PHOSPHOREGULATION OF THE ACTIN-DRIVEN ENDOCYTOSIS IN THE YEAST SACCHAROMYCES CEREVISIAE HUANG BO B.Sc. (Hons, 1st), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I would like to thank my supervisor, A/P. Cai Mingjie, for the patient guidance, encouragement and advice he has provided throughout my time as his student. I have been extremely lucky to have a supervisor who cared so much about my work, and who responded to my questions and queries so promptly. I am also deeply indebted to Dr. Zeng Guisheng, my mentor, the greatest source of encouragement and support both professionally and personally during this endeavor. Not only did he spearhead the effort to help me from day one in the lab, he presented me with every possible assistance and advice in joint projects or projects that I have pursued on my own. In particular, the phenotypic analysis of scd5-1 mutant, in vitro phosphatase assays and live cell imaging data were largely contributed by him. His vast knowledge and sound advice were invaluable in guiding every aspect of my graduate work and were essential in completing this thesis. I must express my gratitude to my supervisory committee members: A/P Edward Manser and Dr. Tang Bor Luen for their generously spending invaluable time in assessing my progress, providing advice and suggestions. I would also like to thank the past and present members in CMJ labs, in particular, Dr. Yu Xianwen for sharing her knowledge and expertise and Dr. Wang Junxia for analyzing the in vivo phosphorylation status of Scd5p. I am also obliged to Ms Neo Suat Peng for her contribution in the in vitro binding assays between Pan1p and Scd5p and an IMCB colleague Mr. Alvin Ng for his excellent job in structural modeling. Ms. Neeyor Bose, Ms. Jin Mingji, Mr. Qiu Wenjie - my peer graduate students were all acknowledged for their moral support and friendship over the years. I am also grateful to Ms. Wang Jun and Ms. Chua Lingling for their superb technical support. I would like to thank IMCB not only for providing the funding and facilities which allowed me to undertake this research, but also for giving me the opportunity to attend conferences. Finally, this work would not have been possible without the constant support and encouragement of my wife, my parents and my brother who experienced all of the ups and downs of my research. Thank God for being my source of strength and inspiration. Huang Bo Jan, 2007 ii TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………… .ii TABLE OF CONTENTS…………………………………………………………… iii LIST OF FIGURES………………………………………………………………… vi LIST OF TABLES………………………………………………………………viii ABBREVIATIONS…………………………………………………………………ix SUMMARY……………………………………………………………………….…xii CHAPTER 1.1 1.2 1.3 1.4 Introduction………………………………………………………… Endocytosis from mammals to yeast……… ……………………… A. Endocytosis in mammals………………………………………… B. Endocytosis in yeast……………………………………………….3 Actin cytoskeleton and endocytosis………………………………………5 A. Evidence of actin’s involvement in endocytosis…………….…….5 B. Actin and endocytosis in mammals……………………………… C. Actin and endocytosis in yeast…………………………………….6 How does actin drive endocytosis in yeast?………………………….….9 A. Yeast endocytic pathway………………………… ………………9 B. Coupling and uncoupling the actin engine with the endocytic coat………………………….……………………………………11 Earlier discoveries made in our lab……………………………………12 CHAPTER Materials and Methods .…………………………………………14 2.1 Materials……………………………………………………………… .15 2.2 Strains and growth conditions………………………………………… .15 2.3 Recombinant DNA methods…………………………………………….18 A. Plasmid DNA preparation and analysis……………………… 18 B. Site-directed mutagenesis………………………………… .18 2.4 Plasmid constructions…………………………………………………19 2.5 Yeast manipulations…………………………………………………… 24 A. Gene disruption and integration .24 B. Two-hybrid assays………………………………………… ……25 C. Endocytosis assays…………………………………… .26 2.6 Microscope imaging…………………………………………………….26 A. Rhodamine-phalloidin staining of actin filaments……………….26 B. Live cell imaging…………………………………………… ….27 2.7 Biochemical assays…………………………………………………… 27 A. Yeast extract preparation………………………………… 27 B. Immunoprecipitation, TCA precipitation and Western blot…… .28 C. In vitro protein-binding assay……………………………………29 D. In vitro kinase and phosphatase assays………………………… 30 2.8 Structural modeling……………………………………… .