COMPARATIVE ANALYSIS OF THE DISCORDANCE BETWEEN THE GLOBAL TRANSCRIPTIONAL AND PROTEOMIC RESPONSE OF THE YEAST SACCHAROMYCES CEREVISIAE TO DELETION OF THE F-BOX PROTEIN, GRR1 Joshua William Heyen Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology, Indiana University May 2010 ii Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. _______________________________________ Mark G. Goebl, Ph.D., Chair _______________________________________ Peter J. Roach, Ph.D. Doctoral Committee _______________________________________ David E. Clemmer, Ph.D. January 15 th , 2010 _______________________________________ Mu Wang, Ph.D. _______________________________________ Jake Yu Chen, Ph.D. iii DEDICATED TO MY WIFE, CANDY, MY SON, NATHANIEL, AND MY DAUGHTER, ADDISON FOR THEIR UNCONDITIONAL LOVE AND SUPPORT iv ACKNOWLEDGMENTS The proceeding volume is the culmination of several years where not only my blood, sweat, and tears were sacrificed in the pursuit of scientific discovery but also the very core of my being. What is the core of one’s being? I define it as an immense network of life instances through which a person’s psyche develops an awareness of who they are and what they stand for. Of course, being the scientist that I am, I believe that one is pre-disposed by genetics at the beginning of life to interpret life circumstances with a certain shade of color or temperament. However, the initial hue of one’s perspective is only the base coat for a lifetime that is susceptible to artistic license from many different painters. In this way a person’s psyche is like a canvas, a sentient, emotionally predisposed canvas that can choose to accept or deny strokes (life instances) of color from any person or situation they may encounter. Thus, I wholeheartedly believe that the people I have met are painters from which many strokes of perception I have received and have attempted to add to my “core”. This volume is the manifestation of a tremendous amount of effort that at times seemed beyond my capacity; the completion of which can only be attributable to not just me but the myriad of people who have contributed to my “core”. I would like to acknowledge each of my immediate and extended family members that have each had to sacrifice in some way for me to pursue this endeavor. “Thank you” seems inappropriate in this instance since the sacrifices made warrant much more than words commonly uttered in passive conversation. At the risk of sounding soft and hokey, which for those that know me is a tremendous risk to take on my part; the only word that seems applicable here is love. So to each of whom I mention here I give my love. To my wife, Candy, who despite her own frustrations, put up with me these past eight years and never waned in her belief that I am exceptional. To my kids, Nathaniel and Addison, whose smiles infect me every day with the energy to do my best in all aspects of life. To my mother and father, who have molded me into the person I am today and have taught me too many things to mention. To my sister and my brother, whom I admire more than they can possibly imagine. To my v grandparents, who always have believed in me and encouraged me to aim high. To my extended family, who all have contributed greatly to my maturation and development as a person. Finally, to my in-laws and friends, who have supported my wife and me through this long journey. To all of you I give my deepest gratitude and love. I would also like to express my extraordinary gratitude to my mentor, Mark Goebl, who showed me what it is to be a real scientist. Also, to my committee I extend my deepest gratitude for our thoughtful discussions and their wise guidance along this journey. vi ABSTRACT Joshua William Heyen COMPARATIVE ANALYSIS OF THE DISCORDANCE BETWEEN THE GLOBAL TRANSCRIPTIONAL AND PROTEOMIC RESPONSE OF THE YEAST SACCHAROMYCES CEREVISIAE TO DELETION OF THE F-BOX PROTEIN, GRR1 The Grr1 (Glucose Repression Resistant) protein in Saccharomyces cerevisiae is an F-box protein for the E3 ubiquitin ligase protein complex known as the SCF Grr1 (Skp, Cullin, F-box). F-box proteins serve as substrate receptors for this complex and in this