Regulation of Sla2p activity in endocytosis and actin organization by the Ark1/Prk1 family of kinases NEEYOR BOSE (B.Sc Hons) NATIONAL UNIVERSITY OF SINGAPORE i ACKNOWLEDGEMENTS “To go through the hardest journey, we need only take one step at a time, but we must keep on stepping”- Chinese proverb At each of my steps, there have been those who’ve helped me, without whom there would be no next step. My heartfelt gratitude goes out to you all. To Asst/P Yeong Foong May and A/P Uttam Surrana: Thank you for rescuing me during a very difficult time and helping me get this far. Dr Yeong, I am deeply grateful for your generosity of time and effort that made this thesis happen. To A/P Cai Ming Jie for giving me the opportunity to pursue my graduate studies. To the former members of CMJ lab: for creating a nurturing and collaborative environment. We began as colleagues but now I am lucky to have you as friends. Thank you all for your support and kindness over the years. To my committee members A/P Edward Manser and A/P Wang Yue: Thank you for nudging me on to the right track and the annual reality checks. To my dear friends Desmond, Karen, Bin, Shal, Bea, Mann, Chris, Ajay, Xian Wen: for always listening, caring and letting me mess up your shoulders with my tears and pointing me towards the direction of the proverbial light at the end of the tunnel. It seems most inadequate to say that I couldn’t have done it without you. To Ma and Baba: for never letting “home” be too far away. Thank you for enveloping me with a sense of security and warmth. I always knew I was very lucky, I now realize how much. To Runtoo: for caring so very much. It was the pep talk that really drove it home. To Vinod: for bringing me back to life. Thank you for stepping with me, for picking me up at every stumble and for never letting me go alone. Above all, I thank God- for giving me so much to be thankful for. Neeyor Bose, August 2008 ii TABLE OF CONTENTS ACKNOWLEDGEMENT .ii TABLE OF CONTENTS .iv SUMMARY .vi LIST OF FIGURES viii LIST OF TABLES ix LIST OF ABBREVIATIONS sx 1. INTRODUCTION .2 1.1ACTIN CYTOSKELETON 1.1.1 Actin cytoskeleton in yeast 1.1.2 Assembly of actin at cortical patches .5 1.1.2.1 Arp2/3 complex . 1.1.2.2 Nucleation promoting factors . 1.1.3 Components of actin patches and proteins associated with them 11 1.1.4 Dynamics of actin patches and their associated proteins .12 1.1.5 Other actin structures in yeast 15 1.1.5.1 Actin cables .15 1.1.5.2 Acto-myosin ring 16 1.2 ENDOCYTOSIS .17 1.2.1 Clathrin mediated endocytosis 17 1.2.2 Endocytic Coat Proteins 20 1.2.2.1 Clathrin 20 1.2.2.2 Sla1p 20 1.2.2.3 Pan1p .21 1.2.2.4 yAP1801 and yAP1802 22 1.2.2.5 Ent1p and Ent2p 22 1.2.2.6 Scd5p .23 1.2.2.7 Sla2p/HIP1/R 23 1.2.2.7.1 Sla2p 23 1.2.2.7.2 HIP1 and HIP1R .27 1.2.2.8 Vesicle Scission 30 1.2.2.9 Proteins regulating Clathrin-mediated endocytosis 33 1.2.3 Endocytic Signals .36 1.2.4 Role of actin dynamics in endocytosis 37 1.3 ACTIN AND ENDOCYTOSIS IN HIGHER EUKARYOTES .39 1.3.1 Clathrin-mediated endocytosis in higher eukaryotes 40 1.3.2 Caveolae-mediated endocytosis 42 1.3.3 Macropinocytosis .43 1.3.4 Phagocytosis 43 1.4 OBJECTIVES .45 2. MATERIALS AND METHODS 46 2.1 MATERIALS .47 2.1.1 Strains .47 2.1.2 Plasmids .48 2.1.3 Antibodies and Reagents .52 ANTIBODIES 52 SOURCE .52 2.2 CULTURE CONDITIONS 53 2.3 RECOMBINANT DNA TECHNOLOGY 53 2.3.1 DNA transformation of E.coli cells 54 2.3.2 Plasmid DNA preparation .55 2.3.3 Site-directed mutagenesis 55 2.4 YEAST G ENETIC MANIPULATIONS .56 2.4.1 Yeast transformation