The Glucose Signal Transduction Pathway Mediated by the Rgt2 and Snf3 Glucose Sensors in the Budding Yeast Saccharomyces cerevisiae by Adhiraj Roy B.Sc. in Microbiology, May 2005, University of Calcutta, India M.Sc. in Microbiology, May 2007, University of Calcutta, India A Dissertation submitted to The Faculty of The Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 18, 2014 Dissertation directed by Jeong-Ho Kim Assistant Professor of Biochemistry and Molecular Medicine The Columbian College of Arts and Sciences of The George Washington University certifies that Adhiraj Roy has passed the Final Examination for the degree of Doctor of Philosophy as of March 18, 2014. This is the final and approved form of the dissertation. The Glucose Signal Transduction Pathway Mediated by the Rgt2 and Snf3 Glucose Sensors in the Budding Yeast Saccharomyces cerevisiae Adhiraj Roy Dissertation Research Committee: Jeong-Ho Kim, Assistant Professor of Biochemistry and Molecular Medicine, Dissertation Director William Weglicki, Professor of Biochemistry and Molecular Medicine, Committee Member Wenge Zhu, Assistant Professor of Biochemistry and Molecular Medicine, Committee Member ii © Copyright 2014 by Adhiraj Roy All rights reserved iii Dedication I dedicate my dissertation to my family, my grandparents, (Late) Sourindra K. Roy and Mrs. Jyotsna Roy, my parents, Mr. Soven Roy and Mrs. Mamata Roy and my beloved sister Ms. Sanjana Roy. iv Acknowledgements This dissertation would not have been possible without the help of so many people in so many ways. It is also a product of a large measure of serendipity, fortuitous encounter with people who changed the course of my academic career. First and foremost, I express my deepest gratitude to my mentor, Dr. Jeong-Ho Kim for providing me the opportunity of being his graduate student and working on this wonderful project. As my “Academic Father”, Dr. Kim inculcated in me the flavor of yeast genetics and molecular biology. His constant guidance, support and encouragement helped me to think “out of the box”, analyze biological phenomenon critically and finally resulted in the growth of my scientific intellect. I would also like to thank my committee members, Dr. Rakesh Kumar, Dr. William Weglicki, Dr. Wenge Zhu and Dr. Paul Brindley for their insightful comments and constant encouragement. A special thanks goes to Dr. Linda Werling, Program Director, IBS for her endless support throughout my graduate career. I will be always indebted to my former colleague Dr. Yong Jae Shin who has been like a mentor to me and taught me many aspects of molecular biology and I am grateful for his constant support. Dr. Shin, Gamsahapnida! Furthermore, I thank my colleague Dr. Yong Bae Kim and Dr. Sujit Nair, Dr. Kazufumi Oshiro from Dr. Rakesh Kumar’s lab for their help and our departmental administrative staffs including Debby, Nichole and Laura. Thank you all. The 5 year long journey for the quest of knowledge would not have been possible without unconditional love and support of my friends. Ananda Banerjee, you have been my best friend ever and I thank you for being with me in the ups and downs of this long v journey. Somenath Chakraborty, how can I forget your support and encouragement which were always needed in my tough times? Kankana, Shubham, your support has always been with me, I thank you all for everything. I must take a moment to express my heartily gratitude to Dr. Ananta K. Das, former Chair, Microbiology department of my undergraduate college at The University of Calcutta. Sir, you have taught me everything, from preparing bacterial media to immunohistochemistry. It was you who has been my inspiration all the time and I am indebted to you forever. I am so lucky to have you as my mentor during the foundation period of my academic career. Most importantly, I would like to thank my family for their unconditional love and moral support. I am forever grateful to my parents for their commitment and dedication they showered upon me in every hardship of my journey. Ma, Bapi, you are the best! My little sister missed me for last few years but nevertheless, always encouraged me. I am blessed to have a sibling like Sanjana. I love you. I thank my grandmother, uncles, aunts and all my cousins for their encouragement. Without their support, I could not have gone this far. vi Abstract of Dissertation The Glucose Signal Transduction Pathway Mediated by the Rgt2 and Snf3 Glucose Sensors in the Budding YeastSaccharomyces cerevisiae Sensing and signaling the presence of extracellular glucose is crucial for yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis, mediated by expression of glucose transporter genes (HXTs). Yeast