capacity Grr1 serves to promote the ubiquitylation and subsequent proteasomal degradation of a number of intracellular protein substrates. Substrates of SCF Grr1 include the G1-S phase cyclins, Cln1 and Cln2, the Cdc42 effectors and cell polarity proteins, Gic1 and Gic2, the FCH-bar domain protein, Hof1, required for cytokinesis, the meiosis activating serine/threonine protein kinase, Ime2, the transcriptional regulators of glucose transporters, Mth1 and Std1, and the mitochondrial retrograde response inhibitor Mks1. Stabilization of these substrates lead to pleiotrophic phenotypic defects in grr1Δ strains including resistance to glucose repression, accumulation of grr1Δ cells in G2 and M phase of the cell cycle, sensitivity to osmotic stress, and resistance to divalent cations. However, many of these phenotypes are not reflected at the gene expression level. We conducted a quantitative genomic vii and proteomic comparison of 914 loci in a grr1Δ and wild-type strain grown to early log-phase in glucose media. These loci encompassed 16.7% of the Saccharomyces proteome of which 22.3% exhibited discordance between gene and protein expression. GO process enrichment analysis revealed that discordant loci were enriched in the processes of “trafficking”, “mitosis”, and “carbon/energy” metabolism. Here we show that these instances of discordance are biologically relevant and in fact reflect phenotypes of grr1Δ strains not evident at the transcriptional level. Additionally, through combined biochemical and network analysis of discordant loci among “carbon and energy metabolism” we were able to not only construct a model for central carbon metabolism in grr1Δ strains but also were able to elucidate a novel molecular event that may serve to regulate glucose repression of genes needed for respiration in response to changes in glucose concentration. Mark G. Goebl, Ph.D., Chair viii SUMMARY OF PROPOSED RESEARCH The goal of my thesis project was to develop and apply a global proteomics strategy to discover novel mechanisms by which the Saccharomyces cerevisiae F-box protein, Grr1, acts to regulate multiple cellular processes in Saccharomyces. The Grr1 protein is a member of a class of proteins known as F-box proteins. F-box proteins are found in all eukaryotic organisms and serve to regulate multiple cellular processes such as development, endocytosis, transcription, translation, and targeted protein degradation. Many of these essential functions for the F-box proteins are carried out through a conserved mechanism by which the F-box protein serves as a receptor to target various protein substrates for ubiquitin modification. Most F-box proteins discovered to date facilitate protein ubiquitylation in conjunction with a well conserved complex of proteins collectively known as the SCF (Skp, Cullin, F-box). The archetype of the SCF complex is the S. cerevisiae SCF composed of the proteins Skp1, Cdc53, Rbx1, Cdc34, and a variable F-box protein. Multiple F-box proteins can associate with this core group of four SCF components adding modularity to the complex and the ability to recognize multiple cellular substrates. The attachment of ubiquitin to SCF substrates has been extensively shown to result in the substrate’s degradation. It is through this targeted degradation that the SCF can control numerous cellular processes including transcription (by targeting transcription factors for degradation), translation, and cell signaling. As one can imagine the function of this complex is critical to the cell and alterations in its function could lead to disease and indeed diseases such as Parkinson’s, Huntington’s, and Alzheimer’s have all been linked to defects in the ubiquitylation machinery. The importance of the SCF complex in maintaining cellular homeostasis underscores the need to characterize each of its components as they relate to the cell as a whole. Recently, through the development of global assays and screens the molecular toolbox available to biologists has expanded allowing researchers to begin to probe the cell and measure its molecular response on a global system wide level. Micro-arrays allow for the measurement of all