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효모에서의 단백질 호모머 형성에 대한 글로벌 분석

By 김연수

Abstract

학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 생명과학부, 2018. 8. 허원기.ABSTRACT<br/> Global analysis of protein homomerization in Saccharomyces cerevisiae.<br/> <br/> Yeonsoo Kim<br/> School of Biological Sciences<br/> The Graduate School<br/> Seoul National University<br/> In vivo analyses of protein-protein interaction (PPI) occurrence, subcellular localization, and dynamics are important issues in functional proteomic researches and various PPI analyzing methods have been developed over the past decades. Bimolecular fluorescence complementation (BiFC) assay has many advantages over other research methods in that it can provide a reliable way to detect PPI in living cells with minimal perturbation of the structure and function of target proteins and it allow the visualization of subcellular localization where PPI occurs. <br/> Previously, to facilitate the application of BiFC assay to the genome-wide analysis of PPIs, we generated a collection of yeast strains expressing full-length proteins tagged with the N-terminal fragment of Venus (VN), a yellow fluorescent protein variant, under the control of their own endogenous promoters. The VN fusion library consists of 5,911 MATa strains (representing ~95% of the yeast proteome) and was successfully exploited to screen SUMO interactome in Saccharomyces cerevisiae. In the present study, we constructed the VC (the C-terminal fragment of Venus) fusion library that consists of 5,671 MAT strains expressing C-terminally VC-tagged proteins (representing ~91% of the yeast proteome) under their native promoters. The reliability of the constructed library was then proved by using it with VN fusion library to genome-widely detect protein homomers in yeast. For a genome-wide analysis of protein homomer formation, we mated each strain of the VC fusion library with its cognate strain of the VN fusion library and performed BiFC assay. Through this analysis, we identified 186 homomer candidates, 104 of which are previously unknown homomers. Subcellular localization, highly enriched gene ontology (GO) and changes of homomeric interaction upon nitrogen starvation was further analyzed to characterize homomer population of yeast. Out of 186 protein homomers identified in this study, Pet10, a protein of unknown function which localize to lipid droplet, was further investigated for its functional relevance of homomerization and lipid accumulation. Our data set represents a useful resource for understanding the physiological roles of protein homomerization. Furthermore, thoroughly examined credibility and feasibility of the VC fusion library together with the VN fusion library in BiFC assay revealed that this system will provide a valuable platform to systematically analyze PPIs in the natural cellular context.<br/>CONTENTS<br/> ABSTRACT i<br/> CONTENTS iii<br/> LIST OF FIGURES vi<br/> LIST OF TABLES viii<br/> ABBREVIATIONS ix<br/> CHAPTER I. Introduction 1<br/> 1. Study of protein-protein interaction 2<br/> 1.1. Interaction study as a tool to understand biological phenomenon 2<br/> 1.2. Bimolecular fluorescence complementation (BiFC) assay 5<br/> 1.3. Advantages and disadvantages of BiFC assay 13<br/> 1.4. Application of BiFC assay for large-scale studies 16<br/> 2. Protein homomerization 22<br/> 2.1. Biological meaning of protein homomerization 22<br/> 2.2. Importance of protein homomerization study 23<br/> 3. Aim of this study 25<br/> CHAPTER II. Construction of VC library to facilitate large-scale BiFC assay 27<br/> 1. Introduction 28<br/> 2. Materials and Methods 30<br/> 2.1. Yeast strains and culture conditions 30<br/> 2.2. Construction of plasmids 30<br/> 2.3. Transformation of yeast cells 34<br/> 2.4. Mating type switching 34<br/> 2.5. Vector elimination 34<br/> 2.6. Western blot analysis 35<br/> 3. Results 36<br/> 3.1. Construction of VC fusion library MATa 36<br/> 3.2. Construction of VC fusion library MAT 38<br/> 4. Discussion 43<br/> CHAPTER III. Global analysis of protein homomerization in S. cerevisiae 45<br/> 1. Introduction 46<br/> 2. Materials and Methods 48<br/> 2.1. Yeast strains and culture conditions 48<br/> 2.2. Amplification of PCR fragment 48<br/> 2.3. Transformation of yeast cells 48<br/> 2.4. Microscopic analysis and fluorescence quantification 55<br/> 2.5. Spot assay 55<br/> 2.6. Western blot analysis 55<br/> 2.7. Co-immunoprecipitation assay 56<br/> 2.8. Flow cytometry 57<br/> 3. Results 58<br/> 3.1. Genome-wide screening of protein homomers by BiFC using the VC and VN fusion libraries 58<br/> 3.2. Characterization of protein homomers 70<br/> 3.3. Changes of homomerization signal upon nitrogen deprivation 82<br/> 4. Discussion 97<br/> CHAPTER IV. Homomerization of Pet10 and its physiological effects 101<br/> 1. Introduction 102<br/> 2. Materials and Methods 104<br/> 2.1. Yeast strains and culture conditions 104<br/> 2.2. Amplification of PCR fragment 104<br/> 2.3. Construction of plasmids 104<br/> 2.4. Transformation of yeast cells 109<br/> 2.5. Microscopic analysis and fluorescence quantification 109<br/> 2.6. Western blot analysis 110<br/> 2.7. Co-immunoprecipitation assay 110<br/> 2.8. FCH and PCH 111<br/> 2.9. TAG quantification assay 112<br/> 3. Results 113<br/> 3.1. Increased homomerization of Pet10 upon nitrogen deprivation 113<br/> 3.2. Finding novel interactors 115<br/> 3.3. Involvement of Pet10 in lipid metabolism 124<br/> 4. Discussion 130<br/> CHAPTER V. Conclusion 133<br/> References 141<br/>Docto

Topics: 570
Publisher: 서울대학교 대학원
Year: 2018
OAI identifier: oai:s-space.snu.ac.kr:10371/143135
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