmotic stress, but also evokes physiological systems commonly engaged in ion homeostasis (chapter 5). The response of yeast to oxidative stress is a reflection of the protection from oxidative damage, which is normally active in yeast cells performing oxidative ATP production in the presence of oxygen (respiration; chapter 6). Protection from oxidative damage encompasses, in conjunction with others, the action of reducing compounds produced by the cell for instance glutathione and thioredoxin as well as enzymatic activities such as superoxide dismutase or catalase. The molecular processes mentioned above are clearly part of the specific responses to a certain type of stress. As indicated above, they may, however, also be activated as an indirect effect of other stress conditions. Hyperosmotic stress, for example, has been shown to induce gene expression, which was thought to be specific for the oxidative stress response. Moreover - probably related to the cross protection phenomenon mentioned above – different stress responses share certain aspects. This significant part of the stress response is variously called the general stress response or environmental stress response of yeast (chapter 2). Phases of the stress response As cells respond to sudden stress, they do so in different phases. In the primary phase, immediate cellular changes occur as a direct consequence of stress exposure and damage, defence processes are triggered in the second phase, and finally adapted cells resume proliferation. Dependent on the type of stress, these different phases can be distinguished more or less clearly. To illustrate this situation, we again use the response to hyperosmotic shock as an example (chapter 4). When yeast cells are exposed to an increase in external osmolarity by the addition of high concentrations of salt or sugar to the medium, they immediately loose intracellular water, which leads to shrinkage of cells. This evokes immediate intracellular changes such as recruitment of water from the vacuole and collapse of the cytoskeleton (actin depolarisation). The response phase consists of an immediate, transient growth arrest at G1 or the G2/M transition of the cell cycle, reduction of transmembrane glycerol transport, triggering of the HOG MAP kinase pathway and activation of gene expression. In the adaptive phase, intracellular glycerol accumulates, cellular energy metabolism is adjusted, the cytoskeleton (actin re-polarisation) is repaired, cell wall architecture is altered, and growth resumes as soon as the critical cell size under those conditions is reached.

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The immediate cellular effect of thermo stress is the (partial) denaturation and aggregation of proteins, the disassembly of protein complexes and other cellular structures and increased fluidity of cell membranes. It is not known which proteins are particularly heat stress susceptible, but it can be predicted that denaturation will have pleiotropic effects on many cellular processes. Formation of heatdenatured proteins evokes the response phase, which induces thermo protective mechanisms. Oxidative stress directly causes oxidative damage to proteins and other macromolecules, in particular lipids and DNA. Some proteins may be more susceptible to oxidative damage than others, but also in this case, multiple direct effects on cellular functioning are likely to occur. Increase of reactive oxygen species induces the secondary response phase leading to protection and adaptation. Sensing and signalling The secondary phase of the stress response is characterised by sensing, signalling, and adjustment of gene and protein expression. The underlying mechanisms of stress sensing are major issues of investigations. In the hyperosmotic stress response, at least one plasma membrane-bound protein, Sln1p, has been shown to play an important role (chapter 4). It is part of a two-component-like sensor histidine kinase – response regulator complex and may sense changes in turgor of the cellular envelope of yeast. However, as discussed in more detail in chapter 4, many potential mechanisms of sensing osmotic changes can be envisaged. Sensing of thermo stress may be based on the recruitment of heat shock proteins by unfolded proteins in the cell (chapter 3). Such recruitment may activate the heat shock transcription factor Hsf1p, which itself interacts with heat shock proteins in its inactive state. Thus, in the heat shock response sensing and gene transcription may be directly coupled. On the other hand, heat shock also elicits the cell integrity MAP kinase pathway, which suggests that thermo stress may also be sensed at the level of the cell wall or plasma membrane. The appearance of free radicals, which trigger the oxidative stress response, most likely is sensed by redox-sensitive signalling proteins, in particular transcription factors (chapter 6). One such factor is Yap1p and possibly other members of the Yap family of transcription factors. To explain the onset of the general stress response, one would expect a critical cellular activity to be susceptible to multiple kinds of stress (chapters 2 and 7). It has been proposed that protein kinase A and Msn2p and Msn4p, the stresscontrolled transcription factors downstream of protein kinase A, might be responsible for this common effect, but conclusive evidence for this hypothesis is still missing. Maybe the “general” signal is fired - for instance at the plasma membrane - through a cellular activity that modulates protein kinase A. On the other hand, recent global expression data and analysis of the promoters of multiple-stress responsive genes have shown that in many instances those genes are controlled by different stress-specific response mechanisms converging on the promoter via different transcription factors. One possibility emerging from global expression stud-

