董桂書臺灣大學：分子與細胞生物學研究所陳貞云Chen, Chen-YunChen-YunChen2007-11-252018-07-062007-11-252018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/49928減數分裂在有性生殖中扮演重要的角色。在營養缺乏的環境中，一個雙倍體的酵母菌細胞會經由減數分裂產生四個單倍體的孢子。整個減數分裂和孢子形成（spore formation）的過程稱之為孢子生成（sporulation）。 酵母菌熱休克蛋白Hsp26被視為一個伴護蛋白（chaperone），它在熱休克情況下和其他熱休克蛋白共同作用抑制不當摺疊的蛋白累積。過去的研究發現，在hsp26突變的細胞株中，酵母菌的產孢率比野生型對照組高，這樣的差異出現在孢子形成時期，而不是減數分裂的過程。為了瞭解Hsp26詳細的調控機制，我們研究兩個可能和Hsp26蛋白質交互作用的蛋白：Ady3和Tem1。Ady3位在原生孢子膜（prospore membrane）的前緣蛋白鞘（leading edge protein coat）上，主要和原生孢子的形成以及孢子壁的合成有關。Tem1是酵母菌有絲分裂MEN pathway的重要分子，控制細胞完成M期和細胞質分離。 我們發現，和野生型相比，在hsp26突變株中可以偵測到較多包含Ady3的前緣蛋白鞘。這些結果顯示，Hsp26可能參與在Ady3定位於前緣蛋白鞘的調控機制。雖然酵母菌雙雜合系統（yeast two-hybrid）顯示Tem1和Hsp26有直接作用，而在免疫共沉澱法中無法確認Tem1和Hsp26的蛋白質交互作用。我們也發現Tem1有磷酸化的現象。Tem1磷酸化在細胞進入到diauxic shift後會增加，並且在孢子生成的過程中一直存在。磷酸化現象可能是由於養分的缺乏，例如葡萄糖的不足而造成。我們利用溫度敏感株tem1-3。在高溫下，tem1-3突變株會產生孢子，表示Tem1可能對於孢子生成並不是必須的。有趣的是，這株溫度敏感株如果處在靜止期（stationary phase）即使在正常生長溫度下也會出現細胞質分離不完全的缺失。tem1-3的磷酸化和在靜止期的存活率也比野生型差。我們推測磷酸化的Tem1可能有助於維持細胞的生命現象。Meiosis plays an important role in sexual reproduction. A diploid yeast cell undergoes meiosis and produces four haploid spores when nutrients are limited. The overall process of meiosis and spore formation is called sporulation. Yeast small heat shock protein Hsp26 is considered as a chaperone cooperating with other heat shock proteins to prevent the aggregation of proteins under heat shock stress. Hsp26 is also induced during sporulation. Previous studies found that the sporulation efficiency of the hsp26 mutant is better than that of the wild type, and the cause seems to be at the stage of spore formation, not meiosis. To understand the molecular mechanism of Hsp26 action during sporulation, we studied two potential Hsp26-interacting proteins: Ady3 and Tem1. Ady3 is a component of the leading edge protein coat of the prospore membrane and is important for prospore formation and spore wall synthesis. Tem1 is a critical component in mitotic MEN pathway to terminate M phase and complete cytokinesis. We found that more Ady3 signals were detected as the leading edge coat in the hasp26 mutant than in the wild-type control. This observation suggests that Hsp26 might be involved in the regulation of Ady3 localization onto the leading edge coat. We were not able to confirm the interaction between Hsp26 and Tem1 by co-immunoprecipitation. However, we discovered an interesting pattern of Tem1 phosphorylation. Tem1 phosphorylation was detected after diauxic shift and maintained through sporulation. The phosphorylation could be caused by nutrient limitation, such as glucose inadequacy. Because TEM1 is an essential gene, we could not study the function of Tem1 in sporulation by a regular knock-out method. We used the conditional tem1-3 ts mutant to study Tem1 function in sporulation. Since the tem1-3 mutant displayed normal sporulation at restriction temperatures, Tem1 is probably not essential to sporulation. It was interesting that the tem1-3 mutant exhibited cytokinesis defects in early stationary phase even at permissive temperatures. Besides, we found that the tem1-3 mutant had impaired phosphorylation and displayed decreased viability in stationary phase. It indicates that Tem1 phosphorylation may be important for cell viability in stationary phase.TABLE OF CONTENTS ABSTRACT i 中文摘要 iii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii CHAPTER 1. INTRODUCTION 1 The Importance of Meiosis 1 The Entry of Meiosis 1 Meiotic Cell Cycle 2 Yeast Small Heat Shock Protein Hsp26 4 Spore Formation of S. cerevisiae 5 Ady3 is Important for Spore Formation 7 Tem1 is the Key Component in MEN Pathway 8 The Spindle Position Checkpoint 10 Specific Aim 11 CHAPTER 2. MATERIALS AND METHODS 12 Strains, Media, and Growth Conditions 12 DNA Preparation 12 Plasmid Constructions 13 Yeast Constructions 19 Protein Extraction and Western Blot Analysis 21 Immunoprecipitation and Alkaline Phosphatase Treatment 22 Co-immunoprecipitation 23 Nuclear Division Analyses 25 Immunofluorescence Staining 26 Spore Viability Analyses 27 Synchronization by Treatment with α Factor and Release 28 Cell Growth Curve 28 Analysis of Cellular Viability 29 CHAPTER 3. RESULTS 30 The Interaction