and integration .56 2.4.2 Gene disruptions and manipulation 57 iii 2.5 YEAST CELL BIOLOGY PROTOCOLS .57 2.5.1 Yeast two-hybrid assay 57 2.5.2 Endocytosis assays .58 2.5.2.1 Lucifer Yellow uptake 58 2.5.2.2 FM4-64 uptake and pulse-chase assay 58 2.5.2.3 Fur4p uptake assay 59 2.5.3 Drop test .60 2.6 FLUORESCENT MICROSCOPY 60 2.6.1 Fixing and staining with rhodamine phalloidin .60 2.6.1 Live-cell, time lapse microscopy .60 2.7 PROTEIN ANALYSIS 61 2.7.1 Crude yeast protein extract .61 2.7.2 Immunoprecipitation and western blot .62 2.7.3 Purification of GST- and His-tagged proteins and in vitro binding assays .63 2.7.4 GST pull down assay .64 2.7.5 In vitro kinase assay 65 2.7.6 In vitro phosphatase assay 65 3. PHOSPHOREGULATION OF SLA2P 67 3.1 BACKGROUND .68 3.2 SLA2P IS A SUBSTRATE OF PRK1P KINASE .69 3.2.1 Identification of Prk1p phosphorylation through p sequence analysis 69 3.2.2 Sla2p is a substrate of Prk1p in vitro .71 3.2.3 Sla2p is a phosphorylated protein in vivo and is likely to be exclusively phosphorylated by the Ark1/Prk1 family of kinases 71 3.3 PHOSPHORYLATION OF SLA2 P AFFECTS ITS FUNCTION AT THE CELL CORTEX .72 3.3.1 Localization and patch dynamics of Sla2p is affected by phosphorylation .72 3.3.2 Localization and patch dynamics of other endocytic proteins are affected by phosphorylation of Sla2p 74 3.3.3 Sla2p phospho-status affects receptor-mediated but not fluid-phase endocytosis 82 3.3.4 Sla2p’s phosphorylation affects its interaction with some of interacting partners but not others 83 3.4 SLA2P INTERACTS WITH SCD5 P AND CAN BE DEPHOSPHORYLATED BY THE PROTEIN PHOSPHATASE1, GLC7 P 88 3.4.1 Sla2p and Scd5p interact with each other using their C-terminal and N-terminal regions respectively 89 3.4.2 Glc7p can dephosphorylate Sla2p, both in vitro as well as in vivo 89 3.5 DISCUSSION 92 3.6 FUTURE WORK AND PERSPECTIVES 97 4. SECOND COILED-COIL DOMAIN OF SLA2P PLAYS A VITAL ROLE IN ITS FUNCTION . 100 4.1 BACKGROUND 101 4.2 SLA2P C-TERMINAL COILED-COIL DOMAIN IS IMPORTANT FOR ITS FUNCTION . 102 4.2.1 Deletion of second coiled-coil domain (amino acid 700-730) causes temperature sensitivity . 102 4.2.2 Localization and dynamics of C-terminal truncation mutants are different from wild-type Sla2p 105 4.2.3 Actin organization and endocytosis is aberrant in some of the C-terminal deletion mutants . 105 4.3 SLA2P C-TERMINAL COILED-COIL MEDIATES INTERACTION WITH ARK1P AND SCD5P AND AFFECTS THEIR LOCALIZATION . 110 4.3.1 Sla2p and Ark1p interaction domain analysis 110 4.3.2 Sla2p and Scd5p interaction domain analysis 114 4.3.3 Sla2p C-terminal affects localization and dynamics of Ark1p . 116 4.3.4 Localization and dynamics of Scd5p in sla2 mutants . 116 4.4 DISCUSSION . 117 4.4.1 Relevance of the Sla2p C-terminal region 117 4.4.2 Phenotype of sla2
CC mutant . 119 4.4.3 Sla2p’s second coiled-coil domain is essential for interaction with Ark1p and Scd5p 123 iv 4.5 FUTURE WORK AND PERSPECTIVES . 124 5. SLA2P AND ABP1P 127 5.1 BACKGROUND 128 5.2 HIGH COPY SUPPRESSOR SCREEN OF SLA2
CC MUTANT 128 5.2.1 Identification and verification of suppressors 128 5.3 ABP1 IDENTIFIED AS A HIGH COPY SUPPRESSOR OF THE SLA2
CC MUTANT . 131 5.3.1 High concentration of ABP1 can suppress the growth defects of the sla2
CC mutant . 131 5.3.2 High concentration of ABP1 can rescue actin defects of the sla2
CC mutant 131 5.2.3 High concentration of ABP1 can rescue endocytic defects of the sla2
CC mutant 134 5.3.4 Physical interaction between Sla2p and Abp1p 137 5.4 DISCUSSION . 139 5.4.1 Over-expression suppression screen . 139 5.4.2 ABP1 is an extragenic high-copy suppressor of the sla2
