FUNCTIONS OF THE DYNAMIN-LIKE PROTEIN VPS1 IN ACTIN ORGANIZATION IN SACCHAROMYCES CEREVISIAE YU XIANWEN INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 FUNCTIONS OF THE DYNAMIN-LIKE PROTEIN VPS1 IN ACTIN ORGANIZATION IN SACCHAROMYCES CEREVISIAE YU XIANWEN (B.Sc., XIAMEN UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENTS Foremost, I would like to express my gratitude to my supervisor A/P Mingjie Cai, for providing me the opportunity to pursue my Ph.D. research work in his laboratory. I am deeply grateful to A/P Cai for his supervision, guidance, tolerance, and support throughout my graduate studies, and for his invaluable amendments to this thesis. My sincere thanks also go to the members of my graduate supervisory committee, A/P Thomas Leung and A/P Walter Hunziker for their constructive comments and encouragement during the course of this work. My special thanks also go to Dr. Alan Munn (Institute for Molecular Bioscience, the University of Queensland) for his invaluable scientific advice and assistance in the endocytosis assay. I would like to thank the past and present members in CMJ laboratory, for their helpful discussion, technique assistance, cooperation, and friendship. Special thanks go to Dr. Hsin-yao Tan, Dr. Guoliang Tian, and Dr. Guisheng Zeng, for their help, advice, and sharing of experience. Thanks also go to Miss Suat Peng Neo and Mr. Jeff Wui Kheng Seow, for their critical reading of my thesis. Many thanks also go to the past and present members in US laboratory, to Dr. Hong Hwa Lim, Dr. Foong May Yeong, Dr. Vaidehi Krishnan, Miss Karen Crasta, Mr. Tao Zhang, and Mr. Saurabh Nirantar, for their interesting discussions and help with the project. Especially, I am deeply grateful to Dr. Padmashree C.G. Rida, for her critical reading of my manuscript and this thesis, and also for her constant and kind help whenever I needed. I would like to express my gratitude to Dr. Lei Lu in HWJ laboratory for his helpful discussions and suggestions pertaining to the project. I also appreciate the excellent services from the various administrative and technical staffs in IMCB which are indispensable to fulfill my studies. Finally, my heartfelt and deepest appreciation goes to my husband, Canhe Chen, for his love, patience, understanding, and support over these years. Last but not the least, this thesis is dedicated to my beloved parents, for their unwavering support and belief in me throughout the journey of my studies. Xianwen Yu January, 2005 Table of Contents ii TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS ii LIST OF FIGURES ix LIST OF TABLES xii ABBREVIATIONS xiii ii SUMMARY CHAPTER I 1.1 1.2 1.3 Introduction General introduction 1.1.1 Endocytosis 1.1.2 Endocytic signals Formation of endocytic vesicle 1.2.1 Vesicle formation in clathrin-mediated endocytosis 1.2.1.1 Clathrin and clathrin adaptor protein AP-2 1.2.1.2 Clathrin accessory factors 1.2.2 Vesicle formation in caveolae-dependent pathway 1.2.3 Vesicle formation in macropinocytosis and phagocytosis 10 Roles of dynamin in endocytic vesicle formation 11 1.3.1 Dynamin and dynamin-related proteins 12 1.3.1.1 14 Domains and properties of dynamins Table of Contents 1.3.1.2 1.3.2 iii Properties of dynamin-related proteins 18 Roles of dynamin in the release of clathrin-coated vesicles (CCVs) 18 1.3.2.1 Dynamins interact with a subset of accessory factors in the formation of CCVs 18 1.3.2.2 Functions of dynamin and its interacting partners in the distinct stages of CCV formation 23 1.3.2.3 Dynamin may function as a force-generating GTPase 26 1.3.2.4 Dynamin may function as a regulatory enzyme 29 1.3.3 Roles of dynamins in clathrin-independent endocytosis 1.4 Roles of actin in endocytosis 31 33 1.4.1 Overview of the connections between actin and endocytosis 33 1.4.2 Actin and dynamic actin polymerization 34 1.4.2.1 Actin treadmilling and its regulators 34 1.4.2.2 Structure of yeast actin cytoskeleton 38 1.4.2.3 Regulation of actin cytoskeleton in yeast 1.4.2.4 1.4.3 1.4.2.3.1 The regulation of cortical patch assembly 42 1.4.2.3.2 Assembly and polarization of actin cables 47 Functions of yeast actin cytoskeleton 1.4.2.4.1 Actin cables in organelle segregation, mRNA inheritance, and polarized secretion 1.4.2.4.2 Cortical actin patch in endocytosis and cell wall morphogenesis Involvement of actin assembly in yeast endocytosis 1.4.3.1 42 Yeast as a model system for the study of endocytosis 49 49 50 53 53 Table of Contents 1.4.4 