請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29002
標題: | 酵母菌Ady3和Tem1在孢子生成中與Hsp26之交互作用 The interaction of Ady3 and Tem1 with Hsp26 in yeast sporulation |
作者: | Chen-Yun Chen 陳貞云 |
指導教授: | 董桂書(Kuei-Shu Tung) |
關鍵字: | 酵母菌,孢子生成,減數分裂, yeast,sporulation,Hsp26,Tem1,Ady3, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29002 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-96-1.pdf 目前未授權公開取用 | 2.21 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。