Immunoisolation and characterization of a subdomain of the endoplasmic reticulum that concentrates proteins involved in COPII vesicle biogenesis
Immunoisolation and characterization of a subdomain of the endoplasmic reticulum that concentrates proteins involved in COPII vesicle biogenesis
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DOI：
10.1091/mbc.9.6.1265
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发表时间：
1998-06-01
影响因子：
3.3
通讯作者：
Farquhar, MG
中科院分区：
文献类型：
--
作者：
Hobman, TC;Zhao, BP;Farquhar, MG
风疹病毒E1糖蛋白通常在内质网（ER）中与E2复合形成异源二聚体，该异源二聚体被转运并保留在高尔基复合体中。在以前的研究中，我们发现，在E2的情况下，未组装的E1亚基积累在一个管状的前高尔基体区室，其形态和生化特性是不同的肥大的ER出口网站，扩大了响应E1的过度表达。在本研究中，我们构建了BHK细胞稳定表达E1蛋白含有一个细胞质处置表位和分离的前高尔基室从这些细胞通过细胞分级和免疫分离。细胞中的双标记间接免疫荧光和免疫分离的管状网络的免疫印迹显示，参与ER衍生的运输囊泡，即p58/ERGIC 53，Sec 23 p和Sec 13 p的形成的蛋白质集中在含E1的前高尔基体隔室。此外，在这些膜配置文件中，出芽结构是明显的，和一个高度丰富的，但未知的65 kDa的蛋白质也存在。通过比较，粗糙的ER，高尔基体和COPI囊泡的标记蛋白在这些膜中没有富集。这些结果表明，管状网络的组成对应于预期的ER出口位点。因此，我们建议将这种结构命名为SEREC（平滑ER出口隔室）。
Rubella virus E1 glycoprotein normally complexes with E2 in the endoplasmic reticulum (ER) to form a heterodimer that is transported to and retained in the Golgi complex. In a previous study, we showed that in the absence of E2, unassembled E1 subunits accumulate in a tubular pre-Golgi compartment whose morphology and biochemical properties are distinct from to hypertrophied ER exit sites that have expanded in response to overexpression of E1. In the present study we constructed BHK cells stably expressing E1 protein containing a cytoplasmically disposed epitope and isolated the pre-Golgi compartment from these cells by cell fractionation and immunoisolation. Double label indirect immunofluorescence in cells and immunoblotting of immunoisolated tubular networks revealed that proteins involved in formation of ER-derived transport vesicles, namely p58/ERGIC 53, Sec23p, and Sec13p, were concentrated in the E1-containing pre-Golgi compartment. Furthermore, budding structures were evident in these membrane profiles, and a highly abundant but unknown 65-kDa protein was also present. By comparison, marker proteins of the rough ER, Golgi, and COPI vesicles were not enriched in these membranes. These results demonstrate that the composition of the tubular networks corresponds to that expected of ER exit sites. Accordingly, we propose the name SEREC (smooth ER exit compartment) for this structure.