actively ix transcribed genes in a cell providing a snapshot of the cell at the transcriptional level. This valuable tool allows scientists to probe the transcriptional framework that dictates genes expression; however the molecular state of the cell at the protein level can only be inferred. Thus, a method to assay global protein expression is needed to complement the gene expression data. Consistencies and paradoxes between these two data sets will aid in our understanding of the cell on a system wide level. Global proteomic strategies based on liquid chromatography followed by mass spectrometry have really just begun to be used as a method to analyze complex protein mixtures. Development in this field has been rapid, still major hurdles are yet to be overcome. First, researchers are still unable to detect and quantify the entire proteome of an organism reliably. This is due to limitations with the current resolving power of liquid chromatography and the sensitivity of widely available mass spectrometers. Second, scoring algorithms for accurately matching experimental MS/MS spectra to the correct peptide are inefficient, leaving many spectra unidentified, and sometimes inaccurate, containing many false positives. Third, quantification of a peptide and/or protein is limited by the fact that post-translational modification of a peptide can skew the relative ratios obtained for the peptide resulting in inaccurate quantification. Finally, software to efficiently and effectively mine the results of the data generated to arrive at interesting biological discoveries are in short supply and those that are available, though useful, fall short of the mark. Thus, a significant part of my thesis will detail the development of a global proteomics strategy that generates valid and accurate LC-MS based results and allows for the efficient and effective analysis of this data to uncover novel scientific discoveries. This method will be applied to discovering novel roles for the F-box Grr1 in S. cerevisiae cell biology. For my thesis I hope to contribute to the development of LC-MS based global proteomic strategies and apply these developments to a significant biological question (the system wide role of the F- box protein Grr1) using the biology to validate my strategy and the strategy to uncover novel biological roles for SCF based functions. x TABLE OF CONTENTS LIST OF TABLES xvi LIST OF FIGURES xvii LIST OF ABBREVIATIONS xix CHAPTER 1: INTRODUCTION TO UBIQUITYLATION AND GRR1 1 1.1. The Process of Ubiquitylation and its Multifarious Role in Eukaryotes 1 1.2. Ubiquitin and the Molecular Mechanism of Ubiquitylation 2 1.3. The SCF (Skp, Cullin, F-Box) Complex 4 1.4. F-Box Proteins 6 1.5. Grr1 7 1.6. The Role of Grr1 in the G1 to S Phase Transition through Targeted Degradation of the G1 Cyclins, Cln1 and Cln2 10 1.7. The Role of Grr1 in Bud Emergence and Polarity through Targeted Degradation of Cln1,2 and Gic1,2 15 1.8. The Role of Grr1 in Cytokinesis through Targeted Degradation of Hof1 17 1.9. The Role of Grr1 in Amino Acid Signaling Through the SPS Sensor 18 1.10. The Role of Grr1 in Mitochondrial Retrograde Signaling through Targeted Degradation of Mks1 22 CHAPTER 2: GLUCOSE TRANSPORT, SIGNALING, AND METABOLISM IN SACCHAROMYCES 27 2.1. Introduction to Glucose Signaling and Metabolism 27 2.2. Grr1 and Glucose Repression 29 2.3. Glucose Transport in S. cerevisiae 33 2.4. Transcriptional Expression of Glucose Transporter Genes in Response to Fluctuating Glucose Concentrations 35 2.5. Glucose Signaling and Control of Hexose Transporters by the Rgt2 and Snf3 Pathway 37 [...]