CC mutant 140 5.5 FUTURE WORK AND PERSPECTIVES . 142 6. CONCLUSIONS 143 v SUMMARY The actin cytoskeleton plays a central role in endocytosis in Saccharomyces cerevisiae. Sla2p is an interesting adaptor molecule that is involved in both these processes and is a vital link between them. Deletion of this protein causes massive accumulation of actin at the cortex as well as a complete block in endocytosis. Sla2p negatively regulates actin polymerization by inhibiting Pan1p, an activator of the Arp2/3 nucleator. However, the regulation of this process is poorly understood. In this study I show that Sla2p is subjected to phosphorylation by Prk1p and possibly Ark1p kinase. These kinases constitute a unique family of actin regulating kinase whose substrates include many components of actin machinery as well as endocytic and membrane trafficking pathways. The phosphorylation on Sla2p exerts subtle regulatory effects leading to longer lifespan at the cortex and endocytic defects and also changes Sla2p’s affinity for Scd5p and Pan1p. I also show that Sla2p can be dephosphorylated by the Scd5p/Glc7p complex, in a manner similar to what is reported for Pan1p. I postulate that this regulatory cycle is important for function of Sla2p in endocytosis and actin organization. Secondly, I studied the C-terminal portion of Sla2p, which contains two additional coiled-coil domains along with an F-actin binding THATCH domain. I found that the C-terminal region of Sla2p is important for binding Ark1p and Scd5p, while the second coiled-coil domain starting from 700 till 730 amino acids is necessary for both these interactions. Deletion of this short domain causes severe temperature sensitivity, actin aberrations and a complete block in endocytosis. I also made a series of truncations in the C-terminal region of Sla2p and studied the effects of such mutations. Different truncations gave different phenotypes while the strongest phenotype was obtained by deleting just the 30 amino acids constituting the coiled- vi coil domain (sla2
CC mutant). I hypothesize that this particular allele is toxic because the 30 amino acids contains a part of the upstream helical domain that regulates binding to F-actin. Thirdly, I performed a high-copy suppressor screening and found that over expressing Abp1p can rescue the temperature sensitivity of sla2
CC. In addition to growth, overexpressing Abp1p can also rescue actin aberrations such as actin clumps and bars seen in the mutant. Receptor-mediated and fluid-phase endocytosis are also significantly improved in cells containing high-copy number plasmid with ABP1 but not in mutant cells containing empty plasmid. No deleterious effects were observed upon overexpression of Abp1p in wild-type cells. vii LIST OF FIGURES Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 Figure 1.7 Figure 1.8 Figure 1.9 Figure 1.10 Figure 1.11 Figure 2.1 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 3.8 Figure 3.9 Figure 3.10 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 5.1 Figure 5.2 Figure 5.3 Schematic of actin polymerization Yeast cytoskeleton through cell cycle Structure and function of Arp2/3 complex Domain organization of nucleation-promoting factors in yeast Endocytic patch maturation and sequence of coat-protein assembly Types of endocytosis in yeast and mammalian cells Complex between Pan1p, Sla1p, End3p and Scd5p Schematic diagram representing Sla2p domains Domain architecture