rsly1 binding to syntaxin 5 is required for endoplasmic reticulum-to-Golgi transport but does not promote SNARE motif accessibility
rsly1 binding to syntaxin 5 is required for endoplasmic reticulum-to-Golgi transport but does not promote SNARE motif accessibility
复制标题
DOI：
10.1091/mbc.e03-07-0535
复制
发表时间：
2004-01-01
影响因子：
3.3
通讯作者：
Hay, JC
中科院分区：
文献类型：
--
作者：
Williams, AL;Ehm, S;Hay, JC
虽然n -乙基丙烯酰亚胺敏感因子附着蛋白受体（SNARE）功能的一些原理已经被很好地理解，但关于sec1/munc18 （SM）蛋白功能及其与SNAREs的关系的细节知之甚少。流行的SM蛋白功能模型认为，这些蛋白促进或维持一个开放的和/或单体的合成蛋白分子池，可用于SNARE复合物的形成。为了研究哺乳动物内质网/高尔基体SM蛋白rsle1及其SNARE结合伙伴syntaxin 5的功能关系，我们制作了一种构象特异性单克隆抗体，该抗体仅结合可用的syntaxin 5，而不结合顺式SNARE络合或分子内封闭形式。免疫染色实验表明syntaxin 5 SNARE基序的可用性是不均匀分布和局部调控的。体外内质网-高尔基转运实验表明，rsly1是转运的迫切需要，而与syntaxin 5的结合是其功能的绝对需要。最后，在体内对rsl1 -syntaxin 5相互作用的操纵表明，它们对syntaxin 5 SNARE基序的可用池的影响非常小。我们的研究结果表明，尽管rsly1似乎不调节syntaxin 5的可用性，但其功能与syntaxin结合密切相关，可能促进SNARE复合物形成或功能的后期步骤。
Although some of the principles of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function are well understood, remarkably little detail is known about sec1/munc18 (SM) protein function and its relationship to SNAREs. Popular models of SM protein function hold that these proteins promote or maintain an open and/or monomeric pool of syntaxin molecules available for SNARE complex formation. To address the functional relationship of the mammalian endoplasmic reticulum/Golgi SM protein rsly1 and its SNARE binding partner syntaxin 5, we produced a conformation-specific monoclonal antibody that binds only the available, but not the cis-SNARE-complexed nor intramolecularly closed form of syntaxin 5. Immunostaining experiments demonstrated that syntaxin 5 SNARE motif availability is nonuniformly distributed and focally regulated. In vitro endoplasmic reticulum-to-Golgi transport assays revealed that rsly1 was acutely required for transport, and that binding to syntaxin 5 was absolutely required for its function. Finally, manipulation of rsly1-syntaxin 5 interactions in vivo revealed that they had remarkably little impact on the pool of available syntaxin 5 SNARE motif. Our results argue that although rsly1 does not seem to regulate the availability of syntaxin 5, its function is intimately associated with syntaxin binding, perhaps promoting a later step in SNARE complex formation or function.