探討第一腺嘌呤核苷二磷酸核糖化因子相似蛋白之鳥糞嘌呤核苷酸交換因子的特性

Characterization of an Arl1p Guanine-Nucleotide Exchange Factor, Syt1p

許俽嘉(Hsin-Chia Hsu)

指導教授 ： 李芳仁

國立臺灣大學/醫學院/分子醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10182

摘要

腺嘌呤核苷二磷酸核醣化因子(ADP-ribosylation factors, ARFs)在真核細胞中的蛋白質運輸與細胞骨架的組織中扮演著很重要的角色，而腺嘌呤核苷二磷酸核糖化相似因子(ARL)就隸屬於ARF這個家族。小鳥糞嘌呤核苷三磷酸結合蛋白(small GTP-binding protein)需要鳥糞嘌呤核苷酸交換因子來協助他從鳥糞嘌呤核苷二磷酸結合的形式轉換為鳥糞嘌呤核苷三磷酸結合的形式，進一步被活化而具有活性。之前的報告指出Syt1p此蛋白質為酵母菌的第二腺嘌呤核苷二磷酸核醣化因子(Arf2p)之鳥糞嘌呤核苷酸交換因子，並且參與在囊泡的運輸過程中。而我們實驗室的研究報告也指出他不只身為Arf2p的鳥糞嘌呤核苷酸交換因子,更是第一腺嘌呤核苷二磷酸核糖化相似因子(Arl1p)的鳥糞嘌呤核苷酸交換因子。在此份報告中，我們進一步去研究Syt1p的特性。首先，Syt1p對Brefeldin A這個藥物並不具敏感性。第二，Syt1p能夠利用許多不同的片段來和Arl1p結合。他的胺基端片段，以及具有催化作用的Sec7區域，和羧基端片段都能夠跟截短胺基端17個胺基酸之Arl1p結合。更重要的是，Syt1p的這三個區域對Arl1p的結合力全都大於全長Syt1p和Arl1p的結合力，暗示著Syt1p可能和其它的鳥糞嘌呤核苷酸交換因子相同，或許都具有分子內部自我抑制的能力。而我們進一步也發現，Syt1p能夠利用其羧基端片段來和Sec7區域做分子內部的結合，而且羧基端片段也可以和另一個羧基端片段進行分子間的結合。在細胞內Syt1p確實能夠相互結合形成二聚體(dimer)或寡聚體(oligomer)，而且Syt1p的胺基端片段對二聚體或寡聚體的形成也扮演著重要的角色。接著我們也利用酵母菌雙雜合(yeast two-hybrid)來大規模搜尋可能身為Syt1p調控者的蛋白質，然而這些可和Syt1p相互結合的蛋白質是否真正參與Syt1p的調控，則需進一步的確認。此外，Syt1p形成二聚體或寡聚體是否在生物上具有生物功能性，還有Syt1p在細胞中是否真的有自我抑制的能力皆需要更深入的探討。

關鍵字

第一腺嘌呤核苷二磷酸核糖化因子相似蛋白 ； 鳥糞嘌呤核苷酸交換因子

並列摘要

ARF-like (ARL) proteins belong to ADP-ribosylation factor (ARF) GTPase family, which are involved in protein trafficking and cytoskeleton organization. Those small G proteins require guanine-nucleotide exchange factors (GEFs) to switch from GDP-bound to GTP-bound form and become active. Previous reports suggested that Syt1p is the GEF of Arf2p and is involved in vesicle trafficking. Recently, our studies showed that Syt1p also acted as a GEF for Arl1p. In this study, Syt1p was further characterized. Firstly, it has been shown that Syt1p belongs to BFA-resistant GEFs. Secondly, Syt1p can use multiple regions to interact with Arl1p. The N-terminus, Sec7 domain, and C-terminus of Syt1p can all interact with Arl1d17N form, whose N-terminal first 17 amino acids are deleted. The interactions between all of the three regions and Arl1d17N are stronger than the interaction between full-length Syt1p and Arl1pd17N. Therefore, it might hint that Syt1p is autoregulated as other GEFs of Small GTPases. Surprisingly, we next found that Syt1p has an intramolecular interaction between the C-terminal region and Sec7 domain and an intermolecular interaction between C-terminal regions, indicating that Syt1p could form dimers or oligomers and might be autoregulated. Syt1p dimerization or oligomerization is also supported by in vivo pull down results, which proved that Syt1p can interact with itself. Moreover, the N-terminus is important for the formation of dimers or oligomers. Yeast two-hybrid screen was also performed to search for putative regulators of Syt1p. However, those candidates remain to be elucidated. Besides, whether dimerization or oligomerization plays an important role in Syt1p activation or membrane tethering and whether autoinhibition truly exists in Syt1p in vivo require further investigation.

並列關鍵字

Arl1p ； Syt1p

參考文獻

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