1.4.3.2 Intact actin cytoskeleton organization is required for endocytosis 55 1.4.3.3 Possible roles of actin cytoskeleton in the endocytic pathway 58 Actin organization in endocytosis of higher eukaryotes Roles of actin in endocytosis of higher eukaryotes: important but not obligatory 59 1.4.4.2 Links between endocytic machinery and actin cytoskeleton 62 65 1.5.1 Similarities in the endocytic pathway of mammals and yeast 65 1.5.2 Differences in endocytosis between the two systems 66 1.6 Research Objectives CHAPTER II 2.2 59 1.4.4.1 1.5 Endocytosis in mammals and yeast: a comparison 2.1 iv Materials and Methods 67 69 Materials 70 2.1.1 Reagents and Antibodies 70 2.1.2 Oligonucleotides 71 2.1.3 Strains 72 2.1.4 Constructs 74 Methods 77 2.2.1 Strains and culture conditions 77 2.2.2 Recombinant DNA methods 79 2.2.2.1 DNA transformation of E.Coli cells 79 2.2.2.2 Plasmid DNA preparation 79 2.2.2.3 Site-directed mutagenesis 80 Table of Contents 2.2.2.4 2.2.3 2.2.4 2.3 Plasmid constructions v 80 Yeast manipulations 80 2.2.3.1 Yeast transformation 80 2.2.3.2 Gene disruption and integration 81 2.2.3.3 Two-hybrid assays 82 2.2.3.4 Uracil uptake assay 82 2.2.3.5 Measurement of the half-life of Ste3p 83 2.2.3.6 Halo assays for Latrunculin-A (LAT-A) sensitivity 84 2.2.3.7 Colony overlay immunoblot 84 Fluorescence microscopy studies 85 2.2.4.1 Staining of F-actin and chitin 85 2.2.4.2 Cellular localization of proteins with fluorescent tags 86 2.2.4.3 FM 4-64 staining 86 Protein Analysis 87 2.3.1 Preparation of yeast cell extracts 87 2.3.2 CHAPTER III 2.3.1.1 Preparation of crude protein extracts using acid-washed glass beads 87 2.3.1.2 Preparation of total protein extracts using TCA precipitation 87 Immunoprecipitation and Western blot Vps1p Is Required for Actin Cytoskeleton Organization 88 90 Table of Contents vi 3.1 Background 91 3.2 Results 92 3.3 3.2.1 Vps1p is required for normal actin cytoskeleton organization 92 3.2.2 The vps1 mutant is defective in bud site selection and chitin deposition 95 3.2.3 The turnover of membrane receptor protein Ste3p is impaired in vps1∆ cells 98 3.2.4 The vps1 mutant shows mild deficiency in receptormediated endocytosis Discussion 101 101 3.3.1 Vps1p is required for normal actin cytoskeleton organization in yeast 101 3.3.2 Vps1p is required for the efficient internalization of some membrane proteins 104 CHAPTER IV Functions of the Putative Vps1p GTPase Mutants in Actin Organization 106 4.1 Background 107 4.2 Results 107 4.2.1 The intact GTPase domain of Vps1p is required for its growth at 37oC 107 4.2.2 The GTPase mutants of Vps1p are defective in the mating projections formation 109 4.2.3 The GTPase mutants of Vps1p are more sensitive to LAT-A 111 4.2.4 The GTPase activity of Vps1p is potentially important for its role in endocytosis 113 Table of Contents 4.3 vii 4.2.5 Overexpression of the GTPase mutants of Vps1p leads to actin defects and cell death at 37oC 114 4.2.6 Overexpression of the GTPase mutants of Vps1p increases sensitivity to LAT-A 116 Discussion 116 4.3.1 The function of Vps1p depends on its intact GTPase domain 116 4.3.2 The dominant-negative effects of vps1 GTPase mutants 119 Genetic and Physical Interactions between SLA1 and VPS1 120 CHAPTER V 5.1 Background 121 5.2 Results 121 5.2.1 Roles of Sla1p in actin organization and endocytosis 121 5.2.2 Genetic interaction between VPS1 and SLA1 124 5.2.3 Physical association between Vps1p and Sla1p 126 5.2.4 Alteration of cellular localization of Sla1p by vps1 GTPase mutations 129 Discussion 130 5.3.1 Genetic interaction between vps1 and sla1 mutants 130 5.3 5.3.2 Vps1p may function in actin cytoskeleton through its interaction with Sla1p CHAPTER VI Functional Characteristics of Vps1p by its Domain Organization 132 135 6.1 Background 136 6.2 Results 136 Table of Contents 6.3 viii 6.2.1 Overexpression of the COOH-terminal region of Vps1p leads to growth defects at 37oC 136 6.2.2 Overexpression of the Vps1p COOH-terminal regions affects 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One possible function of Prk1p is the negative regulation of Pan1p, an actin patch regulatory protein that forms a complex in vivo with at least two other proteins, Sla1p and End3p. In this report, we identified Sla1p as another substrate for Prk1p. The phosphorylation of Sla1p by Prk1p was established in vitro with the use of immunoprecipitated Prk1p and in vivo with the use of PRK1 overexpression, and was further supported by