探討第一腺嘌呤核苷二磷酸核糖化相似因子之鳥糞嘌呤核苷酸交換因子的自相互作用的功能特性

腺嘌呤核苷二磷酸核醣化因子(ADP-ribosylation factors)家族中的小鳥糞嘌呤核苷三磷酸結合蛋白主要在胞膜蛋白運輸的調節與細胞骨架的動態平衡中扮演著很重要的角色。腺嘌呤核苷二磷酸核醣化因子可進一步被活化，藉由鳥糞嘌呤核苷酸交換因子(GEF)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。在之前的報告指出鳥糞嘌呤核苷酸交換因子利用蛋白間的交互作用或是形成寡聚體進行活化，進而參與在調節本身的活性或是改變細胞內座落的位置。近來在我們實驗室的研究中也發現鳥糞嘌呤核苷酸交換因子Syt1p也可幫助第一腺嘌呤核苷二磷酸核糖化相似因子(Arl1p)的活化，進而募集下游的Imh1p到反式高基氏體上，且共同參與在Ypt6p調節液胞型態的能力。在此篇研究中，我們利用活體外結合試驗發現Syt1p能夠利用SEC7區域與pleckstrin同源結構域之間的其中一個羧基端片段(Syt1pC1)來進行分子間的結合。在細胞內若將此羧基端片段去除後確實會影響Syt1p的自相互作用，表示Syt1p確實能夠利用此羧基端片段相互結合形成二聚體(dimer)或寡聚體(oligomer)。而此種不能自相互作用的Syt1p並不影響到下游的Arl1p和Imh1p在細胞中的位置，但是在去除SYT1及YPT6基因的酵母菌中卻會影響到Ypt6p調節液胞型態的能力，且在剔除YPT6基因的細胞中對於溫度的敏感性也有些微的影響。這些發現都暗示著Syt1p形成二聚體(dimer)可能也參與在本身催化鳥糞嘌呤核苷酸交換的活性以外的重要功能。此外Syt1p可藉由胺基端片段的磷酸化進而調控二聚體(dimer)的形成，而這些磷酸化的位置可調節鳥糞嘌呤核苷酸交換因子的活性。所以我們認為Syt1p透過一羧基端片段(Syt1pC1)形成二聚體並參與在Ypt6p調節液胞型態的路徑之中。另一方面，Syt1p的磷酸化幫助形成二聚體，進而活化第一腺嘌呤核苷二磷酸核糖化相似因子並募集下游的Imh1p。

關鍵字

高基氏體；腺嘌呤核苷二磷酸核醣化因子；小鳥糞嘌呤核苷三磷酸結合蛋白；囊泡運輸；鳥糞嘌呤核苷酸交換因子

並列摘要

ADP-ribosylation factor (ARF) family proteins are small G proteins required for the regulation of membrane traffic and cytoskeletal dynamics. They are activated by guanine nucleotide exchange factor (GEF) that can stimulate the exchange of GDP for GTP. Previous reports show that GEF activation through protein–protein interactions or oligomerization, and its regulation can affect GEF activity or alter intracellular localization. Recently, our lab has reported that Syt1p function as a GEF that activates the Arl1p to recruit Imh1p to the late Golgi, which is subsequently involved in synthetic enhancement of Ypt6p-dependent vacuole fragmentation. Here, we identify Syt1p intermolecular interaction region located between SEC7 and PH domain (Syt1pC1) by in vitro pull down. Syt1p∆C1 defined the mutant here affects Syt1p self-interaction in vivo, which indicates that Syt1p can form dimers or oligomers through the C1 region. This mutant Syt1p∆C1 does not affect Arl1p and Imh1p localization. However, Syt1p∆C1 affects Ypt6p-dependent vacuolar fragmentation in syt1∆ypt6∆ strain and causes the inability to complement the temperature sensitivity of ypt6∆. These finding indicates that Syt1p homodimer has another essential functions rather than regulating its GEF activity. Besides, Syt1p forms dimer through its N-terminal phosphorylation, and the phosphorylation sites are required for the GEF activity. Thus, we suggest that Syt1pC1 is involved in the synthetic enhancement of Ypt6p-dependent pathway, and Syt1p dimer formation may be modulated by phosphorylation , which regulates Arl1p activation and Imh1p recruitment.

並列關鍵字

Golgi complex ； ADP-ribosylation factor ； GTPase ； vesicular trafficking ； GEF ； Sec7

參考文獻

Albuquerque, C.P., Smolka, M.B., Payne, S.H., Bafna, V., Eng, J., and Zhou, H. (2008). A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol Cell Proteomics 7, 1389-1396.

Anders, N., and Jürgens, G. (2008). Large ARF guanine nucleotide exchange factors in membrane trafficking. Cellular and Molecular Life Sciences 65, 3433-3445.

Anders, N., Nielsen, M., Keicher, J., Stierhof, Y.D., Furutani, M., Tasaka, M., Skriver, K., and Jurgens, G. (2008). Membrane Association of the Arabidopsis ARF Exchange Factor GNOM Involves Interaction of Conserved Domains. The Plant Cell Online 20, 142-151.

Behnia, R., and Munro, S. (2005). Organelle identity and the signposts for membrane traffic. Nature 438, 597-604.

Behnia, R., Panic, B., Whyte, J.R.C., and Munro, S. (2004). Targeting of the Arf-like GTPase Arl3p to the Golgi requires N-terminal acetylation and the membrane protein Sys1p. Nature Cell Biology 6, 405-413.

國際替代計量

探討第一腺嘌呤核苷二磷酸核糖化相似因子之鳥糞嘌呤核苷酸交換因子的自相互作用的功能特性

全文下載

主題瀏覽