DISSERTATION DETERMINATION OF THE FUNCTIONS OF RAB32, RAB38, AND THEIR EFFECTOR MYOSIN VC IN THE BIOGENESIS OF MELANOSOMES Submitted by Jarred Bultema Department of Biochemistry and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2013 Doctoral Committee: Advisor: Santiago Di Pietro Eric Ross Jennifer DeLuca Chaoping Chen Anireddy Reddy ! ! ABSTRACT DETERMINATION OF THE FUNCTIONS OF RAB32, RAB38, AND THEIR EFFECTOR MYOSIN VC IN THE BIOGENESIS OF MELANOSOMES In mammals, pigment produced within specialized cells is responsible for skin, hair, and eye coloration. Melanocytes are specialized cells that produce pigment within an organelle known as the melanosome. Melanosomes are a member of a specialized class of organelles, known as Lysosome-related organelles (LRO), which are responsible for a number of critical functions in mammals such as pigmentation, blood clotting, lung function, and immune function. LROs are related to the ubiquitous lysosome, and are formed using the same molecular mechanisms as lysosomes that rely upon the Adaptor Protein complexes -1 (AP-1) and -3 (AP- 3), and the Biogenesis of Lysosome-related Organelles Complex (BLOC)-2 (BLOC-2). These protein complexes are critical for the trafficking of specialized cargoes to melanosomes required for proper melanin synthesis. But, these complexes are also used for the formation of lysosomes, and no mechanism is known to distinguish between trafficking to lysosomes and melanosomes. The melanosome serves as a model system to study the formation of LROs, and insights from the study of melanosomes help explain the biogenesis of other LROs. In this dissertation, I present the finding that Rab32 and Rab38 function as melanosome- specific trafficking factors that allow for the use of AP-3, AP-1, and BLOC-2 in melanosome biogenesis. Using biochemical approaches, I show that Rab32 and Rab38 bind directly to AP-3, AP-1, and BLOC-2 on membranes. In microscopy experiments, I demonstrate that Rab32 and Rab38 localize to early endosomal subdomains where AP-3, AP-1, and BLOC-2 function. Using ii ! a combination of biochemical and microscopic approaches, I show that Rab32 and Rab38 serve partially redundant functions in trafficking of specialized cargoes to melanosomes. I report the discovery that Myosin Vc, a class V myosin motor, interacts with Rab32 and Rab38 and serves novel functions in melanosomes trafficking. I show, using biochemical approaches, that Myosin Vc directly binds to several melanosomal Rab proteins and serves as an effector of these proteins in melanosome biogenesis. Using a combination of approaches, I demonstrate that depletion of Myosin Vc from melanocyte cells causes defects in the trafficking of cargoes to melanosomes, but also causes severe defects in the secretion of mature melanosomes. With biochemical and microscopic approaches, I compare the function and localization of Myosin Vc in melanocytes to related proteins Myosin Va and Myosin Vb, and provide evidence to suggest that all three of these proteins function in distinct steps of melanosome trafficking. My results answer outstanding questions about the use of ubiquitous trafficking machinery (AP-3, AP-1, and BLOC-2) in trafficking to a specialized organelle. I provide evidence to answer outstanding questions about the mechanism of action of Rab32 and Rab38 in melanosome trafficking through my studies with Myosin Vc. I also establish new areas of research in the comparison of Myosin Va, Myosin Vb, and Myosin Vc in melanosome trafficking. My results address numerous unknown areas in melanosome biogenesis, expand the knowledge of melanosome biogenesis, and provide numerous new avenues of research to explore to understand specialized trafficking to LROs. iii ! ACKNOWLEDGMENTS Now at the end of my graduate career, I reflect upon the various steps that have brought me to this place. First and foremost, I must thank my advisor Santiago Di Pietro for believing in me and accepting me as one of his first PhD students. Through continuous guidance, advice, and mentorship he has helped shape me into the scientist I am now. My aspiration to be as thoughtful, creative, and dedicated scientist is based on the example set by Santiago. I must also credit Andrea Ambrosio for my development as a scientist. As a core member of the lab and an extremely enjoyable colleague, she was one of my main sources of advice, and in-lab entertainment, and is the reason that the lab has the atmosphere that it does. Daniel Feliciano and I began our PhD studies with Santiago at the same time, and Daniel has been a constant source of support, encouragement, and inspiration. Knowing how dedicated and committed Daniel is to his work inspired me to work harder and push through the difficult times. The early, Di Pietro lab group felt more like a family, and made the time and frustration associated with graduate school infinitely more bearable. Previous lab member Carolyn Burek kept life in lab entertaining, and contributed to my research efforts with several important experiments. It has been a pleasure to see new members join the Di Pietro lab, and see how things have changed since I began. Thank you to all of the Di Pietro lab members, new and old, for all the help and collaboration. I was lucky to have been a part of this group from the start. My family and friends have been an enormous support with the sacrifices and stress that are required in graduate school. Thank you for understanding and supporting me. The frustration of graduate school has brought more to my life than I would have expected. Fellow graduate student, Megan Carter (now Bultema) and I initially bonded by venting about the frustrations of graduate school and the effect that it had on our lives. iv ! Eventually, venting led to dating, an engagement, and a recent wedding. I can say that while earning a PhD is an enormous accomplishment; my proudest moments from graduate school are in meeting and, ultimately, marrying Megan. Thank you Megan for all of your help and support. v ! TABLE OF CONTENTS ABSTRACT.................................................................................................................................... ii Acknowledgments ......................................................................................................................... iv Table of Contents........................................................................................................................... vi List of Figures.............................................................................................................................. viii CHAPTER 1 ................................................................................................................................... 1 Introduction..................................................................................................................................... 1 1.1 Overview............................................................................................................................... 1 1.2 Intracellular trafficking of the endo-lysosomal system ........................................................ 3 1.3 Lysosome-related organelles .............................................................................................. 14 1.4 Diseases of Lysosome-related organelles ........................................................................... 17 1.5 Melanocytes and melanosomes - a model of LRO biogenesis ........................................... 27 1.6 Transmembrane-protein transport to melanosomes............................................................ 35 1.7 Role of Rab GTPases in melanosome biogenesis............................................................... 44 1.8 Role of actin-based Class V Myosin motors in Organelle and vesicle trafficking............. 51 1.9 References............................................................................................................................... 63 CHAPTER 2 ................................................................................................................................. 80 BLOC-2, AP-3, and AP-1 function in concert with Rab38 and Rab32 to mediate protein trafficking to lysosome-related organelles.................................................................................... 80 2.1 Summary............................................................................................................................. 80 2.2 Introduction......................................................................................................................... 81 2.3 Experimental Procedures .................................................................................................... 85 2.4 Results................................................................................................................................. 88 2.5 Discussion......................................................................................................................... 116 2.6 Acknowledgments ............................................................................................................ 122 2.7. References............................................................................................................................ 123 CHAPTER 3 ............................................................................................................................... 126