Gimeno R E, Espenshade P, Kaiser C A
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
J Cell Biol. 1995 Oct;131(2):325-38. doi: 10.1083/jcb.131.2.325.
SEC16 is required for transport vesicle budding from the ER in Saccharomyces cerevisiae, and encodes a large hydrophilic protein found on the ER membrane and as part of the coat of transport vesicles. In a screen to find functionally related genes, we isolated SED4 as a dosage-dependent suppressor of temperature-sensitive SEC16 mutations. Sed4p is an integral ER membrane protein whose cytosolic domain binds to the COOH-terminal domain of Sec16p as shown by two-hybrid assay and coprecipitation. The interaction between Sed4p and Sec16p probably occurs before budding is complete, because Sed4p is not found in budded vesicles. Deletion of SED4 decreases the rate of ER to Golgi transport, and exacerbates mutations defective in vesicle formation, but not those that affect later steps in the secretory pathway. Thus, Sed4p is important, but not necessary, for vesicle formation at the ER. Sec12p, a close homologue of Sed4p, also acts early in the assembly of transport vesicles. However, SEC12 performs a different function than SED4 since Sec12p does not bind Sec16p, and genetic tests show that SEC12 and SED4 are not functionally interchangeable. The importance of Sed4p for vesicle formation is underlined by the isolation of a phenotypically silent mutation, sar1-5, that produces a strong ER to Golgi transport defect when combined with sed4 mutations. Extensive genetic interactions between SAR1, SED4, and SEC16 show close functional links between these proteins and imply that they might function together as a multisubunit complex on the ER membrane.
SEC16是酿酒酵母中内质网产生运输囊泡所必需的，它编码一种存在于内质网膜上且作为运输囊泡包被一部分的大型亲水性蛋白质。在一项寻找功能相关基因的筛选中，我们分离出SED4作为温度敏感型SEC16突变的剂量依赖性抑制因子。Sed4p是一种内质网整合膜蛋白，通过双杂交分析和共沉淀表明，其胞质结构域与Sec16p的COOH末端结构域结合。Sed4p和Sec16p之间的相互作用可能在出芽完成之前就已发生，因为在出芽的囊泡中未发现Sed4p。SED4的缺失降低了内质网到高尔基体的运输速率，并加剧了囊泡形成缺陷的突变，但不影响分泌途径后期步骤的突变。因此，Sed4p对内质网处的囊泡形成很重要，但不是必需的。Sec12p是Sed4p的紧密同源物，也在运输囊泡的组装早期起作用。然而，SEC12执行的功能与SED4不同，因为Sec12p不与Sec16p结合，并且遗传测试表明SEC12和SED4在功能上不可互换。通过分离一个表型沉默的突变体sar1-5强调了Sed4p对囊泡形成的重要性，当与sed4突变结合时，该突变体会产生强烈的内质网到高尔基体的运输缺陷。SAR1、SED4和SEC16之间广泛的遗传相互作用表明这些蛋白质之间存在紧密的功能联系，并暗示它们可能在内质网膜上作为一个多亚基复合物共同发挥作用。
