酵母菌第一腺嘌呤核苷二磷酸核醣化因子相似蛋白的功能性探討

Functional Characterization of ADP-Ribosylation Factor-Like Protein 1 in Saccharomyces cerevisiae

劉雅雯(Ya-Wen Liu)

指導教授 ： 李芳仁

國立臺灣大學/醫學院/分子醫學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.00535

摘要

腺嘌呤核苷二磷酸核醣化因子(ADP-ribosylation factor, ARF)為Ras致癌基因蛋白家族的一支，屬於鳥糞嘌呤核苷三磷酸結合蛋白的一種。在各種不同的真核生物都有ARF家族成員的分布，負責著細胞內的囊泡運輸、磷脂酶的活化、參與磷脂三激酶的訊息調控等等的功能。ARF家族成員可分為ARF與ARF-相似蛋白(ARL)兩大類。目前雖然已知有許多ARL的存在但對其功能卻仍幾乎是一無所知。本文利用酵母菌為模式研究ARL1蛋白在細胞中所扮演的角色。利用螢光染色法，我們發現ARL1蛋白分布在高基氏體上，並利用酵母菌雙雜合篩選法找出與Arl1p蛋白直接作用的蛋白質Imh1p以及Gcs1p。從接下來的功能性探討，我們證明了Imh1p是Arl1p的下游作用基因受其調控，而Gcs1p 是Arl1p的上游調控者會活化其鳥糞嘌呤核苷三磷酸水解能力並繼而影響Arl1p在細胞中的分布。另外，在囊泡運輸路徑的研究中，我們發現ARL1蛋白會參與在醣基化磷脂醯肌醇(glycosylphosphatidylinositol) 連結蛋白Gas1p從高基氏體到細胞膜的運送。無論是ARL1蛋白的上游調控基因或下游作用基因都會影響Gas1p。因此，我們的研究發現了部份ARL1蛋白的結合蛋白及其結合功能以及Arl1p在酵母菌中所參與的囊泡運輸路徑。進一步分析與探討其他與其作用的蛋白質將會使我們對囊泡運輸的分子機轉有更深一層的了解。

關鍵字

腺嘌呤核苷二磷酸核醣化因子 囊泡運輸

並列摘要

ADP-ribosylation factors (ARFs) are highly conserved small GTP-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and have an important role in vesicular transport. Several ARF-like proteins (ARLs) have been cloned from different organisms but the biological functions of ARLs remain largely unknown. In this dissertation, an ARL protein, Arl1p, of yeast Saccharomyces cerevisiae was characterized. Yeast ARL1 encodes a protein that is structurally related to human, rat, and Drosophila ARL1 proteins. Here we identified two interacting proteins of Arl1p, Imh1p and Gcs1p, the former is a tethering factor and the latter is an ARFGAP. From several in vitro and in vivo analysis, we demonstrated that Imh1p is a downstream effector of Arl1p and is recruited by the proper protein-protein interaction; on the other hand, Gcs1p exhibits GAP activity toward Arl1p and is able to regulate subcellular localization of Arl1p thus indicates that Gcs1p is a GAP for Arl1p. To the functional analysis, we found that Arl1p is involved in the anterograde transport from the Golgi to cell surface of the glycosylphosphatidylinositol (GPI)-anchored plasma membrane-resident protein Gas1p, but not those cell wall-localized GPI-anchored proteins Crh1p, Crh2p, and Cwp1p, or non-GPI-anchored plasma membrane-protein Gap1p. We also show that regulators of Arl1p: Sys1p, Arl3p, and Gcs1p; and effector Imh1p, all participate in the transport of Gas1p. From these results, not only the first ARL GAP is identified, but the first endogenous cargo molecule of yeast Arl1p is defined.

並列關鍵字

ARF vesicular trafficking

參考文獻

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