of HIP1 andHIP1R Structure of Prk1p kinase Types of endocytosis in higher eukaryotes Schematic of site-directed mutagenesis Sla2p is a substrate of Prk1 both in vitro and in vivo Lifespan of Sla2p depends on phosphorylation Lifespan of Abp1p depends on phosphorylation state of Sla2p Lifespan of Pan1p depends on phosphorylation state of Sla2p Lifespan of Sla1p depends on phosphorylation state of Sla2p Lifespan of Ark1p depends on phosphorylation state of Sla2p Effect of Sla2p phosphorylation on endocytosis and actin organization Effect of Sla2p phosphorylation on interaction with binding partners Sla2p can be dephosphorylated by Glc7p both in vitro and in vivo Proposed model explaining effects of phosphorylation and dephosphorylation of Sla2p Coiled-coil domains of Sla2p Growth and localization of Sla2p C-terminal truncation mutants Effect of Sla2p C-terminal truncation on endocytosis and actin organization Localization and dynamics of Ark1p in Sla2p mutants Interaction between Scd5p and Sla2p Instrasteric inhibition of actin binding by the upstream helical domain I/LWEQ superfamily Overexpression of Abp1p rescues growth defects of sla2DCC Overexpression of Abp1p rescues endocytic defects of sla2DCC Interaction between Abp1p and Sla2p viii 15 19 21 25 27 34 40 56 70 77 78 79 80 81 85 87 91 96 101 104 108 113 115 121 122 133 136 138 LIST OF TABLES Table 2.1 Table 2.2 Table 2.3 Table 5.1 List of Strains List of Plasmids List of Antibodies Result of high-copy suppressor screen ix 47 49 52 130 LIST OF ABBREVIATIONS a.a. or aa amino acid Abp1p actin-binding protein ADF actin depolymerizing factor ADF-H actin depolymerizing factor homologous region ADP adenosine 5’-diphosphate ALP alkaline phosphatase AMP adenosine 5’-monophosphate ANTH AP180 N-terminal Homology ARK actin-regulating kinase ATP adenosine 5'-triphosphate AP-1/2/3 adaptor protein-1/2/3 BAR BIN-amphiphysin-RVS domain bp base pair BSA bovine serum albumin °C degree Celsius CALM clathrin assembly lymphoid myeloid leukaemia protein cAMP cyclic AMP CBM Clathrin binding motif CC coiled coil CCP clathrin-coated pit CCV clathrin-coated vesicle CFP cyan fluorescent protein CIP calf intestinal phosphatase CME clathrin-mediated endocytosis C-terminus carboxy-terminus CR3 complement receptor C3 cyto D cytochalasin D DKO Double knock out DMSO Dimethyl Sulfoxide x CHAPTER V Sla2p & Abp1p 78. 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Genetics 153, 35-47. 166 [...]... filaments of actin The formation of these actin patches is credited to the actin- polymerization activity of actin nucleators The single most important nucleator in the context of the actin patch is the Arp2/3 complex, which localizes to the patch and is the sole nucleator implicated in their formation (Moreau et al 1996, Winter et al 1997 and 1999) Other actin- nucleators such as the formins - Bni1p and Bnr1p-... NPF is the presence of the acidic domain that facilitates interaction with the complex, most likely via Arp3p or ARPC1 All these proteins bind to either F- or G -actin and have varying dependence on actin binding for their Arp2/3 activator function Las17p binds to G -actin while Myo3p, Myo5p, Pan1p and Abp1p bind to actin filaments The actin binding ability is necessary for function in the case of Pan1p,... portion of Las17p folds into a “WA domain” consisting of the actin binding WH2 motif and the Arp2/3 binding acidic sequence (Figure 1.4) This domain was found to have very potent NPF activity (Moseley and Goode, 2006) 7 CHAPTER I INTRODUCTION Figure 