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ies is that the genuine general stress response is restricted to adjusting cellular metabolism to stressful conditions and hence the signal may be related to the cellular energy status or the cellular proliferation potential. Clearly more work is required to distinguish between these possibilities. Subsequent to sensing of stress, responsive processes are induced. In the osmotic stress response, the HOG MAP kinase cascade is triggered as a consequence of hyperosmotic stress conditions and the cell integrity MAP kinase pathway is elicited by hypo-osmotic stress. In fact, all six MAP kinase pathways in Saccharomyces mediate events that eventually lead to morphological changes or adjustments and osmoregulation is certainly a central part of morphogenesis. Indeed an important outcome of osmostress-induced MAP kinase signalling is the adjustment of cell wall structure. Adjustment of gene expression is a central feature of the stress response of yeast (at least it has attracted most attention so far). Several stress-induced transcription factors have been identified. Transcription factors Msn2p and Msn4p are the key players in the general stress response (chapter 2). Under ambient conditions, when cells proliferate, Msn2/4p are localised in the cytoplasm. Upon stress exposure they are translocated to the nucleus where they bind to so-called STREs - general stress-responsive elements - in the promoters of a large set of genes. Cellular localisation of Msn2/4p is correlated with the cellular level of protein kinase A. Under conditions of low protein kinase A, Msn2/4p is nuclear whereas at high protein kinase A conditions, Msn2/4p are cytoplasmic. Comparison of the transcript profile of general stress-responsive genes revealed large differences. Apparently, the actual contribution of Msn2/4p-mediated transcriptional activation depends on the promoter context and interaction with other stress-specific transcription factors, as indicated above. As mentioned above, the central transcription factor in the heat shock response is Hsf1p, a trimeric protein binding to HSEs - heat shock elements - in the promoters of a large number of genes (chapter 3). Activation occurs by a conformational change, which releases the activating domain of the protein. The oxidative stress response-mediating transcription factor Yap1p is cytoplasmic and upon stress exposure is translocated to the nucleus (chapter 6). Also in this case, a large set of genes is responsive via so-called ARE’s – AP1responsive elements – in the respective promoters. Skn7p is a transcription factor controlling a set of genes overlapping with the Yap1-regulon. Skn7p is a peculiar protein: it is a response-regulator protein controlled by the Sln1p osmosensing histidine kinase and it contains a DNA-binding domain similar to the heat shock transcription factor with which it has been shown to interact. Skn7p interacts with and supports the function of different transcription factors such as Yap1p, Hsf1p, Swi4p/Swi6p (involved in cell cycle dependent gene expression), and Crz1p (mediates calcineurin-dependent calcium-induced responses). Skn7p may, therefore, be a factor involved in integrating different signals at the level of gene expression and the complete understanding of its role deserves some priority.

