Zhang Xiaoyu, Zajac Allison, Zhang Jian, Wang Puyue, Li Ming, Murray John, TerBush Daniel, Guo Wei
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018, USA.
J Biol Chem. 2005 May 27;280(21):20356-64. doi: 10.1074/jbc.M500511200. Epub 2005 Mar 23.
The exocyst complex plays an essential role in tethering secretory vesicles to specific domains of the plasma membrane for exocytosis. However, how the exocyst complex is assembled and targeted to sites of secretion is unclear. Here, we have investigated the role of the exocyst component Exo84p in these processes. We have generated an array of temperature-sensitive yeast exo84 mutants. Electron microscopy and cargo protein traffic analyses of these mutants indicated that Exo84p is specifically involved in the post-Golgi stage of secretion. Using various yeast mutants, we systematically studied the localization of Exo84p and other exocyst proteins by fluorescence microscopy. We found that pre-Golgi traffic and polarized actin organization are required for Exo84p localization. However, none of the exocyst proteins controls Exo84p polarization. In addition, Sec3p is not responsible for the polarization of Exo84p or any other exocyst component to the daughter cell. On the other hand, several exocyst members, including Sec10p, Sec15p, and Exo70p, clearly require Exo84p for their polarization. Biochemical analyses of the exocyst composition indicated that the assembly of Sec10p, Sec15p, and Exo70p with the rest of the complex requires Exo84p. We propose that there are at least two distinct regulatory mechanisms for exocyst polarization, one for Sec3p and one for the other members, including Exo84p. Exo84p plays a critical role in both the assembly of the exocyst and its targeting to sites of secretion.
外排体复合物在将分泌囊泡拴系到质膜的特定区域以进行胞吐作用中起着至关重要的作用。然而，外排体复合物是如何组装并靶向分泌位点尚不清楚。在这里，我们研究了外排体组分Exo84p在这些过程中的作用。我们构建了一系列温度敏感型酵母exo84突变体。对这些突变体的电子显微镜和货物蛋白运输分析表明，Exo84p特别参与分泌的高尔基体后阶段。利用各种酵母突变体，我们通过荧光显微镜系统地研究了Exo84p和其他外排体蛋白的定位。我们发现高尔基体前运输和极化肌动蛋白组织是Exo84p定位所必需的。然而，没有一种外排体蛋白控制Exo84p的极化。此外，Sec3p并不负责Exo84p或任何其他外排体组分向子细胞的极化。另一方面，包括Sec10p、Sec15p和Exo70p在内的几个外排体成员显然需要Exo84p来实现它们的极化。对外排体组成的生化分析表明，Sec10p、Sec15p和Exo70p与复合物其他部分的组装需要Exo84p。我们提出，外排体极化至少有两种不同的调节机制，一种用于Sec3p，另一种用于包括Exo84p在内的其他成员。Exo84p在组装外排体及其靶向分泌位点中都起着关键作用。
