臺灣博碩士論文加值系統

啤酒酵母菌中的兩個同源Slm (Synthetic lethal with Mss4)蛋白Slm1以及Slm2 是磷脂酸肌醇 4,5-雙磷酸結合蛋白，且會跟TORC2 (target of rapamycin complex 2)結合；而Tor為雷帕黴素作用的標的，是一個從酵母菌到哺乳類動物都高度保留的絲胺酸-蘇胺酸蛋白激酶，在細胞中扮演生長代謝調控中樞。Tor與不同細胞蛋白組成兩個多蛋白複合體TORC1以及TORC2，分別去調控細胞生長相關的功能以及肌動蛋白的排列組織。相對於對TORC1調控路徑的了解，我們對於TORC2的訊息傳遞所知甚少。過去Slm蛋白被認為是TORC2下游直接的受質且作為TORC2的周圍組成蛋白，然而Slm蛋白在TORC2訊息傳遞中的角色還不是很清楚。在本論文研究中，我們發現當酵母菌缺失SLM1以及SLM2基因時，許多細胞功能出現問題，包含：細胞壁完整性、肌動蛋白排列組織、和胞吞作用有缺陷，這和TORC2突變株表現型相同。為了進一步了解Slm蛋白在TORC2訊息傳遞中的功能，我們試圖尋找與SLM基因有遺傳交互作用的基因、以及與Slm蛋白有實質交互作用的蛋白。在遺傳交互作用方面，我們發現當細胞表現有自發活性的已知TORC2下游Ypk蛋白激酶時，可以拯救缺失SLM1和SLM2基因所造成的缺陷，並且也觀察到Slm蛋白缺失細胞中，Ypk蛋白激酶的活性會劇烈下降；意外的是，細胞表現有自發活性的已知TORC1受質Sch9蛋白激酶時，也可以拯救缺失SLM基因所導致的表現型。在尋找交互作用蛋白方面，我們以大量表現GST融合Slm重組蛋白並執行共純化實驗的方式來取得可能的交互作用蛋白，由蛋白體學分析獲得的分子互動組顯示可能的交互作用蛋白多半參與在蛋白質轉譯、蛋白質和囊泡運輸以及代謝的過程中，而其中確切的交互作用蛋白仍在被驗證中。綜觀目前的實驗結果，我們結論：Ypk蛋白激酶在訊息傳遞路徑上是位於Slm蛋白的下游；而表現活化態TORC1受質Sch9對Slm蛋白缺失細胞表現型的修飾或許暗示Slm蛋白功能與TORC1的下游傳訊有牽連；另外，由所得候選Slm交互作用蛋白已知的功能來推測，Slm蛋白很有可能參與調節囊泡運輸過程。

Synthetic lethal with Mss4 (Slm) proteins, i.e., Slm1 and Slm2, are two homologous phosphatidylinositol 4,5-bisphosphate-binding proteins which can associate with the target of rapamycin complex 2 (TORC2) in Saccharomyces cerevisiae. Target of rapamycin (TOR), a Ser/Thr kinase highly conserved from yeast to human, serves as a central regulator of growth and metabolism. Tor forms two distinct multi-protein complexes, TORC1 and TORC2, which signal to coordinate growth-related cell processes and regulation of the actin cytoskeleton, respectively. Compared to the understanding about TORC1-linked pathways, little do we know about TORC2 signaling. Slm proteins are considered direct substrates of TORC2 and peripheral components of TORC2, while their role in TORC2 signaling remains to be investigated. In this thesis study, our results demonstrate that cells with deletion of both SLM1 and SLM2 exhibit phenotypes shared among TORC2 mutants. To explore the function of Slm proteins in TORC2 signaling, we set out to search for genetic interactors with SLM alleles and for physical interactors of Slm1 protein. Genetic analyses have revealed that constitutively active Ypk kinases, which are known to function downstream of TORC2, can rescue TORC2-mediated phenotypes caused by depletion of Slm proteins, and the activity of Ypk2 kinase is dramatically reduced in cells without Slm proteins. To our surprise, constitutively active Sch9 kinase, which acts downstream of TORC1, can also rescue the phenotype caused by depletion of Slm proteins. In order to identify the physical interactors of Slm1, GST-pulldown procedures were carried out using lysates from GST-Slm1-overexpressing yeast cells. The obtained interactomes demonstrate that candidate Slm interactors participate in protein translation, trafficking and transport, and metabolism; verification of specific interactions is underway. In summary, our data indicate that Ypk kinases function downstream of Slm proteins; the modification of the phenotypes of Slm proteins-depleted cells by active TORC1 downstream kinase Sch9 may imply a functional crosstalk between Slm proteins and TORC1 signaling; moreover, the identification of many candidate Slm1 interactors with known functions in vesicular transport suggests that Slm proteins may play a significant role in this process.

誌謝...i
摘要...ii
Abstract...iv
Contents...vi
Introduction...1
Discovery of the Target of Rapamycin (TOR) proteins...1
Structure and function of Tor proteins...1
TOR functions in two distinct multi-protein complexes...2
TOR signaling in Saccharomyces cerevisiae...3
Signaling network of TORC1...4
(A) Location of TORC1...4
(B) Upstream signaling of TORC1...4
(C) Downstream signaling of TORC1...5
Signaling network of TORC2...6
(A) Location of TORC2...6
(B) Upstream of TORC2...6
(C) Downstream of TORC2...7
Pkc1 and the cell wall integrity pathway...7
Ypk1 and Ypk2...8
Slm1 and Slm2...10
Aims...13
Materials and Methods...14
Bacterial and yeast culture...14
Bacteria strains...14
Yeast strains...14
Plasmids...14
Trypan blue assay...15
Actin staining...15
Spot assay...15
Growth curve...16
GFP-Snc1 localization...16
E. coli transformation...16
Yeast transformation...17
Flow cytometry...17
GST pulldown assay...18
GO analysis and categorization...19
Co-Immunoprecipitation analysis...19
Results...21
slm1∆ slm2∆ cells showed slow growth at high temperatures and sensitivity to rapamycin...21
SLM1 and SLM2 were required for TORC2-mediated functions...22
Mutation of the AVO3 allele was synthetic lethal with deletion of SLM1 and SLM2...23
Overexpressing components of TORC2 could not rescue the rapamycin sensitivity of slm1∆ slm2∆...24
Only active Bck1, the component in the cell wall integrity pathway rescued the rapamycin sensitivity of slm1∆ slm2∆ cells...25
Ypk1 and Ypk2 appeared to function downstream of Slm proteins...25
Constitutively active Sch9, a TORC1 downstream substrate, also rescued the rapamycin sensitivity of slm1∆ slm2∆ cells...26
Gene Ontology analysis revealed functions of candidate Slm interactors in protein translation, trafficking and transport, and metabolism...27
Candidate Slm interactors involved in vesicular transport showed physical interaction with Slm1 proteins...29
Discussion...31
What are the mechanisms behind the temperature sensitivity in slm1D slm2D cells?...31
What is the direct mediator of rapamycin that causes cells growth inhibition in slm1∆ slm2∆ cells?...32
How can both active Sch9 and Ypk kinases rescue the rapamycin sensitivity of slm1∆ slm2∆ cells?...33
Are phenotypes of slm1∆ slm2∆ cells caused by disruption of upstream or downstream TORC2 signaling?...34
Why components of TORC2 did not co-pulldown with GST-Slm1 in this study?...35
Figures...36
Tables...59
Appendices...66
Supplements...69
References...75

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