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標題: | 人類第一腺嘌呤核苷二磷酸核糖化相似因子與其結合蛋白之功能性探討 Functional characterization of human ADP-ribosylation factor like-1(ARL1) and its interacting proteins |
作者: | Hsiao-Chuan Huang 黃小娟 |
指導教授: | 李芳仁(Fang-Jen Lee) |
關鍵字: | 腺嘌呤核苷,二磷酸核醣化因子,高基氏體, ARF,ARL1,CIB1,GAP,Golgi, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 腺嘌呤核苷二磷酸核醣化因子(ARF)與腺嘌呤核苷二磷酸核醣化因子相似蛋白(ARL)為參與細胞內運輸之ARF家族成員,這些蛋白受到鳥糞嘌呤核苷酸轉換因子(GEF)與鳥糞核苷三磷酸水解脢活化蛋白(GAP)的調控而循環在鳥糞核苷三磷酸(GTP)結合活化態與鳥糞核苷二磷酸(GDP)結合去活化態之間。ARL蛋白之間或與ARF蛋白之序列相同程度大約在40~60%左右。儘管ARL與ARF蛋白有高相似度,其中卻只有ARL1的功能被發現與液泡輸送有關。ARL1位於高基氏體且參與從反式高基氏網(TGN)至細胞膜及從內小體(endosome)至反式高基氏網之蛋白質運輸。在本論文共分為三部份,第一部分主要是利用活化(Q71L)與去活化突變型(T31N)來探討ARL1之功能。與過去研究不同的是,我們發現當大量表現活化型ARL1時,會使Golgin-245於高基氏體的訊號消失。當大量表現去活化型ARL1時,只會影響位於反式高基氏體之蛋白質而不影響位於順式(cis/medial)高基氏體上之蛋白。此外,當大量表現活化型ARL1時,水泡性口炎病毒醣蛋白(VSVG)之運輸會受到阻礙,但這種現象在ARL1弱化(knockdown)的細胞中卻不存在。這意謂著ARL1本身可能不直接參與VSVG的運輸。在第二部份,我們嘗試找尋ARL1的GAP。在三個可能的GAP中,利用酵母菌雙雜交系統(yeast two-hybrid),ARFGAP1不會與ARL1有相互作用;但在細胞中大量表現ARFGAP1會影響ARL1之位置,而我們認為這種影響是因為整個高基氏體都受到破壞的結果所致。Centaurin α1既不會與ARL1作用也不影響ARL1在細胞內之位置。ARAP1會與ARL1Q71LdN作用,但大量表現ARAP1卻看不到ARL1受到影響,ARAP1是否可能為ARL1之GAP需進一歩釐清。在第三部份,我們確認一個活化型ARL1之作用蛋白CIB1且顯示出它們之間的結合可能是仰賴鈣離子的作用。再者,CIB1廣泛分佈於細胞中,尤其在某種狀態下,CIB1在高基氏體的位置與ARL1有明顯重疊的情形。但CIB1位於高基氏體與ARL1之存在與否並無相關,反之亦然。很遺憾地,雖然發現這兩個蛋白之間會相互作用且在細胞中之位置有重疊的情形,目前為止,我們仍無法找出它們在功能上的相關性。 ADP-ribosylation factors (ARFs) and ARF-like proteins (ARLs) are members of the ARF family, which play essential roles in intracellular membrane trafficking. These proteins are regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) to cycle between active GTP-bound form and inactive GDP-bound form. ARLs are ~40–60 % identical to each other and to ARFs. Despite their high similarity to ARFs, only ARL1 has been shown to be involved in vesicular transport. ARL1 is localized in the Golgi complex and may participate in trans-Golgi network (TGN) to plasma membrane or endosome to TGN trafficking pathway. In the first part, we investigated the function of ARL1 by using constitutively active (Q71L) or inactive (T31N) ARL1. Discrepant with previous studies, we found that overexpressed ARL1Q71L specifically disperses Golgin-245 from Golgi apparatus. Over-expressed ARL1T31N disrupted trans-Golgi proteins but not cis/medial Golgi and endosome proteins. Moreover, VSVG transport was blocked in ARL1Q71L overexpressed but not in ARL1 knockdown cells which implied that ARL1 itself may not directly participate in the VSVG transport. In the second part, we attempted to find possible GAP for ARL1. Among our three candidates, ARFGAP1 did not interact with ARL1 in yeast two-hybrid assay but endogenous ARL1 were dissociated from the Golgi in cells with overexpressed ARFGAP1, which we considered an indirect consequence of general effect on Golgi proteins. Centaurin α1 neither interacted with ARL1 nor affected ARL1 localization. ARAP1 interacted with ARL1Q71LdN, but we could not detect the change of ARL1 localization when ARAP1 overexpressed. Whether ARAP1 could be GAP for ARL1 needs to be investigated further. In the third part, we identified an ARL1Q71L interacting protein, CIB1. We showed that ARL1 interacted with CIB1 in a Ca2+-dependent manner. Furthermore, CIB1 was ubiquitously expressed in cells especially colocalized with ARL1 in the Golgi apparatus in particular conditions. In addition, Golgi localization of CIB1 was not dependent on ARL1, and vice versa. Unfortunately, even though the interaction and colocalization of ARL1 and CIB1 were observed, we could not demonstrate the functional relevance between these two proteins. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32002 |
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