cells mediate aerobic glycolysis in part, by the crosstalk between two glucose signaling pathways: 1) the Rgt2/Snf3 glucose induction pathway and 2) the Snf1/Mig1 glucose repression pathway. The yeast Rgt1 repressor inhibits transcription of HXT genes in the absence of glucose by recruiting general transcription repressor Ssn6/Tup1 and the HXT corepressor Mth1. In response to glucose, Rgt1 is phosphorylated by the cAMP-activated protein kinase A (PKA) and dissociates from the HXT promoters and no longer interacts with Ssn6/Tup1, resulting in the HXT gene expression. Glucose regulates Rgt1 function by primarily modulating the Rgt1- Ssn6/Tup1 interaction and Rgt1 removal from DNA occurs but not necessary for expression of HXT genes. Ssn6/Tup1 interferes with Rgt1 DNA-binding activity in the absence of Mth1, and that Rgt1 function abrogated by Ssn6 overexpression is restored by cooverexpression of Mth1. Mth1 acts like a scaffold-like protein to facilitate interaction between Rgt1 and Ssn6/Tup1 by blocking PKA-dependent Rgt1 phosphorylation. The cell surface glucose receptors Rgt2 and Snf3 and the glucose transporter (Hxt1) are stable and functional only in the presence of glucose but are removed from the plasma membrane through ubiquitination and subsequent vacuolar degradation via endocytosis in the absence of glucose. vii Table of Contents Dedication…………………………………………...…………………………………....iv Acknowledgements………………………………………………………………………..v Abstract…………………………………………………………………………………..vii Table of Contents…………………………….............………………….………………viii List of Figures………......................…………………………….….……………………xi List of Tables....................................................................................................................xiv List of Symbols/Abbreviations…………………………………………………...……...xv Chapter I: Introduction………………………………………………………………….....……….…1 1.1.Background…………………………………………………………………....1 1.2. Aerobic Glycolysis in Yeast.............................................................................4 1.3.Key Components of the HXT Gene repressor Pathway.....................................5 1.4. Key Components of the HXT Gene Induction Pathway……………………...8 1.5. Crosstalk Between Glucose Induction and Repression Pathways…………..12 1.6. Research Significance…………………………………………………....….15 Chapter II: Understanding the Mechanism of Glucose-induced Relief of Rgt1- mediated Repression of HXT Gene Expression in Yeast...........................................…....20 2.1 Introduction………………………………………………….….......…….….21 2.2. Materials and Methods………………………………………………........…23 2.3. Results……………………………………………………………....……….26 viii 2.4. Discussion…………………………………………………………………...32 Chapter III: Mth1 Regulates the Interaction between the Rgt1 Repressor and the Ssn6-Tup1 Corepressor Complex by Modulating PKA-dependent Phosphorylation of Rgt1...............................................................................................….40 3.1 Introduction………………………………………….....……………...….….41 3.2. Materials and Methods…………………………………....……...….……...44 3.3. Results………………………………………………………......….….........48 3.4. Discussion………………………………………………………………......59 Chapter IV: Endocytosis and Vacuolar Degradation of the Yeast Cell Surface Glucose Sensors Rgt2 and Snf3………………………………………………............…68 4.1 Introduction…………………………...…………….…………...….……….69 4.2. Materials and Methods………………………………………...………....…71 4.3. Results………………………………………………………………..….….74 4.4. Discussion……………………………………………………………...…...85 Chapter V: Glucose Starvation-induced Turnover of the Yeast Glucose Transporter Hxt1…………………………………………………………...……...….....93 5.1 Introduction………………………………………………….…………........94 5.2. Materials and Methods……………………………………………...……...96 5.3. Results…………………………………………………………………...…99 5.4.Discussion………………………………………………….................…….106 ix Conclusions and Future Direction……………………………………………........…...110 References……………………………………………..………………………..........…112 Appendix I: Construction of Yeast Strains Useful for Screening Drugs that Inhibits Glucose Uptake and Glycolysis…………………………………..…..........…..132 A1.1 Introduction…………….……………………………………..……..………..132 A1.2 Construction and Properties of the HXT-NAT Reporter Strains………….......133 A1.3 Conclusion…………………………………………………....……....………137 x List of Figures Figure 1. The Aerobic Glycolysis, a Hallmark of Cancer...................................................3 Figure 2. Aerobic Glycolysis in Yeast…………………………….…………...........……5 Figure 3. Structures of Glucose and Related Hexoses………….…………………….......9 Figure 4. Rgt2/Snf3 Mediated Glucose Induction Pathway of HXT Gene Expression….…………………………………..........…………............………….13 Figure 5. Schematic Diagram