Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast.
Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast.
复制标题
信号介导的膜蛋白从高尔基体中的膜蛋白回收到酵母中的ER。
DOI：
10.1083/jcb.127.3.653
复制
发表时间：
1994-11
期刊：
影响因子：
--
通讯作者：
Emr SD
中科院分区：
文献类型：
--
作者：
Gaynor EC;te Heesen S;Graham TR;Aebi M;Emr SD
酿酒酵母Wbp 1蛋白是一种内质网（ER）I型跨膜蛋白，含有胞质二赖氨酸（KKXX）基序。该基序先前已显示指导哺乳动物细胞中I型膜蛋白的高尔基体至ER的修复（杰克逊，M. R.，T. Nilsson和P.A.彼得森1993. 121：317-333）。为了分析该基序在酵母中的作用，我们构建了SUC 2-WBP 1嵌合体，该嵌合体由正常分泌的糖蛋白转化酶的编码序列与Wbplp的COOH末端（包括跨膜结构域和16个氨基酸的胞质尾）的编码序列融合组成。使用甘露糖连接特异性抗血清的转化酶-Wbp 1融合蛋白的碳水化合物分析表明，融合蛋白被早期高尔基体初始α 1，6甘露糖基转移酶（Och 1 p）有效地修饰。亚细胞分级分离显示，> 90%的α 1，6甘露糖修饰的融合蛋白与ER（Wbp 1 p）共定位，而不与高尔基体Och 1 p-含有室或其他膜组分共定位。dilysine基序内的氨基酸变化（KK->QK、KQ或QQ）没有改变融合蛋白的初始α 1，6甘露糖修饰的动力学，但确实显著增加了更远端的高尔基体（延长α 1，6和α 1，3）甘露糖基转移酶的修饰速率。然后将这些突变体融合蛋白直接从晚期高尔基体隔室递送到液泡，在那里它们以PEP 4依赖性方式被蛋白水解裂解。虽然围绕二赖氨酸基序的氨基酸在保留能力中仅起次要作用，但改变赖氨酸相对于融合蛋白的COOH末端的位置的突变也产生了ER保留的显著缺陷。总的来说，我们的研究结果表明，KKXX基序并不简单地保留蛋白质在ER，而是指导他们快速检索从一个新的，Och 1 p-含有早期高尔基室。与哺乳动物细胞中的观察结果相似，在适当的COOH末端位置存在两个赖氨酸残基，这代表了该分选决定簇的最重要特征。
The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317-333). To analyze the role of this motif in yeast, we constructed a SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbp1 fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revealed that > 90% of the alpha 1,6 mannose-modified fusion protein colocalized with the ER (Wbp1p) and not with the Golgi Och1p-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ, or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating alpha 1,6 and alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Och1p-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important features of this sorting determinant.