... Characterized Roles for Grr1 that are not Reflected at the Transcriptional Level 155 4.3.4 Manual Curation and Comparative Analysis of the Transcriptional and Proteomic Response to GRR1 Deletion 161 4.3.5 Characterization of Discordance between Protein and Gene Expression Levels in grr1 Cells 166 CHAPTER 5: PROTEIN AND GENE EXPRESSION DISCORDANCE IN grr1 CELLS AND ITS IMPLICATIONS FOR GRR1 s IN GLUCOSE... rate of respiratory growth 68, defects in high affinity glucose transport 69,70, defects in divalent cation 7 8 Figure 1.2 The SCFGrr1 Complex, Substrates, and Regulated Processes The SCFGrr1 E3 ubiquitin ligase consists of the cullin, Cdc53, Skp1, the Ring Finger, Rbx1, the E2, Cdc34, and the F-box protein, Grr1 Together these proteins recognize and catalyze the covalent Grr1 and has been shown to mediate... of impaired cytokinesis due to Hof1 stabilization and hyper-polarization due to Gic2 stabilization The substrates identified for the SCFGrr1 complex involved in cell cycle progression implicate Grr1 s role in the cell cycle as diversified and complicated Despite the complicated nature of the role of Grr1 in the cell cycle it has been hypothesized that the sequential and timely degradation of each of. .. stabilization of substrates normally targeted for ubiquitylation by the SCF 1.8 The Role of Grr1 in Cytokinesis through Targeted Degradation of Hof1 Before the mother cell can re-enter G1 of the cell cycle, separation of the mother from the daughter cell must occur through cytokinesis Most eukaryotic cells facilitate cytokinesis through the formation of an actomyosin-based contractile ring, however S cerevisiae. .. role for Grr1 in the processes of budding and cytokinesis Upon the transition from G1 to S phase of the cell cycle yeast cells begin to polarize the actin cytoskeleton to produce the daughter cell This process is highly regulated and mainly facilitated by the small GTPase known as Cdc42 100 Through a signaling cascade that remains enigmatic the Cln1/2/3-Cdc28 complex stimulates the formation of the active... through the SPS sensor 63, and mitochondrial retrograde response 57 Each of these functions for Grr1 will be discussed in later sections 14 1.7 The Role of Grr1 in Bud Emergence and Polarity through Targeted degradation of Cln1,2 and Gic1,2 For some time the role of Grr1 in cell cycle progression was believed to be limited to its control of G1 to S phase progression; however, it has become clear that there... to the mother bud neck Once the spindle pole reached the mother bud neck, mitosis or M phase begins In this phase the spindle elongates, segregating chromosomes to mother and bud The end of M phase and the beginning of G1 of the next cell cycle is marked by cytokinesis and cell separation C Progression through the cell cycle is regulated by modulating the kinase activity of the cyclin dependent kinase,... mutations at the GRR1 locus are viable (null mutations of CDC4 as well as MET30 are lethal) making grr1 cells particularly amenable to biochemical and genetic analysis Substrates for the SCFGrr1 complex include the cyclins, Cln1 and Cln2 52, the meiosis activating kinase Ime2 53, the Hof1 protein required for cytokinesis 54, the Cdc42 effectors Gic2 and Gic1 55, the glucose responsive transcriptional. .. cells grow and divide at rates specific to the available nutrient profile Thus, yeast not only coordinate growth and division strictly to the availability of nutrients but also to the quality of these nutrients For example, the average doubling time of yeast grown in the presence of glucose (high quality carbon source) is ~75 minutes with cell volumes reaching ~38um3 before the transition from G1 to S However,... level, the chronological initiation of each of these events is tightly controlled through a control system that monitors and integrates environmental and intracellular cues to insure cell cycle fidelity The initial phase of the cell cycle is considered to be “Gap 1” or G1 and is the phase in which Saccharomyces spends most of its time During G1 yeast cells undergo cell growth in response to the availability . Mixtures 10 5 3 .11 .3. Stage 3: Electrospray Ionization and Mass Spectrometry 11 4 3 .11 .4. Stage 4: Data Analysis and Validation 12 0 3 .11 .4 .1. Peptide Identification Utilizing SEQUEST™ 12 0 3 .11 .4.2 Sample Preparation 83 3 .11 .1. 1. Experimental Question and Approach 83 3 .11 .1. 2. Factors Influencing Strains and Media Conditions 85 3 .11 .1. 3. Protein extraction 95 3 .11 .1. 4. Determination of. Pipeline 12 4 3 .11 .4.3. Peptide Prophet 12 6 3 .11 .4.4. Protein Prophet 12 7 3 .11 .4.5. Determination of Peptide and Protein Relative Abundance Using ASAPratio 12 8 3 .11 .4.6. Generation of a Final