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Adaptation to stress The stress-responsive phase is followed by the adaptation phase, at which integration of all stress-induced cellular activities leads to resumption of growth and proliferation. After a hyperosmotic challenge, intracellular glycerol levels are increased, as a combined result of the diminished efflux and the enhanced synthesis (chapter 4). This leads to uptake of water and swelling of cells up to a certain size, which is critical for resumption of growth and division. In general, the growth rate in the presence of high external osmolarity is reduced as compared to that of cells not challenged by osmotic stress. Similarly, adaptation to hypo-osmotic stress is achieved when a new osmotic balance between intracellular medium and the environment and hence an appropriate cellular turgor has been achieved. It has been demonstrated that both hyper- and hypo-osmotic stress conditions lead to adaptations in the structure of the cell wall, which most likely reflect the need to ensure cellular integrity under the new circumstances. After ionic stress - apart from the mechanisms in common with osmotic stress – energy metabolism is adjusted in order to ensure detoxification by sodium export (chapter 5). The same holds for oxidative stress with respect to the intracellular concentration of non-enzymatic and enzymatic defence components (chapter 6). Adjustment to increased growth temperature encompasses enhanced levels of protective functions (proteins, trehalose) and stimulated energy production. Remarkably, a sudden transfer of yeast cells from 24°C to 37°C is experienced as stress, while after adaptation to the elevated temperature the growth rate is higher. Yeast as a model Saccharomyces cerevisiae is generally appreciated as a model eukaryote for fundamental and applied studies. Indeed, from both the genetic and physiological point of view, yeast is a favourite organism for molecular cell biologists. But how well are stress response mechanisms conserved in evolution? Principally, the general strategies with which cells respond and adapt to stress are well conserved but the molecular details may differ somewhat. For instance, the accumulation of compatible protective molecules is common to probably all cells but the actual compounds produced (trehalose and glycerol in yeast) may be different. However, even beyond general strategies, many molecular details are similar. Heat shock chaperones are well conserved, as is the heat-shock transcription factor, although the exact mechanisms controlling that factor may be somewhat different (chapter 3). The mechanisms to combat reactive oxygen species as well as the molecules and proteins involved are well conserved and redox-sensitive transcription factors are widespread. Finally, eukaryotic cells frequently employ MAP kinase pathways in stress responses. It appears, however, that different cells utilise those MAP kinase pathways in different ways. For instance, the S. cerevisiae HOG pathway seems to specifically mediate responses to osmotic stress while the related pathways in fission yeast and mammalian cells are well documented to also mediate protective responses to other stress conditions. Perhaps, many of these differences

1 Introduction

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may actually turn out to be far less important when the details of the function of those pathways are better understood. In any case, the studies of yeast stress responses with budding and fission yeast are complementary and reveal a large deal of information relevant also for mammalian and plant cells. In fact, stress research has a large impact on medical issues. For instance, stress responses are related to ageing, apoptosis, cancer, and immunological responses. In addition, stress plays an important role in applied biotechnology, for example in studies aimed at improvement of the resistance of plant crops against saline conditions, heat, frost, or drought. In these fields, yeast has been widely used as a model in recent years. Industrial applications of yeast also benefit from stress research such as osmotolerance or cryotolerance of baker’s yeast, ethanol tolerance of wine yeast, or protection of foodstuffs from spoilage. Taken together, there is good reason why yeast stress responses are a highly active research field and this volume provides a snapshot of our present knowledge and sets the scene for further rapid advances in the years to come. This book is based on a previously published volume within a different series (Hohmann S, Mager WH: Yeast stress responses. In Molecular Biology Intelligence Unit; Landes Company. 1997). All chapters have been completely rewritten, some even by different authors and with different emphasis to accommodate recent developments. We would like to dedicate this volume to the memory of Helmut Ruis, Vienna, a good friend and colleague, who died prematurely in fall 2001. Helmut has contributed to the previous volume on stress responses but even more so to the study of yeast stress responses in general. In recent years, his work on stress responses had focused on the role of protein kinase A and the transcription factors Msn2p and Msn4p. His spirit and ideas have influenced our own research to a large extent and we are grateful for his many stimulating discussions and friendship.

