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Molecular Mechanism and Regulation of Autophagy in Saccharomyces Cerevisiae Molecular Mechanism and Regulation of Autophagy in Saccharomyces cerevisiae by Zhiyuan Yao A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular and Developmental Biology) in the University of Michigan 2018 Doctoral Committee: Professor Daniel J. Klionsky, Chair Professor Laura Olsen Professor Lois S. Weisman Professor Haoxing Xu Zhiyuan Yao [email protected] ORCID iD: 0000-0002-5759-9822 © Zhiyuan Yao 2018 Dedication This thesis is dedicated to my beloved wife Jie Li, and to my parents Cai Yao and Hexiang Liu for their unconditional support. This thesis is also dedicated to Saber, Artoria Pendragon, who always inspires me with her strong will and noble spirit. ii Acknowledgements First I want to thank my mentor, Dr. Daniel J. Klionsky, for his continuous support and patient guidance during my Ph.D. works. Dan is a great scientist as well as a wise advisor. His passion in science and helpful advice on my projects always encouraged me to plan ahead and consider potential problems before they develop. Five years’ experience in one’s 20s will greatly shape his view in both career and life, and I feel so lucky to have Dan as my example during my graduate life. I also want to thank my thesis committee members, Professor Haoxing Xu, Professor Lois S. Weisman and Professor Laura Olsen. Your generous support and immense knowledge provided me with invaluable suggestions and criticisms for my research. Without all this help, I could not finish this thesis. Here I would like to express my sincere thanks for all of the efforts you made. I would like to thank all current members and alumni in the Klionsky lab—thanks for your help both inside or outside the lab. I am so happy to have you guys around for the past five years. I enjoyed scientific discussions we had and lab activities we held. Specifically I want to thank Dr. Meiyan Jin and Dr. Xu Liu. Meiyan taught me most of the experimental methods when I was new in the lab and provided helpful suggestions when I encountered obstacles in my research. Xu provided countless suggestions during my graduate studies and offered generous help when I faced problems. Discussion with Xu is always a happy experience regardless of the topic. I learned a lot from both of them. iii Finally I would like to thank my family for their unconditional support. Specifically I want to thank my wife Dr. Jie Li, who is playing Dota2 beside me while I am writing this acknowledgement. We have known each other over 16 years. Over this time you were my schoolmate, my classmate, my competitor, my girlfriend and finally you are my life mate. Thanks for supporting me both in life and in game, my thanks and love to you are far beyond what words can express. We shared a similar taste in food, and similar opinions of life, even though our Dota2 rank is not similar, I will be happy to help you rise up. Also, I want to thank our pet cat, Batman, for the happiness she gave us during these years. iv Table of Contents Dedication ................................................................................................................................... ii Acknowledgements ................................................................................................................... iii List of Tables ............................................................................................................................ vii List of Figures .......................................................................................................................... viii Abstract ........................................................................................................................................ x Chapter 1 Introduction ................................................................................................................. 1 1.1. Terms and structures .......................................................................................................... 1 1.2. The history of discovery ..................................................................................................... 2 1.3. The types of autophagy ...................................................................................................... 4 1.4. Identification of the PAS and the origin of the phagophore membrane ............................. 4 1.5. Core molecular machinery of autophagosome formation .................................................. 6 1.6. Regulation of autophagy .................................................................................................. 12 1.7. References ........................................................................................................................ 16 Chapter 2 Atg41/Icy2 regulates autophagosome formation ...................................................... 26 2.1. Abstract ............................................................................................................................ 26 2.2. Introduction ...................................................................................................................... 27 2.3. Result ................................................................................................................................ 28 v 2.4. Discussion ........................................................................................................................ 41 2.5. Materials and Methods ..................................................................................................... 43 2.6. References ........................................................................................................................ 45 Chapter 3 Dhh1 promotes autophagy-related protein translation and autophagy during nitrogen starvation ..................................................................................................................... 69 3.1. Abstract ............................................................................................................................ 69 3.2. Introduction ...................................................................................................................... 70 3.3. Result ................................................................................................................................ 71 3.4. Discussion ........................................................................................................................ 78 3.5. Materials and Methods ..................................................................................................... 81 3.6. References ........................................................................................................................ 85 Chapter 4 Summary ................................................................................................................... 99 4.1. Atg41/Icy2 regulates autophagosome formation ............................................................. 99 4.2. RNA helicase Dhh1 functions in translational regulation of Atg proteins to promote autophagy ................................................................................................................................. 101 4.3 . Contribution of the thesis ................................................................................................ 102 vi List of Tables Table 1.1. Atg/ATG proteins in the core machinery of autophagosome formation. .............. 25 Table S2.1. Detailed analysis of TEM data. .............................................................................. 49 Table S2.2. Strains used in this study. ....................................................................................... 50 Table S2.3. Amino acid, nucleoside and vitamin stock solutions. ............................................ 52 Table S2.4. Primers used in qPCR, ChIP and promoter replacement. ...................................... 53 Table S3.1. Yeast strains used in this study .............................................................................. 88 Table S3.2. Primers for RT-qPCR analysis in the RNA immunoprecipitation assay ............... 90 vii List of Figures Figure 1.1. Schematic depiction of the macroautophagy in yeast. ............................................ 24 Figure 2.1. Icy2 is required for nonselective autophagy. .......................................................... 55 Figure 2.2. The Atg41 expression level increases after autophagy is induced. ......................... 56 Figure 2.3. The increase in Atg41 protein level is required for autophagy. .............................. 57 Figure 2.4. Atg41 localizes to peripheral Atg9-containing sites. .............................................. 58 Figure 2.5. The C-terminal region of Atg41 is important for its function in autophagy. .......... 60 Figure 2.6. Gcn4 activates the transcription of ATG41 during nitrogen starvation. ................. 62 Figure S2.1. Exogenous ICY2 can restore autophagy activity in the icy2∆ strain. ................... 64 Figure S2.2. The Atg41-GFP chimera is functional and the protein level increases after starvation ................................................................................................................................... 67 Figure S2.3. Atg41 and the corresponding deletion mutants are stable under nitrogen starvation conditions .................................................................................................................. 68 Figure 3.1. Dhh1 positively
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