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標題: | 探討腺嘌呤核苷二磷酸核醣化相似因子ARL4A
與其結合蛋白ESCRT複合體次單元之作用途徑 Characterization of ARL4A and its interacting protein ESCRT subunit |
作者: | Ming-Ting Tsai 蔡明庭 |
指導教授: | 李芳仁(Fang-Jen Lee) |
關鍵字: | 腺嘌呤核苷,二磷酸核醣化相似因子, ARL4A, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 腺嘌呤核苷二磷酸核醣化相似因子4A (ARL4A),隸屬於腺嘌呤核苷二磷酸核醣化因子 (ARF) 的子家族腺嘌呤核苷二磷酸核醣化相似因子 (ARL) 家族的其中一員。這個蛋白質家族廣為人知的功能主要參與在囊泡傳遞運輸、細胞骨架重組、和胞器的形成等等。其中ARL4A又特別被報導了參與在細胞膜皺褶的形成、細胞骨架的調控、高基氏體的形成等等功能之中;然而ARL4A在囊泡傳遞運輸中扮演何種角色,目前還不明白。為了找出ARL4A可能的新功能,我們運用了酵母菌雙雜交篩選法,以一個新的ARL4A鳥糞嘌呤核苷二磷酸 (GDP) 結合態突變株ARL4A T51N為誘餌,來篩選人類胚胎時期腦的去氧核醣核酸基因庫。在十二個新篩選出的ARL4A結合蛋白中,發現了ESCRT 蛋白複合體的其中一個單元分子。ESCRT 蛋白複合體包含了複合體0,複合體I,複合體II,複合體III和 VPS4等成員。其功能為將泛素化 (ubiquitinated) 的膜蛋白由初級內體 (early endosome) 的膜上轉入內體空腔中 (intraluminal) 以形成多囊體 (multivesicular body),接著引導其走向降解的命運。因此,ESCRT複合體的功能在調控膜蛋白降解的途徑中是重要的存在。我們首先運用酵母菌雙雜交系統,以及利用穀胱苷肽轉移酶 (GST) 融合蛋白進行體外結合實驗,確認了ESCRT複合體單元分子和ARL4A之間有專一性的交互作用。並且發現ESCRT複合體單元分子偏好和GDP 結合態的ARL4A結合。接著也利用免疫螢光染色法在細胞中觀察到了ESCRT複合體次單元分子和ARL4A共同坐落於細胞膜和細胞核周圍區域。更進一步地,我們發現細胞在有表皮生長因子 (EGF) 刺激的條件下,剃除ARL4A的細胞將加速表皮生長因子受體 (EGFR) 降解之速度。反之,過量表現ARL4A的細胞將會減緩表皮生長因子受體的降解速度。另外我們利用N端共價結合螢光體之磷脂酰乙醇胺 (NRhPE) 標定了多泡體的位置和大小,並且發現在細胞中過量表現ARL4A將造成多泡體的分布不正常以及大小不均勻。這些實驗結果暗示了ARL4A可能在表皮生長因子受體透過多泡體進行降解的作用途徑中扮演著一個負向調控者的角色。 ADP-ribosylation factor-like protein 4A (ARL4A) is a member of the ARF/ARL small GTPases family. The ARF/ARL family is well-known for its functions on vesicular trafficking, cytoskeleton reorganization and organelle morphogenesis. ARL4A has been found to participate in regulations of membrane ruffling, cytoskeleton remodeling and Golgi organization; however, whether ARL4A is directly involved in vesicular trafficking remains unclear. In order to explore the new functions of ARL4A, we used a more accurate inactive GDP-bound mutant ARL4A T51N as bait and performed yeast two-hybrid screening to identify its novel interacting proteins. We have identified 12 potential interacting proteins of ARL4A T51N, and one of them is the subunit of Endosomal Sorting Complex Required for Transport (ESCRT) complex. ESCRT complexes, including ESCRT-0, -I, -II, -III, and VPS4, are involved in sorting of ubiqutinated endosomal membrane proteins into the multivesicular body (MVB), thus are required for their degradation. We first demonstrated the interaction between ARL4A and ESCRT subunit in vitro using yeast two-hybrid assay and γGTP-, GDP-loaded GST pull-down assay. Next, we studied their co-localization in vivo by immunofluorescence staining assay, and followed with monitoring the MVB pathway using epidermal growth factor (EGF) -stimulated EGF receptor (EGFR) degradation assay. Finally, we also studied the MVB biogenesis using NRhPE labeling assay. We found that the ESCRT subunit prefers interact with GDP-bound ARL4A in vitro and co-localized with membrane-associated ARL4As in perinuclear region and plasma membrane in vivo. We also found that ARL4A is involved in MVB biogenesis and EGF internalization as well as EGFR turnover in HeLa cells. Depletion of ARL4A in HeLa cells speeds up the EGF-stimulated EGFR degradation. By contrast, overexpression of ARL4A in HeLa cells delays the degradation of EGFR and defects the formation of MVB. These results imply that ARL4A may play a negative regulatory role in MVB-dependent EGFR degradation pathway through interacting with the ESCRT subunit. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17106 |
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顯示於系所單位: | 分子醫學研究所 |
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