the finding that immunoprecipitated Sla1p contained PRK1- and ARK1-dependent kinase activities. Stable complex formation between Prk1p and Sla1p/Pan1p in vivo could be observed once the phosphorylation reaction was blocked by mutation in the catalytic site of Prk1p. Elevation of Prk1p activities in wild-type cells resulted in a number of deficiencies, including those in colocalization of Pan1p and Sla1p, endocytosis, and cell wall morphogenesis, likely attributable to a disintegration of the Pan1p/Sla1p/End3p complex. These results lend a strong support to the model that the phosphorylation of the Pan1p/Sla1p/End3p complex by Prk1p is one of the important mechanisms by which the organization and functions of the actin cytoskeleton are regulated. INTRODUCTION The rapid assembly and disassembly of actin filaments at specific subcellular locations provide the mechanistic basis for various dynamic activities such as cell motility, change of cell shapes, and translocation of intracellular organelles (Carlier and Pantaloni, 1997; Mermall et al., 1998; Cooper and Schafer, 2000). An important part of our current knowledge on the actin cytoskeleton dynamics comes from studies of the actin cytoskeleton organization in the yeast Saccharomyces cerevisiae. The major actin cytoskeletal structures in yeast are the cortical patches and the cytoplasmic cables, both of which display a conspicuous pattern of dynamics during the cell cycle. The pattern of cellular distribution of these actin structures has long been noticed to correlate with that of the localized surface growth (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Novick and Botstein, 1985). The actin patches and cables are distributed evenly in unbudded cells undergoing isotropical surface expansion. At the time of bud emergence and during the entire period of bud formation, the yeast cell assumes an apical growth pattern, with most if not all of the actin patches mobilized first to the bud site and later in the bud. The mother cell * Corresponding author. E-mail address: mcbcaimj@imcb.nus. edu.sg. © 2001 by The American Society for Cell Biology exhibits essentially no enlargement during this time and contains only the actin cables, which are all aligned toward the bud (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Lew and Reed, 1993). Despite the correlation in the patterns of actin distribution and bud formation, the exact roles of the actin cytoskeleton, especially the cortical patches, in promoting cell growth have remained largely unknown. Although cytoplasmic cables may serve as paths for myosin molecules to transport secretion vesicles to the cell surface (Novick and Botstein, 1985; Govindan et al., 1995; Ayscough et al., 1997; Pruyne et al., 1998), a polarized distribution of the cortical patches does not appear to be a necessity for bud growth, because mutants that failed to maintain a polarized localization of the cortical actin patches could still form bud efficiently (Karpova et al., 2000). Among a large number of proteins that have been identified to play direct or indirect roles in the function of the actin cytoskeleton in yeast are a group of actin patch proteins (Pruyne and Bretscher, 2000). These proteins reside on the cell cortex as clusters and partially colocalize with the actin patches. Three of them, Pan1p, End3p, and Sla1p, have been known to form a complex in vivo (Tang et al., 2000), and to be required for the actin patch morphology, membrane protein endocytosis, and cell wall synthesis (Holtzman et al., 1993; Be´ne´detti et al., 1994; Tang and Cai, 1996; Tang et al., 1997, 2000; Ayscough et al., 1999). Recently, the role of Pan1p 3759 Research Article 3839 The yeast dynamin-related GTPase Vps1p functions in the organization of the actin cytoskeleton via interaction with Sla1p Xianwen Yu and Mingjie Cai* Institute of Molecular and Cell Biology, National University of Singapore, 61 Biopolis Drive, Proteos, Singapore 138673, Rep. of Singapore *Author for correspondence (e-mail: mcbcaimj@imcb.nus.edu.sg) Accepted 25 March 2004 Journal of Cell Science 117, 3839-3853 Published by The Company of Biologists 2004 doi:10.1242/jcs.01239 Summary Recent studies have suggested that the function of the large GTPase dynamin in endocytosis in mammalian cells may comprise a modulation of actin cytoskeleton. The role of dynamin in actin cytoskeleton organization in the yeast Saccharomyces cerevisiae has remained undefined. In this report, we