Regulation of membrane traffic between the ER and Golgi by SNARE-associated proteins, ZW10/RINT-1

SNAREs（可溶性NSF附着蛋白受体）参与真核细胞囊泡运输途径中的膜融合。我们之前报道了syntaxin 18，一种哺乳动物er定位的SNARE，与其他SNARE （BNIP1， p31和Sec22b）和外周膜蛋白ZW10和RINT-1)形成复合物。在本研究中，我们检测了ZW10/RINT-1复合物在内质网和高尔基体之间的膜运输中的功能，得到了以下结果。1) ZW10与动力蛋白的相互作用蛋白质从内质网转运到高尔基体是由微管运动蛋白动力蛋白-动力蛋白介导的。我们发现dynactin复合物的一个亚基dynamictin和RINT-1以竞争的方式结合到ZW10的n端区域。ZW10的n端区域与动力蛋白在体内相互作用的证据是通过使用去二氢愈创木酸获得的，一种已知可以刺激动力蛋白-动力蛋白介导过程的试剂。这些结果提出了内质网膜上的ZW10通过与动力蛋白的相互作用被招募来运输载体的可能性。2)在哺乳动物细胞中，ZW10与涂层亚单位scopi包被囊泡的相互作用负责蛋白质从内质网转运到高尔基体。结合实验显示，β、γ和δ-COPI与ZW10存在弱相互作用，支持ZW10存在于copi包被的囊泡上并作为动力蛋白-动力蛋白受体的观点。3) NAG（神经母细胞瘤扩增基因）产物与ZW10/ rint -1相互作用。我们发现NAG是syntaxin 18复合物的一个组成部分。NAG的n端区域与p31的n端区域相互作用，NAG的c端区域与ZW10/RINT-1结合。

SNAREs (Soluble NSF attachment protein receptors) are involved in membrane fusion in the vesicular transport pathways in eukaryotic cells. We previously reported that syntaxin 18, a mammalian ER-localized SNARE, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and peripheral membrane proteins ZW10, and RINT-1). In the present study, we examined the function of a ZW10/RINT-1 complex in membrane traffic between the ER and the Golgi apparatus, and obtained the following results.1) Interaction between ZW10 and dynamitinProtein transport from the ER to the Golgi is mediated by the microtubule motor protein dynein-dynactin. We found that dynamitin, a subunit of the dynactin complex, and RINT-1 bind to the N-terminal region of ZW10 in a competitive manner. Evidence for the in vivo interaction of the N-terminal region of ZW10 with dynamitin was obtained by using nordihydroguaiaretic acid, a reagent that is known to stimulate dynein-dynactin-mediated processes. These results raise the possibility that ZW10 on the ER membrane is recruited to transport carriers through the interaction with dynamitin.2) Interaction of ZW10 with coatomer subunitsCOPI-coated vesicles are responsible for protein transport from the ER to the Golgi in mammalian cells. Binding experiments revealed that β,γ, and δ-COPI, weakly interact with ZW10, supporting the idea that ZW10 exists on COPI-coated vesicles and acts as a dynein-dynactin receptor.3) The NAG (Neuroblastoma amplified gene) product interacts with ZW10/RINT-1We discovered that NAG is a component of the syntaxin 18 complex. The N-terminal region of NAG interacts with the N-terminal region of p31, and the C-terminal region of NAG bind ZW10/RINT-1.