1.4 Domain organizations of various nucleation-promoting factors in yeast a) The five NPFs in yeast and their domain structures The actin binding domains are... coating the actin cables, and bind actin filaments together to form a cable The exact mechanism and the regulation of their activities are not known The yeast capping proteins Cap1/2 bind to barbed ends of filaments, preventing assembly or disassembly of the filament, thereby stabilizing the cable size The exact mechanism of actin cable dynamics, size and positioning are not known 1.1.5.2 Acto-myosin... INTRODUCTION Figure1.2 Yeast actin cytoskeleton through the cell cycle The organization of the yeast actin cytoskeleton changes with the cell cycle The actin patches gathering at the presumptive bud site mark the initiation of budding As the bug grows, actin patches preferentially localized at the small bud and few are seen in the mother cell Actin cables run along the motherbud axis As the bud reaches it optimal... one of the first actin binding proteins discovered in yeast (Drubin et al, 1988) It is tightly associated with actin patches and it is arrives at the endocytic complex along with Myo3p and Myo5p and its presence signifies the approach of the actin machinery to the endocytic machinery Upon the onset of the rapid patch movement, type I myosins dissociate, but Abp1p remains attached to the vesicle and. .. described that the actin in yeast manifested in three primary structures: actin patches at cell cortex, actin cables running along the long axis of the cell, and the acto-myosin ring at the bud neck (Moseley and Goode, 2006) Since then, the interest in yeast cytoskeleton has scarcely abated Subsequent studies showed that the distribution of these various actin structures is 3 CHAPTER I INTRODUCTION Figure1.2... of proteins is characterized by the presence of an N-terminal lipid binding motif known as the ANTH domain (AP180 N-terminal homology), which is structurally similar to the ENTH domain found in epsins and their homologues The yeast AP180 proteins also contain 5 EH domain-binding NPF (asparagine-prolinephenylalanine) motifs and a clathrin-binding motif (CBM) These proteins are found to localize to endocytic... burst of actin polymerization, bending the membrane Once the vesicle has left the cortex, it begins rapid, inward movement along actin cables, which guide the endocytic vesicles to the early endosomes The proteins still associated with the vesicle at this point are Abp1p, Arp2/3 complex, capping proteins and Sac6p, an actin bundling protein (Moseley and Goode, 2006) The various steps of the maturation of. .. detaching from the plasma membrane How the inhibition by Sla2p is overcome is yet to be determined (iii) Myo3p and Myo5p: are the yeast type-1 myosins, which function both as actin independent motor molecules as well as NPFs These molecules comprise of an Nterminal motor domain, a lipid-binding TH1 domain, an F -actin binding TH2 domain, an SH3 domain and an Arp2/3 binding acidic motif (Moseley and Goode, . these various actin structures is Figure 1.1 Schematic of actin polymerization Actin monomers bind to profilin and are attached to the barbed end of the growing actin filament. Actin has very. patches Actin patches are known to contain short, highly branched filaments of actin. The formation of these actin patches is credited to the actin- polymerization activity of actin nucleators. The. i Regulation of Sla2p activity in endocytosis and actin organization by the Ark1/ Prk1 family of kinases