TerBush D R, Maurice T, Roth D, Novick P
Yale University School of Medicine, Department of Cell Biology, New Haven, CT 06520-8002, USA.
EMBO J. 1996 Dec 2;15(23):6483-94.
In the yeast Saccharomyces cerevisiae, the products of at least 15 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Previously, we have shown that three of these genes, SEC6, SEC8 and SEC15, encode components of a multisubunit complex which localizes to the tip of the bud, the predominant site of exocytosis in S. cerevisiae. Mutations in three more of these genes, SEC3, SEC5 and SEC10, were found to disrupt the subunit integrity of the Sec6-Sec8-Sec15 complex, indicating that these genes may encode some of the remaining components of this complex. To examine this possibility, we cloned and sequenced the SEC5 and SEC10 genes, disrupted them, and either epitope tagged them (Sec5p) or prepared polyclonal antisera (Sec10p) to them for co-immunoprecipitation studies. Concurrently, we biochemically purified the remaining unidentified polypeptides of the Sec6-Sec8-Sec15 complex for peptide microsequencing. The genes encoding these components were identified by comparison of predicted amino acid sequences with those obtained from peptide microsequencing of the purified complex components. In addition to Sec6p, Sec8p and Sec15p, the complex contains the proteins encoded by SEC3, SEC5, SEC10 and a novel gene, EXO70. Since these seven proteins function together in a complex required for exocytosis, and not other intracellular trafficking steps, we have named it the Exocyst.
在酿酒酵母中，至少15个基因的产物专门参与从高尔基体到质膜的囊泡运输。此前，我们已经表明，其中三个基因SEC6、SEC8和SEC15编码一种多亚基复合物的组分，该复合物定位于芽的顶端，这是酿酒酵母中胞吐作用的主要部位。另外三个基因SEC3、SEC5和SEC10的突变被发现会破坏Sec6-Sec8-Sec15复合物的亚基完整性，这表明这些基因可能编码该复合物的一些其余组分。为了检验这种可能性，我们克隆并测序了SEC5和SEC10基因，破坏了它们，并对它们进行表位标记（Sec5p）或制备针对它们的多克隆抗血清（Sec10p）用于共免疫沉淀研究。同时，我们对Sec6-Sec8-Sec15复合物中其余未鉴定的多肽进行了生化纯化，用于肽微测序。通过将预测的氨基酸序列与从纯化的复合物组分的肽微测序获得的序列进行比较，鉴定了编码这些组分的基因。除了Sec6p、Sec8p和Sec15p外，该复合物还包含由SEC3、SEC5、SEC10和一个新基因EXO70编码的蛋白质。由于这七种蛋白质在胞吐作用所需的复合物中共同发挥作用，而不是在其他细胞内运输步骤中，我们将其命名为外排体。
