Luo Guangzuo, Zhang Jian, Guo Wei
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018.
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018
Mol Biol Cell. 2014 Nov 15;25(23):3813-22. doi: 10.1091/mbc.E14-04-0907. Epub 2014 Sep 17.
During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.
在膜运输过程中，囊泡载体在可溶性N - 乙基马来酰亚胺敏感因子附着蛋白（SNARE）介导的膜融合之前，被运输并拴系到它们对应的受体区室。外排体复合物被认为在胞吐作用期间将高尔基体后分泌囊泡靶向并拴系到质膜。然而，没有确凿的实验证据支持这一观点。我们在酵母中开发了一种异位靶向测定法，其中八个外排体亚基中的每一个都在线粒体表面表达。我们发现大多数外排体亚基能够在那里招募复合物的其他成员，并且外排体的错误靶向导致细胞中的分泌缺陷。另一方面，只有异位定位的Sec3p亚基能够将分泌囊泡招募到线粒体。我们的测定还表明，胞质扩散和基于细胞骨架的运输在胞吐作用期间介导外排体亚基和分泌囊泡的招募。此外，Rab GTP酶Sec4p及其鸟嘌呤核苷酸交换因子Sec2p调节外排体复合物的组装。我们的研究有助于确定外排体亚基在拴系中的作用，并允许研究胞吐作用期间调节囊泡拴系的机制。
