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Dissertation Investigating New Protein Components Of DISSERTATION INVESTIGATING NEW PROTEIN COMPONENTS OF THE ENDOCYTIC MACHINERY IN SACCHAROMYCES CEREVISIAE Submitted by Kristen Farrell Graduate Degree Program in Cell and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Spring 2016 Doctoral Committee: Advisor: Santiago Di Pietro James Bamburg Diego Krapf Michael Tamkun Copyright by Kristen Farrell 2016 All Rights Reserved ABSTRACT INVESTIGATING NEW PROTEIN COMPONENTS OF THE ENDOCYTIC MACHINERY IN SACCHAROMYCES CEREVISIAE Clathrin-mediated endocytosis is an essential eukaryotic process which allows cells to control membrane lipid and protein content, signaling processes, and uptake of nutrients among other functions. About 60 proteins have been identified that compose the endocytic machinery in Saccharyomes cerevisiae, or budding yeast. Clathrin-mediated endocytosis is highly conserved between yeast and mammals in terms of both protein content and timing of protein arrival. First, there is an immobile phase in which clathrin and other coat components concentrate at endocytic sites. Second, another wave of proteins assembles about 20 seconds before localized actin polymerization. Third, a fast mobile stage of endocytosis occurs coinciding with local actin polymerization and culminates with vesicle scission. Fourth, most coat proteins disassemble from the internalized vesicle. Despite the knowledge of so many endocytic proteins, gaps still remain in the complete understanding of the endocytic process. We attempt to fill some of these gaps with a screen of the yeast GFP library for novel endocytic-related proteins using confocal fluorescence microscopy. We identified proteins colocalizing with RFP-tagged Sla1, a clathrin adaptor that serves as a well-known marker of endocytic sites. Ubx3 and Tda2, two unstudied proteins, were selected for further investigation based on high degree of colocalization with Sla1. Ubx3 shows fluorescent patch dynamics similar to an endocytic coat protein. Ubx3 is dependent on clathrin for patch lifetime and binds clathrin via a W-box, the first identification of this clathrin binding ii motif in a non-mammalian species. Uptake assays performed in a knockout strain of Ubx3 display a reduction in both bulk endocytosis by fluorescent dye Lucifer Yellow and cargo- specific endocytosis by methionine transporter Mup1. The Ubx3 knockout cells also show a significant increase in lifetime of early endocytic protein Ede1, and removing its UBX domain alone results in similar defects to the Ubx3 knockout. The endocytic defect may be due to lack of recruitment of ubiquitin regulator AAA ATPase Cdc48 to the endocytic site. Inactivation of Cdc48 reduces Lucifer yellow uptake to minimal levels and causes aggregates of early endocytic protein Ede1-GFP. This is the first identification of a UBX domain-containing protein in clathrin-mediated endocytosis. Tda2 appears at the tail end of each endocytic site, suggesting a function in late stage endocytosis. A Tda2 knockout strain shows similar reductions in bulk and cargo dependent endocytosis through Lucifer yellow and Mup1 uptake assays. Tda2 appears unaffected by clathrin disruption, but is no longer recruited to the endocytic site when cells are treated with the actin depolymerizing agent LatA, suggesting it is associated with the actin cytoskeleton. A crystal structure of Tda2 reveals it is a homolog of mammalian dynein light chain TcTex-1. Tda2 is associated with a larger protein complex in the cytosol but does not co-purify with dynein and is unaffected by addition of the microtubule depolymerizing drug Nocodazole. Tda2 has similar localization to actin capping proteins Cap1/2 which localize to the plus end of actin filaments near the plasma membrane. Tda2 deletion increases Cap1 patch lifetime but reduces its fluorescent intensity. Aim21 fluorescent intensity at endocytic sites is reduced to half without Tda2. When Aim21 is deleted, Tda2 is no longer recruited to endocytic sites and the large Tda2- containing complex is no longer present in the cytosol. Tda2 is a newly identified component of the actin cytoskeleton in stable complex with Aim21. This is the first identification of a TcTex iii type dynein light chain in yeast and the first dynein light chain associated with clathrin-mediated endocytosis. Thus, we have identified two novel components of endocytic machinery by screening the yeast GFP library. The successful identification of previously uncharacterized endocytic proteins indicates the unique advantages of the GFP library screening. Many previous screens for endocytic proteins rely on the yeast knockout library or cargo accumulation, which have many disadvantages. The GFP library screening method has potential for use with other cellular processes that have distinct cellular localizations and established fluorescent markers. The GFP library also has potential for use in a screen for cargo proteins dependent on clathrin-mediated endocytosis. Additionally, more proteins of the endocytic machinery may be further characterized from the list of Sla1-colocalizing proteins identified in our screen. iv ACKNOWLEDGMENTS I would like to thank all of the Di Pietro lab members, who have always provided helpful discussions and suggestions for experiments especially when working through confusing data. My yeast counterparts have been especially helpful, including Thomas Tolsma and Lauren Barry. The undergraduates who have worked with me, Caitlin Grossman and Colette Worchester, have also been extremely helpful in contributing to this project. I would also like to thank my advisor Santiago Di Pietro for accepting me into his lab and teaching me to become a successful scientist. The entire Biochemistry and Molecular Biology department earns my thanks as well, for welcoming a Cell and Molecular Biology program student into their ranks. I would also like to thank everyone outside of the academic community who has contributed to my graduate career. My family has always been a constant support, despite being far in distance they have provided me with life advice and aid whenever I needed it. My friends, also both near and far, for keeping me sane during my studies and always providing weekend entertainment and mountain exploration companions. Finally I would like to thank Andrew Burkhart for always supporting me and pushing me to better myself in many aspects. v TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1: Introduction ..............................................................................................................1 1.1 Overview ........................................................................................................................1 1.2 Clathrin-mediated endocytosis.......................................................................................2 1.3 Yeast and the GFP library ............................................................................................34 REFERENCES ..............................................................................................................................40 CHAPTER 2: A screening of the yeast GFP library reveals Ubx3 as a novel protein of the endocytic machinery ......................................................................................................................49 2.1 Summary ......................................................................................................................49 2.2 Introduction ..................................................................................................................50 2.3 Results ..........................................................................................................................51 2.4 Discussion ....................................................................................................................80 2.5 Experimental Procedures .............................................................................................82 REFERENCES ..............................................................................................................................86 CHAPTER 3: Tda2 is a TcTex type dynein light chain with a role in the actin cytoskeleton during clathrin-mediated endocytosis ............................................................................................89 3.1 Summary ......................................................................................................................89 3.2 Introduction ..................................................................................................................90 3.3 Results ..........................................................................................................................91 3.4 Discussion ..................................................................................................................107 3.5 Experimental Procedures ...........................................................................................112
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