2 The environmental stress response: a common yeast response to diverse environmental stresses Audrey P. Gasch Department of Genome Science, Lawrence Berkeley National Laboratory, USA

Abstract Unicellular organisms require specific internal conditions for optimal growth and function, however sudden changes in the external environment can perturb the internal milieu, disrupting normal processes. Therefore, cells must maintain their internal system despite fluctuations in the external surroundings. One mechanism that yeast cells use to protect the internal system from the effects of environmental variation is to initiate a common gene expression program that generally protects the cell during stressful times. This program, referred to as the environmental stress response, includes ~900 genes whose expression is stereotypically altered when yeast cells are shifted to stressful environments. The coordinated expression changes of these genes is a common feature of the responses to many different environments, however the regulation of these expression changes is gene-specific and condition-specific, indicating that initiation of the program is precisely controlled in response to each new environment. This review will focus on recent developments in defining and characterizing the genes that participate in the environmental stress response and the regulatory mechanisms that the cell utilizes to orchestrate this program.

2.1 Introduction Microorganisms must have specific and delicately balanced internal conditions for optimal growth and function. The internal milieu of the cell is maintained to promote proper operation of the cell, however fluctuations in the external environment can result in a variety of cellular perturbations that can disrupt the internal environment. These perturbations can prevent optimal enzyme activities, disrupt metabolic fluxes, destabilize cellular structures, perturb chemical gradients, etc., leading to overall instability. Thus, cells must be able to protect and maintain the

Topics in Current Genetics, Vol. 1 S. Hohmann/P.W.H. Mager (Eds.) Yeast Stress Responses Springer-Verlag Berlin Heidelberg 2003

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critical features of the internal homeostasis in the face of variable external conditions. Yeast cells have evolved to be exceptionally proficient at surviving sudden and often harsh changes in their external environment. In the wild, yeast cells must contend with fluctuations in temperature, osmolarity, and acidity of their environment, the presence of radiation and toxic chemicals, and long periods of nutrient starvation. Growth under these various conditions requires maintenance of the internal system, however the cellular program required for its maintenance differs depending on the external challenges that the cell must deal with. Thus, when environmental conditions change abruptly, the cell must rapidly adjust its internal milieu to that required for growth at the new conditions. Details regarding the mechanisms that the yeast S. cerevisiae uses to adapt to new environments have been emerging over the years. Yeast cells gain cross protection against different stresses, evident by the fact that cells exposed to a mild dose of one stress become resistant to large, normally lethal doses of other stresses (for example Mitchel and Morrison 1982; Blomberg et al. 1988; Wieser et al. 1991; Flattery-O'Brien et al. 1993; Lewis et al. 1995). This observation sparked the idea that yeast cells use a general mechanism of cellular protection that is provoked when cells are exposed to stressful stimuli. Concordant with this model was the realization that a set of so-called “heat shock” genes was induced not only by temperature shock but also by other stressful environmental changes, hinting that the genes played a more general role in protecting the cell in response to stressful environments (Kurtz et al. 1986; Werner-Washburne et al. 1989; Kobayashi and McEntee 1990; Susek and Lindquist 1990). Although these observations suggested a general stress response in yeast, the role and regulation of this response remained obscure. Subsequently, it became apparent that the expression of the stress-induced genes was controlled by a common mechanism. A number of studies identified a sequence element common to the promoters of the stress-induced genes, referred to as the Stress Response Element (STRE), strongly suggesting that these genes were coregulated by a common factor (Kobayashi and McEntee 1990; Kobayashi and McEntee 1993; Marchler et al. 1993). The hypothetical STRE binding factor was proven to be either of two related zinc-finger transcription factors, Msn2p and Msn4p (Martinez-Pastor et al. 1996; Schmitt and McEntee 1996). Deletion of these factors renders cells sensitive to a variety of stressful conditions, and it was shown that Msn2p and Msn4p govern the induction of a large number of genes in response to many different stresses (Martinez-Pastor et al. 1996; Schmitt and McEntee 1996; Boy-Marcotte et al. 1998; Moskvina et al. 1998). Thus, these factors became known as the “general stress” transcription factors and were proposed to be generically activated in response to cellular stress to induce a set of genes that defend against environmental insult. However, it was noted that under certain conditions the genes identified as targets of these factors were normally induced regardless of MSN2 and MSN4 deletion, thereby hinting that the regulation of the stress response was more complicated than the initial model suggested (Schuller et al. 1994).