between Ady3 and Hsp26 30 The 9Xmyc tagged Ady3 is functional 30 Ady3 is expressed and phosphorylated during sporulation 30 Ady3 physically interacts with Hsp26 31 The genetic interaction between ADY3 and HSP26 in sporulation 32 Subcellular localization of Hsp26 and Ady3 32 More Ady3 is located on the leading edge protein coat in the hsp26 mutant 34 Ady3 Phosphorylation 35 Ady3 phosphorylation depends on the progression of meiotic cell cycle 35 Cdc28 is a potential kinase of Ady3 35 Phosphorylation sites prediction 36 Tem1 Phosphorylation 39 Epitope tagging of Tem1 39 Tem1 is expressed and phosphorylated during sporulation 39 Nutrient limitation could cause Tem1 phosphorylation 40 Glucose limitation may be one of the factors that cause Tem1 phosphorylation 41 Tem1 was phosphorylated at log phase in YPAacetate medium 41 Tem1 was not phosphorylated in vegetative cell-cycle 42 The tem1-3 mutant displays incomplete cytokinesis in early stationary phase 43 Phosphorylation of the tem1-3 mutant is impaired 43 The tem1-3 mutant cells display no obvious growth defect 44 The tem1-3 mutation causes a decrease in viability of cells at stationary phase 44 The tem1-3 mutant can not reach the maximal density as the wild-type does in YPAacetate medium 45 Tem1 in Sporulation 46 Tem1 may be not essential for sporulation 46 CHAPTER 4. DISCUSSION 47 Hsp26 may play a Regulatory Role of Ady3 Localization onto the Leading Edge Protein Coat 47 Phosphorylation of Ady3 in Sporulation 48 Tem1 Phosphorylation may be Important for Cell Viability 50 The Relationship between Hsp26 and Tem1 52 Conclusion 53 REFERENCES 54 LIST OF TABLES Table 1 Yeast strains 60 2 Plasmid list 62 3 Primer list 64 4-1 Complementation test of 9Xmyc-tagged Ady3 65 4-2 Types of asci in ady3 complementation test 65 5-1 Sporulation efficiency of wild-type, hsp26 mutant, ady3 mutant and hsp26 ady3 double mutant in liquid sporulation medium 66 5-2 Ascus types of wild-type, hsp26 mutant, ady3 mutant and hsp26 ady3 double mutant in liquid sporulation medium 66 6-1 Sporulation frequency of wild-type, hsp26 mutant, ady3 mutant and hsp26 ady3 double mutant on sporulation plates at 30℃ 67 6-2 Sporulation frequency of wild-type, hsp26 mutant, ady3 mutant and hsp26 ady3 double mutant on sporulation plates at 32.5℃ 67 7-1 Complementation test of ady3 mutants based on Cdc28 consensus target sites 68 7-2 Ascus types of ady3 mutants at 32.5℃ 68 8-1 Complementation test of ady3 mutants which were confirmed according to the PredPhospho website prediction at 32.5℃ 69 8-2 Ascus types of ady3 mutants at 32.5℃ 69 9 Complementation test of 6Xmyc-tagged Tem1 70 10 Morphology of wild type and the tem1-3 mutant in early stationary phase 71 11 Sporulation efficiency of wild-type, tem1-3mutant, hsp26 mutant and tem1-3 hsp26 double mutant at 25℃, 34℃, or 25℃ with a shift to 34℃ 72 LIST OF FIGURES Figure 1 Ady3 expression and phosphorylation pattern 73 2 Co-immunoprecipitation of Ady3 and Hsp26 74 3 Hsp26 was localized in nuclei in meiosis 75 4 Ady3 was localized on the leading edge protein coat 76 5 Hsp26 and Ady3 were not co-localized 77 6 Comparison of Ady3 localization in wild type and the hsp26 mutant cells 78 7 Phosphorylation of Ady3 in the ndt80-arrested cells 79 8 Ady3 phosphorylation in the cdc28-63 mutant 80 9 The phosphorylation of ady3 mutants 81 10 The phosphorylation of ady3 mutants 82 11 Tem1 expression and phosphorylation pattern 83 12 Time course of Tem1 phosphorylation 84 13 Addition of glucose increased the nphosphorylated Tem1 85 14 The effect of carbon source on Tem1 phosphorylation 86 15 Tem1 was not phosphorylated during vegetative cell cycle 87 16 Incomplete cytokinesis in the tem1-3 mutant 88 17 The tem1-3 mutant displayed a phosphorylation defect 89 18 Cytokinesis defect of the tem1-3 mutant at 37℃ heat shock 90 19 Growth curve of the tem1-3 mutant in YPAD medium 91 20 Cell viability in stationary phase 92 21 Growth curve of the tem1-3 mutant in YPAacetate medium 93 22 Tem1 expression under the CLB2 promoter 942258002 bytesapplication/pdfen-US酵母菌孢子生成減數分裂yeastsporulationHsp26Tem1Ady3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49928/1/ntu-96-R94b43003-1.pdf