……………31 iii CHAPTER 3.1 Characterization of the Substrate Specificities for Prk1p-Family Kinases……………………………………… .33 Introduction…………………………………………………………… .34 3.2 Results……………………………………………………………… .35 A. Hydrophobic residues at P-5 are required for Prk1p recognition…………………………………………………… …35 B. Identification of N, T and S as additional P-2 residues………… 39 C. Prk1 targets threonine but not serine residues … ………………44 D. Prk1p tolerates alterations in amino acids at P-2……………… .45 E. Determining the substrate specificities of Prk1p analogues: Ark1p and Akl1p………………………………………………48 F. Structural modeling of the Prk1p kinase domain……………… .53 G. Validation of the structural model of the Prk1p kinase domain.59 3.3 Discussion…………………………………… .62 A. Characterization of substrate specificity for Prk1p….….………62 B. Similarities and differences among Prk1p family kinases.…… 64 C. Autophosphorylation of Prk1p and Akl1p……………….………65 CHAPTER 4.1 Prk1p Regulates Type I Phosphatase Targeting Factor Scd5p by Phosphorylation……………………………………………… .68 Introduction……………………………………………………………69 4.2 Results…………………………………………………………………70 A. Prk1p phosphorylates Scd5p…………………………………….70 B. Suppression of the scd5-1 mutant by prk1Δ .73 C. Constitutive dephosphorylation of Scd5p is unable to suppress scd5-1 mutant…………………………………………………78 4.3 Discussion……………………………………………………………….80 A. Putative substrates of Prk1p……………….……………………80 B. Scd5p is a regulatory target of Prk1p in vivo…………………….88 CHAPTER Scd5p is the Switch to Initiate Dephosphorylation of Pan1p… 90 5.1 Introduction……………………………………………………………91 5.2 Results…………………………… …………………………………….93 A. Scd5p interacts with Pan1p genetically and physically………….93 B. Scd5p patch coincides with Pan1p patch spatiotemporally…… .95 C. Scd5p can not bind to phosphorylated Pan1p…………… .…….95 D. Scd5p binds to End3p-Pan1p complex………………………… .98 E. Glc7p-Scd5p-End3p mediates dephosphorylation of Pan1p in vivo……………………………………….………………… 101 F. Glc7p is the upstream phosphatase for Pan1p and Scd5p………105 G. Glc7p dephosphorylates Pan1p and Scd5p in vitro…………… 110 H. Phosphoregulation of Scd5p by Prk1p and Glc7p…………… .113 iv I. Steady-state phosphorylation level correlates with patch life-time of Pan1p…………………………………………………………115 5.3 Discussion……………………………………………………………119 A. Working model for the phosphoregulation of Pan1p during endocytosis…………………………………………………… .120 B. In vitro reconstitution of the phospho-regulation of Pan1p by Glc7p……………………………………………………………123 C. Dephosphorylation of other phospho-endocytic proteins………125 D. Alternative dephosphorylation pathway……………………… .126 E. Possible role of Glc7p-related phosphatases in endocytosis……126 F. Phosphorylation status of Pan1p may affect its stability………127 CHAPTER Overview…… ………………………………………….… .130 REFERENCES…………………………………………………………………….133 PUBLICATIONS….……………………………………………………………… 142 v LIST OF FIGURES Fig. 1-1 Actin-implicated processes in yeast and mammalian cells. Fig. 1-2 Role of actin cytoskeleton in yeast endocytic internalization. Fig. 1-3 The sequential assembly of proteins at endocytic sites. 10 Fig. 2-1 Schematic diagram of site-directed mutagenesis by overlap extension. 19 Fig. 3-1 Distribution of QxTG motifs in Pan1p and Sla1p. 36 Fig. 3-2 Analysis of sequence requirement at P-5 for Prk1p recognition. 37 Fig. 3-3 The requirement for leucine at P-5 for Prk1p recognition. 38 Fig. 3-4 Identification of N, T and S as additional P-2 residues for Prk1p recognition. 40 Fig. 3-5 Comparison of phosphorylation [L/I/V/M]xx[Q/N/T/S]xTG motifs. 43 Fig. 3-6 Prk1p was unable to phosphorylate LxxQxSG. 44 Fig. 3-7 Distribution of [L/I/V/M]xxxxTG motifs in Pan1p and Sla1p. 46 Fig. 3-8 Hydrophobic residues at P-2 support Prk1p phosphorylation. 47 Fig. 3-9 Aspartic acid at P-2 does not interfere with Prk1p recognition. 48 Fig. 3-10 Homology among the kinase domains of the Prk1 family kinases of budding yeast and the human orthologue adaptorassociated kinase AAK1. 48 Fig. 3-11 Ark1p does not phosphorylate Pan1p motifs in LR1 and LR2. 51 Fig. 3-12 Akl1p shares the same phosphorylation site preference with Prk1p. 52 Fig. 3-13 Interactions between Prk1p and its recognition motifs depicted by homology based modeling. 54 Fig. 3-14 Structural modeling in agreement with biochemical assays. 57 Fig. 3-15 Ile117 is the most critical residue in P-5 binding pocket. 61 Fig. 3-16 Prk1p and Akl1p, but not Ark1p, directly phosphorylate the LxxTxTG motif in vitro. Prk1p and Akl1p may phosphorylate themselves or each other. 