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The recent increase in popularity of whole-genome studies is expanding our definition and understanding of yeast stress responses. Studies characterizing genomic transcript abundance and global protein synthesis levels allow the exploration of these aspects of the cellular responses of yeast cells to environmental changes. Using DNA arrays, the relative transcript levels of all genes in an organism’s genome can be rapidly quantified, and computational analysis of the resulting genomic expression data can implicate gene function and regulation while providing insights into the overall physiological response of the cell (Fodor et al. 1993; Pease et al. 1994; Shalon et al. 1996; Eisen et al. 1998; Brown and Botstein 1999). Large-scale changes in protein synthesis can be measured by twodimensional electrophoresis of pulse-labeled proteins, complementing gene expression studies and adding additional levels of detail about the protein repertoire in the cell (Blomberg 1995; Norbeck and Blomberg 1996; Godon et al. 1998; Lee et al. 1999a; Appella et al. 2000). These types of global studies have provided insights into the mechanisms that yeast use to defend themselves against environmental insult. Many of the observed cellular responses are specifically triggered to counteract features that are unique to each environment. The reader is directed to other chapters in this book that review the specialized yeast responses to a number of environmental stresses that are prevalent in nature. In addition to these specialized responses, global studies have identified the players in a common response to environmental stresses while providing insights into the complicated regulation of this cellular program. This review will focus on recent advances in defining and studying the common yeast response to stressful environments while summarizing existing literature on the genes and proteins that participate in and regulate this program.

2.2 The environmental stress response Characterization of the genomic expression programs in yeast responding to different environmental conditions revealed that a substantial fraction of each of the responses is not specific to the stimulus but instead represents a common response to all of the conditions tested. In a study conducted with colleagues, we used DNA microarrays to identify approximately 900 genes whose expression was stereotypically altered in S. cerevisiae responding to a variety of stressful environmental transitions (Gasch et al. 2000). (The complete list of the genes that participate in this response can be found at http://www-genome.stanford.edu/yeast_stress). These genes fell into two groups based on their expression patterns (Fig. 2.1): one group consisted of genes whose transcript levels increased in abundance in response to the environmental changes, and the other group was comprised of genes whose transcript levels decreased following environmental stress. (For the purposes of this review, genes whose transcript levels increase in response to environmental change will be referred to as induced, while genes whose transcript levels decrease will be referred to as repressed. It is important to note that the observed changes in transcript levels can be mediated by alterations in transcript

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Fig. 2.1. Gene expression patterns in the ESR. The average gene expression changes of the genes whose expression is induced ( ) and repressed ( ) in the ESR in response to 25oC to 37oC heat shock, 0.3 mM hydrogen peroxide, 1.5 mM diamide, 1M sorbitol, 0.02% methylmethane sulfonate, and 170 Gray of ionizing radiation. The same scale is used for all of the plots shown. Data were taken from Gasch et al. 2000 and Gasch et al. 2001. The complete list of the genes that participate in this response can be found at http://www-genome.stanford.edu/ yeast_stress

synthesis as well as transcript degradation; therefore, the terminology used here to indicate the increases or decreases in gene expression is not intended to imply any mechanism in the alteration of transcript abundance.) The two groups of genes that participate in the common gene expression response displayed nearly identical but opposite patterns of expression in response to the environmental shifts. This strongly suggests that the expression changes were coordinately regulated. A similar common gene expression response was also identified in a study by Causton et al. (2001). Since then, these gene expression changes have been observed in the cellular response to many environmental conditions, corroborating the commonality of the program (Table 2.1). Remarkably, the genes that participate in this response amount to ~14% of the currently predicted genes in the yeast genome (Ball et al. 2000; Blandin et al. 2000). Exploration of the genes involved in this response revealed that many of the induced genes are targets of Msn2p and/or Msn4p (Msn2/4p) and had already been implicated in a general stress response in yeast (Martinez-Pastor et al. 1996; Schmitt and McEntee 1996). However, characterization of the common gene expression program distinguished it from the previously described Msn2/4pdependent response in a number of ways. First, the common gene expression program encompasses nearly 900 genes and includes not only induced genes but also hundreds of genes whose expression is repressed in response to environmental changes. Second, as discussed below in a subsequent section, although many of the induced genes are regulated by Msn2/4p under certain conditions, the coordinate expression changes of these genes extends beyond Msn2/4p control. Furthermore, detailed characterization of the regulation of this response revealed that

