Han S Y, Park D Y, Park S D, Hong S H
School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
Biochem J. 2000 Nov 15;352 Pt 1(Pt 1):165-73.
In this study we show the interaction of N-ethylmaleimide-sensitive fusion protein (NSF) with a small GTP-binding protein, Rab6. NSF is an ATPase involved in the vesicular transport within eukaryotic cells. Using the yeast two-hybrid system, we have isolated new NSF-binding proteins from the rat lung cDNA library. One of them was Rab6, which is involved in the vesicular transport within the Golgi and trans-Golgi network as a Ras-like GTPase. We demonstrated that the N-terminal domain of NSF interacted with the C-terminal domain of Rab6, and these proteins were co-immunoprecipitated from the rat brain extract. This interaction was maintained preferentially in the presence of hydrolysable ATP. Recombinant NSF-His(6) can also bind to C-terminal Rab6-glutathione S-transferase under the conditions to allow the ATP hydrolysis. Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx. 2-fold as does alpha-soluble NSF attachment protein receptor. Anti-Rab6 polyclonal antibodies significantly inhibited the Rab6-stimulated ATPase activity of NSF. Furthermore, we found that Rab3 and Rab4 can also associate with NSF and stimulate its ATPase activity. Taken together, we propose a model in which Rab can form an ATP hydrolysis-regulated complex with NSF, and function as a signalling molecule to deliver the signal of vesicle fusion through the interaction with NSF.
在本研究中，我们展示了N - 乙基马来酰亚胺敏感融合蛋白（NSF）与一种小GTP结合蛋白Rab6的相互作用。NSF是一种参与真核细胞内囊泡运输的ATP酶。利用酵母双杂交系统，我们从大鼠肺cDNA文库中分离出了新的NSF结合蛋白。其中之一是Rab6，它作为一种类Ras GTP酶参与高尔基体和反式高尔基体网络内的囊泡运输。我们证明了NSF的N末端结构域与Rab6的C末端结构域相互作用，并且这些蛋白可从大鼠脑提取物中进行共免疫沉淀。这种相互作用在可水解ATP存在的情况下优先维持。重组NSF - His(6)在允许ATP水解的条件下也能与C末端Rab6 - 谷胱甘肽S - 转移酶结合。令人惊讶的是，Rab6刺激NSF的ATP酶活性约2倍，α - 可溶性NSF附着蛋白受体也有同样的效果。抗Rab6多克隆抗体显著抑制Rab6刺激的NSF的ATP酶活性。此外，我们发现Rab3和Rab4也能与NSF结合并刺激其ATP酶活性。综上所述，我们提出了一个模型，其中Rab可以与NSF形成一种受ATP水解调节的复合物，并作为一种信号分子，通过与NSF的相互作用传递囊泡融合信号。