65 Fig. 3-17 vi efficiency of the 67 Fig. 4-1 Identification of Scd5p as a new phosphorylation target of Prk1p. 72 Fig. 4-2 Suppression of the scd5-1 mutation by prk1Δ. 74 Fig. 4-3 Constitutively unphosphorylated scd5-1 is still TS. 79 Fig. 4-4 Verified or potential substrates of Prk1p in yeast proteome. 84 Fig. 5-1 Interactions of Scd5p with Pan1p. 94 Fig. 5-2 Live cell imaging of Pan1p, Scd5p and Prk1p patches. 96 Fig. 5-3 Scd5p cannot bind to phosphorylated Pan1p. 97 Fig. 5-4 Scd5p interacts with End3p genetically and physically. 100 Fig. 5-5 In vivo phosphorylation level of Pan1p is regulated by Prk1p, Scd5p and End3p. 103 Fig. 5-6 Phosphorylation level of Pan1p in end3 truncation mutants. 105 Fig. 5-7 Glc7p is the upstream phosphatase for Pan1p in vivo. 107 Fig. 5-8 Glc7p is the upstream phosphatase for Scd5p in vivo. 109 Fig. 5-9 Glc7p dephosphorylates phospho-LR1-Pan1p in vitro. 111 Fig. 5-10 Glc7p dephosphorylates phospho-LR2-Pan1p in vitro. 112 Fig. 5-11 Glc7p dephosphorylates phospho-Scd5p in vitro. 112 Fig. 5-12 Effects of Scd5p phosphorylation by Prk1p on protein-protein interactions. 114 Fig. 5-13 The effect of SCD5AAA mutation on Prk1p-induced Pan1p phosphorylation. 115 Fig. 5-14 Dephosphorylation increases Pan1p patch lifetime significantly. 117 Fig. 5-15 Live cell imaging of Pan1p, Abp1p and Prk1p patches. 120 Fig. 5-16 Model of the dynamic assembly and disassembly of the actindriven endocytic machinery with an emphasis on phosphoregulation of Pan1p by Prk1p and Glc7p. 124 Fig. 5-17 Hyperphosphorylation induces rapid degradation of Pan1p. 129 vii LIST OF TABLES Table Yeast strains used in this study 16 Table Plasmids used in this study 20 viii ABBREVIATIONS a.a. amino acid ABS actin binding sequence AP180 clathrin adaptor protein 180 ATP adenosine 5'-triphosphate bp base pair BSA bovine serum albumin °C degree Celsius CFP cyan fluorescent protein CH calponin homology domain CIP calf intestinal phosphatase C-terminus carboxy-terminus degron degradation signal DHFR dihydrofolate reductase DNA deoxyribonucleic acid DTT dithiothreitol ECL enhanced chemiluminescence E. coli Escherichia coli EDTA ethylenediamine tetraacetic acid EGTA ethylene-bisoxyethylenenitrilo tetraacetic acid EH Eps15 homology EM electron microscopy F-actin filamentous actin G6PDH glucose-6-phosphate dehydrogenase G-actin globular actin GFP green fluorescent protein GST glutathione S-transferase ix h hour HA haemagglutinin HEPES hydroxyethylpiperazine ethanesulfonic acid HIP1R Huntingtin-interacting protein 1R HRP horseradish peroxidase IgG immunoglobulin G IP immunoprecipitation IPTG isopropyl-1-thio-β-D-galactopyranoside kb kilobase(s) Kd kilodalton KD kinase domain LB Luria-Bertani medium LR1 N-terminal first long repeat of Pan1p LR2 N-terminal second long repeat of Pan1p LY Lucifer Yellow M Molar MF Mutagenic forward primer μg Microgram Minute ml milliliter μl Microliter mM Millimolar MOPS morpholinepropanesulfonic acid MR Mutagenic reverse primer nm Nanometer NPF nucleation promoting factor NPF motif Asp-Pro-Phe tripeptide N-td td being mutated to WT N-terminus amino-terminus OD optical density ONPG o-nitrophenyl β-D-galactopyranoside x 128 Chapter with an elevation in protein phosphorylation status in these two mutants in comparison to WT cells. Shown in Fig. 5-17, total cell lysates for immunoprecipitation were normalized by housekeeping protein G6PDH. At permissive temperature, scd5-1 and end3Δ mutants express only a 23% and 17% of full-length Pan1p level in WT. Upon shifting to 37oC, further loss of full-length Pan1p protein to a mere 2% of WT level was observed for both mutants. In order to detect the protein band, 5-fold more concentrated lysates [visualized by α-G6PDH loading] were used for the immunoprecipitation of Pan1p-myc in these two mutants than WT cells. In contrast, temperature shift had little effect on Pan1p level in WT cells. Likewise in end3 truncation mutants [Fig. 5-6] and PRK1-overexpressing cells [Fig. 5-13], Pan1p expression level apparently reversely correlates with the extent of hyperphosphorylation. All these data suggest that hyperphosphorylation due to unchecked phosphoregulation led to rapid turnover of Pan1p. However the mechanism of this hyperphosphorylation-induced protein degradation is unclear. Conversely, Pan1p