Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex.
Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex.
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DOI：
10.1091/mbc.e12-05-0343
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发表时间：
2012-12
影响因子：
3.3
通讯作者：
Merz AJ
中科院分区：
文献类型：
--
作者：
Lobingier BT;Merz AJ
Vps33 是可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE) 伴侣 Sec1/Munc18 家族的成员，是同型融合和蛋白质分选以及 C 类核心液泡/内体束缚复合物的亚基，对于内溶酶体运输至关重要。在这项研究中，使用遗传和生化方法研究了 Vps33 与 SNARE 蛋白的相互作用。可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE) 蛋白催化分泌和内溶酶体系统中的膜融合事件，所有 SNARE 介导的融合过程都需要 Sec1/Munc18 (SM) 家族的辅助因子。 Vps33 是一种 SM 蛋白，是 Vps-C 复合物 HOPS（同型融合和蛋白质分选）和 CORVET（C 类核心液泡/内体束缚）的亚基，它们是内吞运输的中央调节因子。在这里，我们提出了酿酒酵母液泡 SNARE 与 HOPS 全复合物或单独的 Vps33 之间相互作用的生化研究。 HOPS 结合 Qa 家族 SNARE Vam3 的 N 端 Habc 结构域，但这种相互作用不需要 Vps33。相反，Vps33 结合 Vam3、Vam7 和 Nyv1 的 SNARE 结构域。 Vps33 直接结合液泡四元 SNARE 复合物，并且 Vps33 对 SNARE 复合物的亲和力大于对单个 SNARE 的亲和力。通过靶向突变分析，我们发现了 Vps33 的错义突变，这些突变会产生一系列新的缺陷，包括货物错误分类和 Vps33-HOPS 关联的丧失。这些数据共同提出了膜对接的工作模型：HOPS 通过 Vps33 独立的相互作用与 Vam3 和 Vam7 的 N 端结构域相关联，随后 Vps33（HOPS SM 蛋白）与 SNARE 结构域结合，最后与四元 SNARE 复合物结合。我们的结果还强化了这样的假设：SNARE 复合物结合是 SM 蛋白功能的核心属性。
Vps33, a member of the Sec1/Munc18 family of soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) chaperones, is a subunit of the homotypic fusion and protein sorting and class C core vacuole/endosome tethering complexes and essential for endolysosomal transport. In this study, Vps33 interactions with SNARE proteins are investigated using genetic and biochemical approaches. Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal Habc domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function.