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it is not controlled by a general regulatory mechanism, but rather is mediated by condition-specific signaling pathways. Despite similarities to the previously defined “general” stress response, the coordinate increases and decreases in the expression of the genes in this program were referred to as the environmental stress response (ESR) (Gasch et al. 2000). For consistency, the terminology will be maintained in this review.

2.3 Responsiveness of ESR gene expression Each genomic expression program triggered by environmental change is unique to the specific features of the new conditions in terms of the genes affected and the magnitude and choreography of their expression, indicating that the cell precisely responds to the distinctive challenges of each new environment (see other chapters in this book). Nonetheless, the bulk of each genomic expression program is accounted for by the genes in the ESR. The ESR is initiated in response to a wide variety of environmental transitions, as indicated by the stereotyped alterations in expression of the genes in this response (Table 2.1). Although this program is commonly initiated in response to these diverse conditions, the precise levels and timing of the gene expression changes appear to be specific to the features of each new environment (Fig. 2.2), hinting at the sensitivity with which the program is regulated. Like the overall genomic expression responses, initiation of the ESR is often transient: immediately after the shift to a new environment, the cell responds with large changes in the expression of genes in the ESR, however over time the differences in expression usually subside, and transcript levels return to near pre-stress levels (Fig. 2.1) (Gasch et al. 2000; Causton et al. 2001). This observation is in line with previous observations of transient gene expression changes in response to stress (for example Parrou et al. 1997; Parrou et al. 1999; Rep et al. 1999). The transient changes in gene expression may help the cell to rapidly adjust the concentrations of the corresponding gene products to the levels required for growth at the new conditions (discussed further below, see Fig. 2.9). According to this model, the transient pattern of gene expression represents an adaptation phase during which the cell initiates the optimization of its internal milieu before resuming growth. An important exception to this observation is the case of nutrient starvation, in which the cells do not resume growth but enter a quiescent state until nutrients become available (see Chapter 7); consistently, the gene expression response to nutrient starvation involves large gene expression changes that are not transient but instead persist until starvation is alleviated (Gasch et al. 2000). The magnitude of the expression changes of genes in the ESR is graded to the severity of the environmental shock. Populations of cells experiencing larger doses of stress respond more strongly than cells experiencing subtle environmental changes (Gasch et al. 2000). For example, cells exposed to a 25oC to 37oC heat shock will show larger and more prolonged changes in gene expression before ad-

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Table 2.1. Environmental transitions that lead to ESR initiationa Environment Temperature Shocks Heat shock

Ethanol Shock pH Extremes Acid Alkali Oxidative and Reductive Stress Hydrogen Peroxide Menadione Diamide Cadmium DTT Hyper-Osmotic Shock Sorbitol Potassium Chloride Sodium Chloride

Starvation Stationary Phase Amino Acid Starvation Nitrogen Starvation Phosphate Starvation Zinc Starvation Respiration Petite mutants Diauxic Shift Transition Nonfermentable Carbon Sources Diverse Drug Treatments Long-term Exposure to alpha Factor DNA-Damaging Agents Alkylating Agents

