A sorting nexin-1 homologue, vps5p, forms a complex with vps17p and is required for recycling the vacuolar protein-sorting receptor
A sorting nexin-1 homologue, vps5p, forms a complex with vps17p and is required for recycling the vacuolar protein-sorting receptor
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DOI：
10.1091/mbc.8.8.1529
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发表时间：
1997-08-01
影响因子：
3.3
通讯作者：
Emr, SD
中科院分区：
文献类型：
--
作者：
Horazdovsky, BF;Davies, BA;Emr, SD
许多酿酒酵母空泡蛋白分选（vps）突变体表现出改变的空泡形态。与含有1-3个大空泡结构的野生型细胞不同，B类vps 5和vps 17突变体细胞含有10-20个较小的空泡样区室。为了探索这些VPS基因产物在液泡生物发生中的作用，我们克隆并测序了VPS 5，并对其蛋白产物进行了表征。预测VPS 5基因编码675个氨基酸的非常亲水的蛋白质，其显示与哺乳动物分选连接蛋白-1的显著序列同源性。针对VPS 5基因产物的多克隆抗血清检测丝氨酸残基特异性磷酸化的单个细胞质蛋白。亚细胞分级分离研究表明，Vps 5 p是相关的外围与致密的膜部分不同的高尔基体，内体，和空泡膜。发现这种关联依赖于另一种B类VPS基因产物Vps 17 p的存在。生化交联研究表明，Vps 5 p和Vps 17 p物理相互作用。基因破坏实验表明，VPS 5基因产物对细胞活力不是必不可少的;然而，携带无效等位基因的细胞含有片段化的空泡，并表现出与vps 17无效突变体相似的空泡蛋白分选缺陷。超过95%的羧肽酶Y以高尔基体修饰的p2前体形式从这些细胞中分泌。此外，Vps 10 p空泡蛋白分选受体在vps 5突变细胞中错误定位于空泡。在这些和其他观察的基础上，我们提出，Vps 5 p/Vps 17 p蛋白复合物可能参与Vps 10 p分选受体的细胞内运输，以及其他晚期高尔基体蛋白。
A number of the Saccharomyces cerevisiae vacuolar protein-sorting (vps) mutants exhibit an altered vacuolar morphology. Unlike wild-type cells that contain 1-3 large vacuolar structures, the class B vps5 and vps17 mutant cells contain 10-20 smaller vacuole-like compartments. To explore the role of these VPS gene products in vacuole biogenesis, we cloned and sequenced VPS5 and characterized its protein product. The VPS5 gene is predicted to encode a very hydrophilic protein of 675 amino acids that shows significant sequence homology with mammalian sorting nexin-1. Polyclonal antiserum directed against the VPS5 gene product detects a single, cytoplasmic protein that is phosphorylated specifically on a serine residue(s). Subcellular fractionation studies indicate that Vps5p is associated peripherally with a dense membrane fraction distinct from Golgi, endosomal, and vacuolar membranes. This association was found to be dependent on the presence of another class B VPS gene product, Vps17p. Biochemical cross-linking studies demonstrated that Vps5p and Vps17p physically interact. Gene disruption experiments show that the VPS5 gene product is not essential for cell viability; however, cells carrying the null allele contain fragmented vacuoles and exhibit defects in vacuolar protein-sorting similar to vps17 null mutants. More than 95% of carboxypeptidase Y is secreted from these cells in its Golgi-modified p2 precursor form. Additionally, the Vps10p vacuolar protein-sorting receptor is mislocalized to the vacuole in vps5 mutant cells. On the basis of these and other observations, we propose that the Vps5p/Vps17p protein complex may participate in the intracellular trafficking of the Vps10p-sorting receptor, as well as other late-Golgi proteins.