Autophagy: From Membrane Movement to Nuclear Regulation by Melinda Anne Lynch Day 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 2012 Doctoral Committee: Professor Daniel J. Klionsky, Chair Professor Laura J. Olsen Professor Lois S. Weisman Associate Professor Anuj Kumar © Melinda Anne Lynch Day 2012 In Memory of Geoff Whose death drove my desire to study biology, but whose life inspires me to live with conviction and passion. ii Acknowledgements I would first like to thank my wonderful husband, Dave, who has been patiently waiting for me to finish my doctoral studies. His love and encouragement have helped carry me through the last five years. I would also like to thank my parents, whose help has been invaluable. Thank you for teaching me the value of hard work. Appreciation is also due to others of my family including my in-laws and my aunt and uncle, all of whom have provided encouragement through it all. A special thank you is owed to my advisor, Dan Klionsky. He has been a wonderful mentor and has taught me the critical thinking skills needed to become an independent scientist. He challenged me and encouraged me, and I couldn’t have asked for a better mentor. I would also like to thank my committee members, Lois Weisman, Laura Olsen, and Anuj Kumar. Thank you all for your patience and your invaluable advice. I am grateful for all of your support in working with me to finish my dissertation and helping me to find a post-doctoral position. Lastly, I would like to thank the members of the Klionsky laboratory. It has been a pleasure working and learning with all of you over the last several years. I would especially like to thank Clinton Bartholomew who was always there to offer help and advice when I needed it most, Steven Backues who aided me in my post doctoral search, and Katie Parzych, who was my writing buddy as I put this dissertation together and who didn’t think it strange when I would be “gaily tripping” throughout the lab. iii Chapter 2 is a review article that I authored on the cytoplasm to vacuole targeting pathway: Melinda A. Lynch-Day and Daniel J. Klionsky. The Cvt pathway as a model for selective autophagy. FEBS Letters, 584: 1359-1366, 2010. It has been reprinted here with permission from Elsevier with minor modifications. Chapter 3 is a primary research article that I co-first authored on the role of Trs85 and Ypt1 in autophagy: Melinda A. Lynch-Day, Deepali Bhandari, Shekar Menon, Ju Huang, Huaqing Cai, Clinton R. Bartholomew, John H. Brumell, Susan Ferro-Novick, and Daniel J. Klionsky. Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore to promote autophagy. Proc Natl Acad Sci USA, 107: 7811-7816, 2010. It has been reprinted here with permission. I was responsible for figures 3.3, 3.4, 3.5, 3.6, S3.5, S3.6, S3.7, and S3.8B and Table S3.1. Chapter 4 is a manuscript that has been submitted to Science. I was responsible for all of the yeast work in the paper. Chapter 5 is a first author manuscript that has been submitted to Science. I was responsible for all of the figures. Appendix A is a review article that I first authored on the role of autophagy in Parkinson’s Disease: Melinda A. Lynch-Day, Kai Mao, Ke Wang, Mantong Zhao, and Daniel J. Klionsky. The role of autophagy in Parkinson’s disease. Cold Spring Harb Perspect Med, 2: a009357, 2012. It has been reprinted here with permission from Cold Spring Harbor with minor modifications. I was responsible for the writing of the section “Early discoveries: α-SYNUCLEIN and autophagy”, the organization of the section “Recent studies: controversies abound”, and general editing. iv Appendix B is a research study on mitophagy: Tomotake Kanki, Ke Wang, Misuzu Baba, Clinton R. Bartholomew, Melinda A. Lynch-Day, Zhou Du, Kai Mao, Zhifen Yang, Wei-Lien Yen, and Daniel J. Klionsky. A genomic screen for yeast mutants defective in selective mitochondria autophagy. Mol Biol Cell, 20:4730-4738, 2009. It has been reprinted here with permission from The American Society for Cell Biology. I was responsible for the screening of mitophagy defective mutants for defects in the Cvt pathway (Ape1 maturation) and non-specific autophagy (GFP-Atg8 cleavage), figures SB.2B and SB.4. Appendix C is a research study on the transcriptional inhibition of ATG8: Clinton R. Bartholomew, Tsukasa Suzuki, Zhou Du, Steven K. Backues, Meiyan Jin, Melinda A. Lynch-Day, Midori Umekawa, Avani Kamath, Mantong Zhao, Zhiping Xie, Ken Inoki, and Daniel J. Klionsky. Ume6 transcription factor is part of a signaling cascade that regulates autophagy. Proc Natl Acad Sci USA, 2012. It has been reprinted here with permission. I was responsible for figure C.2. v Table of Contents Dedication ........................................................................................................................... ii Acknowledgements ............................................................................................................ iii List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii List of Appendices ............................................................................................................. xi CHAPTER 1. The study of autophagy is important for understanding human disease ............1 2. The Cvt pathway as a model for selective autophagy ......................................41 3. Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore to promote autophagy .........................................................................................................75 4. The histone acetyltransferase hMOF regulates the outcome of autophagy ...118 5. Histone acetylation regulates autophagy by controlling Atg8 expression .....155 6. Summary and Perspectives ............................................................................188 Appendices .......................................................................................................................205 vi List of Tables Table S3.1. Summary of localization data .....................................................................104 S3.2. Strains .........................................................................................................112 S4.1. Primary antibodies used in this study .........................................................149 S4.2. Reagents used in this study .........................................................................149 S4.3. ON-TARGET plus SMART pool small interfering RNAs used in this study ..............................................................................................................................150 S5.1. Strains .........................................................................................................181 SB.1 Strains used in this study .............................................................................241 SB.2 Initial screen results for all strains tested ....................................................255 SB.3 Summary of autophagy analyses.................................................................259 SC.1 Strains used in this study .............................................................................313 vii List of Figures Figure 2.1. Three main types of autophagy .......................................................................43 2.2. Classification of Atg protein according to function ........................................52 2.3. Cvt vesicle formation ......................................................................................59 3.1. Trs85 does not coprecipitate with Trs130, Trs120, and Trs65 .......................82 S3.1. Epitope tagging Trs85 does not interfere with autophagy function .............83 3.2. Identification of a third TRAPP complex that activates Ypt1 ........................86 S3.2. Trs85 is not a component of the TRAPPI complex ......................................87 S3.3. Loss of Trs85 does not block ER-Golgi traffic ............................................88 S3.4. Subunit composition of the three TRAPPI complex ....................................89 3.3. Ypt1 is involved in nonspecific autophagy in yeast .......................................92 S3.5. Ypt1 is involved in nonspecific autophagy in yeast .....................................93 S3.6. Ypt1 and Trs85 function at the stage of autophagosome formation .............95 3.4. The ypt1 mutants are defective for the Cvt pathway ......................................97 3.5. Ypt1 overexpression enhances autophagy and constitutively active Ypt1 bypasses the requirement for its GEF ....................................................................99 3.6. Ypt1 and Trs85 localize to the PAS ..............................................................103 S3.7. The GFP tag on Trs85 does not result in aggregate formation ...................105 S3.8. Subcellular fractionation and localization of Trs85 ....................................106 4.1. SIRT1-dependent and –independent autophagy is associated with a reduced acetylation of histone H4 lysine 16 ......................................................................125 viii S4.1. Starvation does not induce H4K16ac downregulation in Sirt1-/- MEF cells ......................................................................................................................126