Hutagalung Alex H, Coleman Jeff, Pypaert Marc, Novick Peter J
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Mol Biol Cell. 2009 Jan;20(1):153-63. doi: 10.1091/mbc.e08-02-0157. Epub 2008 Oct 22.
The exocyst consists of eight rod-shaped subunits that align in a side-by-side manner to tether secretory vesicles to the plasma membrane in preparation for fusion. Two subunits, Sec3p and Exo70p, localize to exocytic sites by an actin-independent pathway, whereas the other six ride on vesicles along actin cables. Here, we demonstrate that three of the four domains of Exo70p are essential for growth. The remaining domain, domain C, is not essential but when deleted, it leads to synthetic lethality with many secretory mutations, defects in exocyst assembly of exocyst components Sec5p and Sec6p, and loss of actin-independent localization. This is analogous to a deletion of the amino-terminal domain of Sec3p, which prevents an interaction with Cdc42p or Rho1p and blocks its actin-independent localization. The two mutations are synthetically lethal, even in the presence of high copy number suppressors that can bypass complete deletions of either single gene. Although domain C binds Rho3p, loss of the Exo70p-Rho3p interaction does not account for the synthetic lethal interactions or the exocyst assembly defects. The results suggest that either Exo70p or Sec3p must associate with the plasma membrane for the exocyst to function as a vesicle tether.
外排体由八个杆状亚基组成，这些亚基并排排列，将分泌囊泡拴系到质膜上，为融合做准备。两个亚基Sec3p和Exo70p通过一条不依赖肌动蛋白的途径定位于胞吐位点，而其他六个亚基则沿着肌动蛋白丝束搭在囊泡上。在此，我们证明Exo70p的四个结构域中的三个对生长至关重要。剩下的结构域C并非必需，但缺失时会导致与许多分泌突变产生合成致死性，外排体组分Sec5p和Sec6p在外排体组装上出现缺陷，以及丧失不依赖肌动蛋白的定位。这类似于Sec3p氨基末端结构域的缺失，它会阻止与Cdc42p或Rho1p的相互作用，并阻断其不依赖肌动蛋白的定位。这两个突变是合成致死的，即使存在高拷贝数的抑制子，它们可以绕过单个基因的完全缺失。尽管结构域C结合Rho3p，但Exo70p与Rho3p相互作用的丧失并不能解释合成致死相互作用或外排体组装缺陷。结果表明，为使外排体作为囊泡拴系发挥作用，Exo70p或Sec3p必须与质膜结合。