Ionizing Radiation Double-strand Breaks a

References

(Boy-Marcotte et al. 1999; Gasch et al. 2000; Causton et al. 2001) (Alexandre et al. 2001) (Causton et al. 2001) (Causton et al. 2001) (Godon et al. 1998; Gasch et al. 2000; Causton et al. 2001) (Gasch et al. 2000) (Gasch et al. 2000) (Momose and Iwahashi 2001) (Gasch et al. 2000; Travers et al. 2000) (Gasch et al. 2000; Rep et al. 2000; Causton et al. 2001) (S.M. O’Rourke and I. Herskowitz, personal communication) (Posas et al. 2000; Rep et al. 2000; Causton et al. 2001; Yale and Bohnert 2001; S.M. O’Rourke and I. Herskowitz, personal communication) (Fuge et al. 1994; Gasch et al. 2000) (Gasch et al. 2000; Natarajan et al. 2001) (Gasch et al. 2000) (Ogawa et al. 2000) (Lyons et al. 2000) (Traven et al. 2001) (Fuge et al. 1994; DeRisi et al. 1997) (Kuhn et al. 2001) (Hughes et al. 2000b) (Spellman et al. 1998) (Jelinsky and Samson 1999; Jelinsky et al. 2000; Gasch et al. 2001; Natarajan et al. 2001) (Gasch et al. 2001) (Lee et al. 2001b)

This table lists conditions that trigger initiation of the ESR, as monitored in global studies of genomic expression and translation initiation.

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Fig. 2.2. Initiation of the ESR is often transient. The average gene expression changes of the genes whose expression is induced ( ) and repressed ( ) during ESR initiation in response to a 25oC to 37oC heat shock and a 29oC to 33oC heat shock. Data taken from Gasch et al. 2000

apting to their new steady-state expression program, relative to cells exposed to a mild temperature shift of 29oC to 33oC (Fig. 2.2). Furthermore, conditions that result in high levels of cell death usually provoke a substantial initiation of the ESR, with some of the ESR transcript levels changing more than 100-fold (A.P. Gasch and P.O. Brown, unpublished data). Thus, the ESR is initiated in response to a wi-

Fig. 2.3. Reciprocal expression of the ESR genes in response to reciprocal environmental changes. The average gene expression changes of genes whose expression is nor) and repressed ( ) during ESR initiation are shown as cells remally induced ( sponded to a 25oC to 37oC shock (A, left panel), 37oC to 25oC shock (A, right panel), a shift from YPD medium to YPD supplemented with 1M sorbitol (B, left panel), and YPD supplemented with 1M sorbitol to standard YPD medium (B, right panel)

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de range of environmental transitions, from subtle changes in conditions to lethal environmental shocks, in a manner that is graded to the severity of the environment-al stress. The ESR is not initiated in response to any environmental shift but appears to represent a response to suboptimal environments. This is evident from the genomic expression program of cells shifted back and forth between two environments. For example, when cells adapted to growth at 25oC were transferred to 37oC, they responded with large and transient changes in the expression of the ESR genes (Fig. 2.3A) (Gasch et al. 2000). In contrast, when cells adapted to 37oC were shifted to 25oC, they showed reciprocal changes in the expression of these genes: genes whose expression is normally induced during ESR initiation showed decreased expression in response to the reverse temperature shift, and genes whose expression is normally repressed in response to stressful environments became induced under these conditions. This observation indicates that initiation of the ESR is relieved when cells that are adapted to 37oC are shifted to 25oC. Furthermore, the cells immediately (within 5 minutes) adjusted their transcript levels to the final steady-state required for growth at 25oC, with no observable transient features. Thus, while a shift from 25oC to 37oC triggered initiation of the ESR, the reciprocal shift rapidly relieved the ESR gene expression differences within a very short period (Gasch et al. 2000).

2.4 Transcript levels versus protein synthesis levels The cell goes to great lengths to alter the expression of its genome, presumably to alter the abundance of the corresponding gene products. Indeed, many of the changes in ESR transcript levels correlate with changes in protein synthesis. Proteomic studies have identified proteins whose translation increases or decreases following starvation, osmotic shock, oxidative stress, and heat shock (Fuge et al. 1994; Norbeck and Blomberg 1997; Godon et al. 1998; Boy-Marcotte et al. 1999; Norbeck and Blomberg 2000). Although in each study, only a subset of these changes (