protein level markedly increased when overexpressing Glc7p or its two targeting factors End3p and Scd5p [data now shown]. Shown in Fig. 5-14, Pan1p is protected from being phosphorylated in these cells, suggesting that hypo-phosphorylated Pan1p seems to be protected from degradation. However, not all data agree with this assumption. Full-length Pan1p expression level was slightly reduced in prk1Δ and sla1Δ mutant in comparison with WT cells [data now shown], although the Pan1p phosphorylation status has dropped to 15 % of the level of WT in both mutants [Fig. 5-5 and data not shown]. Chapter Fig 129 5-17 Hyperphosphorylation induces rapid degradation of Pan1p. Endogenously expressed Pan1-Myc was immunoprecipitated from cells and sequentially immunoblotted with anti-PThr and anti-Myc antibodies. The full-length Pan1p-myc level of each lane was measured by densitometer and normalized against its G6PDH loading control. The relative protein expression level were then calculated and presented as bar graphs [right panels]. A. Pan1-Myc from YMC441 [lanes and 3, 5-fold dilution], and YMC479 [lanes and 4] cells at either 25°C [lanes 1-2] or 37°C for h [lanes 3-4]. B. Pan1-Myc from YMC441 [lanes and 3, 5-fold dilution] and YMC480 [lanes and 4] cells at either 25°C [lanes 1-2] or 37°C for h [lanes 3-4]. Chapters Overview Chapter 131 Here is a summary of my thesis work described in Chapters to 5. This thesis work has been carried out in the context of endocytic regulation. Referring to Fig. 3-16, an endocytic process is initiated by coat proteins that are responsible for selecting endocytic sites and stimulating clathrin assembly. Actin polymerization is subsequently launched and coupled with the invaginating vesicle. Shortly after the nascent vesicle is formed by scission, actin polymerization activity is disengaged from the vesicle and eventually shut off. Proteinaceous vesicle coat then has to be disintegrated and removed from the vesicle to enable its fusion with the endosome. Work from our lab (Zeng & Cai, 1999; Zeng et al., 2001) and others (Toshima et al., 2005) has corroborated an important role of the threonine kinase Prk1p in the disassembly of the vesicle coat. Prk1p was shown to phosphorylate coat proteins Pan1p and Sla1p on threonine residues lying in the motif LxxQxTG and promote the disassembly of Pan1p-Sla1p-End3p trimeric complex (Zeng & Cai, 1999; Zeng et al., 2001). In Chapter 3, I first explored the limitation of the original Prk1p phosphorylation consensus motif LxxQxTG (Zeng & Cai, 1999). Efforts using the directed mutagenesis approach and in vitro kinase assays were made to determine the amino acid requirement at P-5 and P-2 positions respectively. Thus the minimal phosphorylation motif for Prk1p was established as [L/I/V/M]xxxxTG. In Chapter 4, I then searched the yeast proteome for putative Prk1p substrates containing the newly defined Prk1p target sequence. Not only new sites were found in known substrates, but also numerous novel substrates were predicted. Furthermore, endocytic protein Scd5p was selected and tested to be a true Prk1p target in vivo. Chapter 132 In Chapter 5, I went on to demonstrate that Scd5p is the key to one of the most long-standing questions in the field of endocytic regulation. What was of great interest and yet of little knowledge is that how the endocytic factors including Pan1p and Sla1p can be relieved from phospho-inhibition inflicted by the kinase Prk1p. Scd5p, forming a complex with type I phosphatase [encoded by GLC7], is able to bridge the gap between phosphatase activity to the phosphorylated Pan1p. Two targeting routes for the Glc7p-Scd5p complex to Pan1p were uncovered. 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PUBLICATIONS Publications 143 Huang, B., et al. (2003). Identification of novel recognition motifs and regulatory targets for the yeast actin-regulating kinase Prk1p. Mol Biol Cell, 14(12), 4871-4884. Huang, B., and Cai M. (2007). Pan1p: an actin director of endocytosis in yeast. Int J Biochem Cell Biol. [in press]. [...]