Novel Functions of the Flippase Drs2 and the Coat Protein COPI in Protein Trafficking By Hannah Marcille Hankins Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Sciences May, 2016 Nashville, Tennessee Approved: Todd R. Graham, Ph.D Katherine L. Friedman, Ph.D. Kendal S. Broadie, Ph.D. Anne K. Kenworthy, Ph.D. Charles R. Sanders, Ph.D. 1 ACKNOWLEDGEMENTS It takes a village to raise a graduate student, and I have many villagers to thank for getting me to this point. I would never have considered doing research if it were not for my professors at Henderson State University, particularly Martin Campbell and Troy Bray, who were very supportive and encouraging throughout my undergraduate studies. I was also fortunate to be able to do full time research during the summers of my sophomore and junior years in the lab of Kevin Raney at the University of Arkansas for Medical Sciences. If it were not for the support system and these wonderful research experiences I had as an undergraduate, I never would have pursued graduate school at Vanderbilt and would not be the scientist I am today. I would like to thank my graduate school mentor, Todd Graham, for giving me the opportunity to join his lab and for introducing me to the wonderful world of yeast. Todd is an amazing and enthusiastic scientist; his guidance and support throughout my graduate career has been invaluable and I am truly grateful to have such a wonderful mentor. I must also thank Todd for his “constructive” criticism of my wine bottle opening technique (or lack thereof) during my short-lived career as a bartender during the Southeastern Regional Yeast Meeting. I like to think that under Todd’s mentorship I have grown both as a scientist and as a normal adult capable of opening bottles of wine. I thank my committee members Chuck Sanders, Kendal Broadie, Anne Kenworthy, and my chair Kathy Friedman for all of their helpful input and guidance over the years. Kathy is also my lab neighbor and yeast comrade, and I must also thank her for sharing countless reagents and incubator space over the years. My research would not have been possible without my wonderful collaborators Anant Menon and Yves Sere at Cornell University and Howard and Isabelle Riezman at the University of Geneva. I also thank Puck Ohi and Jason MacGurn for generously allowing me to use their microscopes. The people at the BRET office, especially Ashley Brady, have also been an invaluable resource for my career development. ii I must also thank all of my labmates for their support and friendship over the years. To my baymate Mehmet Takar, your humor defies all logic and reason, but it is amazing and I hope you never change. Thank you for putting up with me all these years even though I’m a blue-eyed devil and can’t be trusted. Peng Xu, thank you for all your help and advice over the years and, most importantly, always reminding us of the two most important phrases in science: “it is what it is” and “let it go.” Bart Roland, thanks for being a good friend to me, despite my constant criticism of your “unique” color choices at lab meetings. To the newest member of the Graham lab, Jordan Best, thank you for bringing humor and what I hesitate to call fashion to the lab. I would also like to acknowledge all of the talented visiting students, Lisa Theorin, Susanne Prokop, and Cayetana Arnaiz, and undergraduates, Katie Ivy, Christina Snider, Deanna Tiek, Nick Diab, and Sam Erlinger, we have had come through the lab during my time here. Funding from the National Institutes of Health supported all of my dissertation research. Funding from the Vanderbilt Graduate School and the Gisela Mosig Travel Fund allowed me to present my research at national conferences in Birmingham and Philadelphia as well as an international conference in Corsica. In particular, the trip to Corsica was my first time traveling abroad (alone, no less!) and it was truly an unforgettable experience with great people and great science. I am truly fortunate to have a great group of friends in the graduate program. In no particular order, I would like to thank Arwen, Alex, Ushashi, Abigail, Sophia, Jen, Amy, Lehanna, Adrian, Nathan, Joel, Patrick, and Andrew for their friendship over the years. I can’t imagine what my graduate school experience would have been like without all of you in it. To my parents, Jim and Gloria, thank you for the support throughout my graduate studies despite you not really knowing what it is I do here and probably wondering why I am just now finishing up college in my 9th year. To my older brothers, Caleb and Noah, thank you for being so wonderful and always so supportive of everything I do. My childhood would not have been nearly as fun without the two of you. Finally, to my boyfriend, Rubin Baskir, thank you for the unending love and support. You and your family have been a huge part of my life these past three years and I look forward to the years to come. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .......................................................................................................................... ii LIST OF TABLES ...................................................................................................................................... vii LIST OF FIGURES ................................................................................................................................... viii LIST OF ABBREVIATIONS ....................................................................................................................... x CHAPTER I. INTRODUCTION ..................................................................................................................................... 1 Overview ................................................................................................................................................... 1 Vesicle-mediated protein transport in Saccharomyces cerevisiae ........................................................... 2 Transport between the Endoplasmic Reticulum and the Golgi ....................................................... 2 Transport to the vacuole .................................................................................................................. 4 Endocytosis ..................................................................................................................................... 5 Recycling pathway .......................................................................................................................... 6 Exocytosis ....................................................................................................................................... 7 Role of flippases in membrane asymmetry and protein trafficking ......................................................... 8 Flippases belong to the P-type ATPase family ............................................................................... 8 Mechanism of phospholipid translocation ..................................................................................... 11 Flippases as regulators of membrane asymmetry ......................................................................... 17 Importance of flippase activity in protein trafficking ................................................................... 20 Influence of Drs2 and Kes1 on sterol homeostasis ................................................................................. 25 Budding yeast lateral membrane organization ............................................................................. 25 Non-vesicular sterol transport by Kes1 ......................................................................................... 27 Antagonistic relationship between Drs2 and Kes1 in sterol homeostasis ..................................... 30 II. PHOSPHATIDYLSERINE TRANSLOCATION AT THE YEAST TRANS-GOLGI NETWORK REGULATES PROTEIN SORTING INTO EXOCYTIC VESICLES .............................................. 33 Abstract ................................................................................................................................................... 33 Introduction ............................................................................................................................................. 34 Results .................................................................................................................................................... 36 Compartmentalization of the plasma membrane is significantly perturbed in cho1Δ cells but not drs2Δ cells ............................................................................................................................... 36 Pma1 and Can1 mislocalize to the vacuole when PS flippase activity is disrupted ...................... 37 Drs2 and Kes1 work antagonistically in the sorting of Pma1 and Can1 ........................................ 39 iv Role of PS in ergosterol enrichment at the plasma membrane ...................................................... 41 Pma1 and Can1 are missorted from the TGN to the vacuole in drs2Δ and cho1Δ cells................ 44 Missorting of Pma1 and Can1 is not due to drs2Δ AP-1/Rcy1 trafficking defects ....................... 45 Discussion ............................................................................................................................................... 47 Materials and Methods ..........................................................................................................................