found that one of the yeast dynamin-related proteins, Vps1p, is required for normal actin cytoskeleton organization. At both permissive and non-permissive temperatures, the vps1 mutants exhibited various degrees of phenotypes commonly associated with actin cytoskeleton defects: depolarized and aggregated actin structures, hypersensitivity to the actin cytoskeleton toxin latrunculinA, randomized bud site selection and chitin deposition, and impaired efficiency in the internalization of membrane receptors. Over-expression of the GTPase mutants of vps1 also led to actin abnormalities. Consistent with these actinrelated defects, Vps1p was found to interact physically, and partially co-localize, with the actin-regulatory protein Sla1p. The normal cellular localization of Sla1p required Vps1p and could be altered by over-expression of a region of Vps1p that was involved in the interaction with Sla1p. The same region also promoted mis-sorting of the vacuolar protein carboxypeptidase Y upon over-expression. These findings suggest that the functions of the dynamin-related protein Vps1p in actin cytoskeleton dynamics and vacuolar protein sorting are probably related to each other. Introduction Dynamin and dynamin-related proteins are an evolutionally conserved family of large GTPases engaged in a diversity of cellular processes, including endocytosis, intracellular protein trafficking, and organelle partitioning (Hinshaw, 2000; Danino and Hinshaw, 2001). The role of dynamin in clathrin-mediated endocytosis is first suggested in the analysis of a temperaturesensitive mutant of dynamin in Drosophila. At the nonpermissive temperature, the mutant, shibire, exhibits a paralytic phenotype due to a block in endocytosis at the presynaptic membranes (Kosaka and Ikeda, 1983). The unusual accumulation of long invaginations at these membranes indicates a failure in vesicle detachment (Kosaka et al., 1983; Koenig and Ikeda, 1989). One conspicuous feature of dynamin is its ability of self-assemble into spiral-like structures around lipid tubules, which has led to the proposal that dynamin acts as a mechano-enzyme to release the clathrincoated vesicles using its GTPase-dependent conformational changes (Sweitzer and Hinshaw, 1998; Stowell et al., 1999; Marks et al., 2001; McGavin et al., 2001; Song and Schmid, 2003). Recent studies reveal that the function of dynamins in endocytosis might depend on their roles as actin cytoskeleton regulators. Actin cytoskeleton has been known for quite some time to be important for endocytosis in the yeast Saccharomyces cerevisiae. Yeast mutants with an abnormal or perturbed cortical actin cytoskeleton are often found to be defective in endocytosis (Munn, 2001). The evidence for actin cytoskeleton participating in endocytosis in mammalian cells has also been accumulating in recent years. Numerous studies have established the ability of dynamin to interact with various actin regulatory factors including profilin (Witke et al., 1998), and the actin-binding protein Abp1 (Kessels et al., 2001), as well as syndapin, intersectin, and cortactin, which link dynamin to the Wiskott Aldrich Syndrome protein (WASP) and the Arp2/3 complex, the major actin assembly promoters (Qualmann et al., 1999; McGavin et al., 2001; Schafer et al., 2002). Despite these findings, however, the exact function of dynamin in endocytosis remains unresolved. In addition to conventional dynamins, there are other proteins from the dynamin family that share high homology with dynamins in their N-terminal GTPase domain but show less or no sequence conservation in other regions. These dynamin-related proteins are generally also found to have functions distinct from dynamins. For example, one of the better-studied dynamin-like proteins, DLP1, is known to be required for organelle morphology in mammalian cells (Shin et al., 1997; Yoon et al., 1998; Imoto et al., 1998; Kamimoto et al., 1998; Smirnova et al., 1998; Sever et al., 1999). So far, there is no documentation yet to suggest that these dynaminlike proteins have an actin-related function similar to the conventional dynamins. There are three dynamin-like proteins in yeast that are structurally more related to DLP1 than to conventional Key words: GTPase, Dynamin, Actin, Vps1p, Sla1p [...]