... are also present in the budding yeast Studying endocytosis in yeast with powerful molecular genetics technique has made one of the many groundbreaking discoveries - the important role of actin cytoskeleton in the endocytic process The first line of evidence came from the observation that the internalization step of the endocytosis of mating pheromone α-factor was defective in an actin mutant [act1-1]... the regulatory complexity of endocytosis By far, the best understood mechanism of turning off actin polymerization machinery at the nascent vesicle is phosphorylation by Prk1p family kinases One of the phosphorylation targets is the multivalent protein Pan1p, which is responsible for organizing the vesicle coat, tethering the coat to the actin meshwork by physically binding to F -actin, and activating... endocytic machinery An important advance in understanding the specific role of actin Fig 1-2 Role of actin cytoskeleton in yeast endocytic internalization This schematic diagram illustrates putative functions of different actin- cytoskeleton proteins during endocytic internalization in S cerevisiae Las17p, the yeast homology of Wiskott–Aldrich syndrome protein [WASP] together with the myosins Myo3p and... leads to the formation of a cone of crosslinked actin filaments tethered to the endocytic coat This growing actin network cooperates with myosin motor activity (Sun et al., 2006) to pull the attached coat inwards and invaginates the underlying membrane [Fig 1-2] As the vesicle coat continues growing, the amphiphysins Rvs161p and Rvs167p are recruited to the endocytic site for the release of the forming... filaments are further crosslinked by Sac6p [fimbrin] The crosslinked actin network is linked to the underlying vesicle coat by actin- binding proteins such as Sla2p and Pan1p, which are represented by green hand-like structures The growth of the actin network leads to the invagination of the coated membrane [Figure reproduced with permission from (Kaksonen et al., 2006) © 2006 the Nature Publishing Group.]... Budding yeast has served as an excellent model organism for studying endocytosis in eukaryotes One of the most debated topics in this field has been the role of actin in the endocytic process Only when the high-resolution, dual-labeling and realtime fluorescence microscopy emerged, has the direct contribution of actin to endocytosis been observed A growing body of evidence indicates that a dynamic actin. .. receptor-mediated endocytosis (EngqvistGoldstein & Drubin, 2003) The majority of these genes encode proteins that either have homologues in mammalian cells or have domains that share homology with domains present in mammalian proteins Chapter 1 1.2 5 Actin cytoskeleton and endocytosis 1.2.A Evidence of actin s involvement in endocytosis Most of the proteins required for mammalian endocytic internalization... cytoskeleton is indispensable for plasma membrane remodeling, invagination and vesicle scission during the clathrin-mediated endocytosis Furthermore, actin polymerization that drives the internalization process is tightly regulated, as transient bursts of actin polymerization are precisely coordinated with the recruitment of other endocytic proteins Hence studies on the control of the actin dynamics will... these sites The cells then must know how to target this delivery and how to choose the sites of growth The actin cytoskeleton has been implicated in these processes Cortical structures containing actin filaments cluster at sites of cell growth during the cell cycle (Adams & Pringle, 1984), as previously described in Section 1.2C Mutations in actin gene itself, actin- cross linker or capping proteins all... activate the Arp2/3 complex at the cell surface Myosins might also generate force on the actin network or anchor the actin filaments to the plasma membrane through their motor domains The activated Arp2/3 complexes form branched actin filaments that grow through the addition of ATP actin monomers near the plasma membrane Older filaments are capped at their barbed ends by capping proteins [Cap1/2p] The branched . spatiotemporal map of phosphoregulation of actin-driven endocytosis [Chapter 5]. This finding completes the other half of the cycle of phosphoregulation of endocytosis and, therefore, is an important. important role of actin cytoskeleton in the endocytic process. The first line of evidence came from the observation that the internalization step of the endocytosis of mating pheromone α-factor. PHOSPHOREGULATION OF THE ACTIN-DRIVEN ENDOCYTOSIS IN THE YEAST SACCHAROMYCES CEREVISIAE HUANG BO B.Sc. (Hons, 1st), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR