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研究生:李信毅
研究生(外文):Hsin-Yi Lee
論文名稱:探討出芽酵母菌中RhoGAPs在Tsc11下游訊息傳遞扮演之角色
論文名稱(外文):Exploring the roles of RhoGAPs in Tsc11p signaling in Saccharomyces cerevisiae
指導教授:陳美瑜陳美瑜引用關係
指導教授(外文):Mei-Yu Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:144
中文關鍵詞:TOR2TSC11RhoGAPase
外文關鍵詞:TOR2TSC11RhoGAPase
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出芽酵母菌之TSC11/AVO3是一維持生存所必須之基因,其所表現出來的蛋白質是TOR Complex 2 (TORC2)中的一個成員。之前有研究報告指出,TORC2會透過Rho1來調控細胞的Cell wall integrity以及Actin之架構。在之前實驗室的研究結果指出Tsc11會藉由不同的訊息傳導路徑-可能包括Cell integrity pathway,來調控actin之架構和Cell wall integrity。但是目前還不清楚Rho1是否參與在Tsc11下游訊息傳遞之中。Rho GTPase activating proteins (RhoGAPs)是像Rho1這種Samll GTPase的負向調控者(negative regulator),而且不同的RhoGAPs會調控不同Rho1下游的訊息傳導路徑以及相關功能。為了去研究Rho1或是其他的Rho GTPase是否參與在Tsc11下游之訊息傳遞之中,我們採取了系統化的基因剔除實驗(Systemic deletion),在tsc11ts的突變株中個別剔除所有的RhoGAPs,再藉由觀察其phenotypes有無遭受影響來決定RhoGAPs與tsc11ts之間是否有genetic interaction。在所有九個測試的RhoGAPs中,我們發現SAC7, BAG7, BEM2, BEM3, LRG1以及RGD1會對tsc11ts的phenotype有所影響。SAC7是唯一可以拯救所有phenotype的deletion,包括對高溫及咖啡因的敏感性,還有不正常的actin分佈及cell wall異常。BAG7以及LRG1則是分別解救不同的phenotype,前者只解救不正常之actin分佈,而後者則是只拯救cell wall異常情形。BEM3則是只拯救tsc11ts突變株對咖啡因的敏感性。相反的,BEM2及RGD1之deletion則會造成tsc11ts突變株的某些phenotype變得更加嚴重。以上的結果指出,不同的RhoGAPs deletion會對tsc11ts突變株的phenotype造成不同的影響。我們更進一步研究SAC7之deletion之所以能解救tsc11ts突變株的原因,結果支持是移除Sac7之GAP acitivity的原因。總結來說,我們的研究也指向Tsc11會經由Rho1來調控cell integrity pathway的可能性。
Saccharomyces cerevisiae TSC11/AVO3 is an essential gene encoding one component of TORC2, a multi-protein complex of yeast Tor2 that controls actin cytoskeleton. It has been shown that Tor2 acts through Rho1 to modulate the cell integrity pathway and actin organization. Our genetic data suggest that Tsc11 acts through different signaling pathways, including the cell integrity pathway, to regulate actin polarization and cell wall integrity. However, whether Rho1 participates in linking Tsc11 to the cell integrity pathway is still unclear. Rho GTPase activating proteins (RhoGAPs) are negative regulators of Rho-type small GTPases like Rho1, and furthermore, different RhoGAPs in yeast control distinct Rho1 functions. In order to find out whether Rho1 or other Rho GTPases participate in TSC11/AVO3 signaling, we investigated the genetic interaction between TSC11 and different RhoGAPs by performing deletions of individual RhoGAPs in two different tsc11ts strains and examined the effect of each deletion upon mutant phenotypes. Among the 9 RhoGAPs we tested, deletions of SAC7, BAG7, BEM2, BEM3, LRG1 and RGD1 had effects. SAC7 deletion suppressed phenotypes of both tsc11ts mutants, including temperature and caffeine sensitivities, actin polarization defects, and cell wall abnormalities. BAG7 deletion partially restored the actin organization but not other phenotypes, whereas LRG1 deletion improved cell wall integrity but not actin defects of tsc11ts mutants. Deletion of BEM3 only rescued the caffeine sensitivity of tsc11ts mutants. In contrast, deletions of BEM2 and RGD1 worsened some phenotypes of tsc11ts mutants. These results show that deletions of distinct RhoGAPs have differential effects on rescuing the phenotypes of our mutants. We further investigated the reason why SAC7 deletion rescued tsc11ts, and the results indicated that the removal of GAP activity of Sac7 led to suppression of tsc11ts. Taken together, our findings also suggest that Tsc11 regulates cell integrity pathway through Rho1.
中文摘要 --------------------------------------------------------------------------------------- 1
ABSTRACT ----------------------------------------------------------------------------------- 2
INTRODUCTION ---------------------------------------------------------------------------- 3
METERIALS AND METHODS ----------------------------------------------------------- 6
Yeast strains ------------------------------------------------------------------------------ 6
Deletion of RhoGAPs ------------------------------------------------------------------- 6
Plasmids ----------------------------------------------------------------------------------- 6
Trypan blue assay ----------------------------------------------------------------------- 7
Actin staining ------------------------------------------------------------------------------ 7
RESULTS -------------------------------------------------------------------------------------- 8
Six out of 9 RhoGAPs genetically interact with TSC11 -------------------------- 8
Deletion of SAC7 rescues tsc11ts by removing the GAP activity ------------ 10
DISCUSSION -------------------------------------------------------------------------------- 11
REFFERENCE ------------------------------------------------------------------------------ 15
TABLES AND FIGURES ------------------------------------------------------------------ 21
Table 1. Rho proteins and their regulators --------------------------------------- 21
Table 2. Yeast strains used in this study ---------------------------------------- 22
Table 3. Oligonucleotide primers used in this study --------------------------- 23
Table 4. Plasmids used in this study ---------------------------------------------- 25
Table 5. Effects of RhoGAP deletions on the temperature and caffeine
sensitivity of tsc11ts mutants ------------------------------------------- 26
Figure 1. Effects of RhoGAP deletions on the temperature sensitivity of
tsc11ts mutants ------------------------------------------------------------ 27
Figure 2. Effects of RhoGAP deletions on the caffeine sensitivity of tsc11ts
mutants ---------------------------------------------------------------------- 28
Figure 3. Effects of RhoGAP deletions on the cell wall defect of tsc11ts
mutants ---------------------------------------------------------------------- 29
Figure 4. Effects of RhoGAP deletions on the actin defect of tsc11ts
mutants ---------------------------------------------------------------------- 30
Figure 5. SAC7R173A allele failed to reverse the suppression of tsc11ts by
SAC7 deletion ------------------------------------------------------------- 31
APPENDIX ----------------------------------------------------------------------------------- 33
A1.實驗材料及方法 --------------------------------------------------------------------- 34
A2. Plate assays原始照片 ------------------------------------------------------------ 40
A3.其餘plate assays ------------------------------------------------------------------- 44
A4. Actin assay原始照片 -------------------------------------------------------------- 49
A5. Trypan blue原始照片 ----------------------------------------------------------- 110
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