... undergoing conformational changes upon GTP hydrolysis Dynamin- interacting proteins including syndapin, intersectin and cortactin, serve as bridge molecules to connect actin cytoskeleton to the endocytosis via interacting with actin assembly activators such as WASP and Arp2/3 complex to modulate the actin organization at the endocytic sites In budding yeast Saccharomyces cerevisiae, interestingly, the. .. Las17p, Profilin, Abp1p, and Cortactin 36 1.13 A regulatory model of WASP protein 39 1.14 The organization of yeast actin cytoskeleton through the cell cycle 40 1.15 The yeast actin- associated proteins can be organized into three functional complexes 45 1.16 Schematic representation of the domain organization in yeast formin homologues Bni1p and Bnr1p 48 1.17 Summary of the protein- protein interactions... vesicles in different endocytic pathways will be further discussed in the following section 1.3.1 Dynamin and dynamin- related proteins Dynamin was originally identified as a microtubule-binding protein from bovine brain extracts (Obar et al., 1990;Shpetner and Vallee, 1989) Three related isoforms of dynamin, dynamin I, II, and III (referred to as dynamins in the following text) have been identified in mammals... domain (GED), and the COOH-terminal proline- and arginine-rich domain (PRD) found in these dynamins also share some sequence similarities (Fig 1.6A) In addition to the above conventional dynamin proteins, there is another group of proteins that has been identified as dynamin- related proteins (Table 1) Similarly to dynamins, they all have an NH2-terminal GTPase domain, a middle coiled-coil domain, and... important binding partner of Eps15 The central domain of Epsin associates with AP-2, and its COOH-terminal region interacts with Eps15 Epsin localizes to clathrin coats in vivo, and the loss of the Epsin functions results in a block in RME (Chen et al., 1998) These lines of evidence suggest that epsin, together with Eps15, is an accessory protein of the clathrin coats which assists the clathrin-mediated... protein sorting 145 7.1 Roles of Vps1p in the protein trafficking at the trans-Golgi network (TGN) 150 List of Tables xii LIST OF TABLES Table: 1 Dynamin- like proteins and their proposed functions 14 2 Yeast strains used in this study 72 3 Plasmids used in this study 74 4 The Budding Pattern Distribution in vps1 and wild type cells 97 5 Two-hybrid interaction between Sla1p and Vps1p domains 137 Yeast proteins... Intersectin, Amphiphysin, Endophilin Grb2, Nck (B) GTPase Middle GED Chapter I Introduction - 14 - Figure 1.6 Domain structures of dynamins and dynamin- like proteins (A) The domains and their respective functions of mammalian dynamin I (see text for details) The four white stripes in the GTPase domain, labeled G1~G4, are the conserved GTP-binding elements in dynamin I and are numbered according to its sequence... (Liu et al., 1996;Tuma and Collins, 1995) GTP hydrolysis domain (GTPase domain): The GTPase domain possesses the highest degree of sequence identity among dynamins The structural insights of this domain therefore can be provided by the solved crystal structure of the GTPase domain of dynamin A (Niemann et al., 2001) Dynamin A is one of the dynamin- related proteins from the lower eukaryote Dictyostelium... according to its sequence PH, pleckstrin homology domain; GED, GTPase effector domain; PRD, proline arginine rich domain (B) The domain structure of dynamin- like proteins 1.3.1.1 Domains and properties of dynamins Dynamins are different from the small GTPases in that they are large, multidomain proteins and have a higher GTPase activity (1-20 min-1) and lower affinity for GTP (10~100 µM) (Maeda et al.,... Yeast proteins with dual functions in actin organization and protein trafficking 153 6 Abbreviations ABBREVIATIONS a.a or aa amino acid Abp1p actin- binding protein 1 ADF actin depolymerizing factor ADF-H actin depolymerizing factor homologous region ADP adenosine 5’-diphosphate ALP alkaline phosphatase AMP adenosine 5’-monophosphate AMP-PNP 5’-adenylylimidodiphosphate ARK actin- regulating kinase ASH . through anchoring to the neck of membrane invaginations and undergoing conformational changes upon GTP hydrolysis. Dynamin- interacting proteins including syndapin, intersectin and cortactin, serve. representation of the domain organization in yeast formin homologues Bni1p and Bnr1p 48 1.17 Summary of the protein- protein interactions identified between dynamin and the components of actin cytoskeletal. FUNCTIONS OF THE DYNAMIN- LIKE PROTEIN VPS1 IN ACTIN ORGANIZATION IN SACCHAROMYCES CEREVISIAE YU XIANWEN INSTITUTE OF MOLECULAR AND CELL BIOLOGY