Saraste J, Palade G E, Farquhar M G
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Cell Biol. 1987 Nov;105(5):2021-9. doi: 10.1083/jcb.105.5.2021.
A 58-kD cis-Golgi protein has been identified by generating polyclonal antibodies against heavy (cis) Golgi subfractions. Total microsomes isolated from rat pancreatic homogenates were subfractionated to yield a rough microsomal fraction (B1) and three smooth membrane subfractions (B2-B4) enriched in cis-, middle, and trans-Golgi elements, respectively. The heavy (cis) subfraction, B2 (d = 1.17 g/ml), was fractionated by Triton X-114 phase separation, and the proteins recovered in the detergent phase were used to immunize rabbits. One of the anti-B2 antibodies obtained gave a "Golgi"-staining pattern when screened by immunofluorescence on normal rat kidney cells and mouse RPC 5.4 myeloma cells. In rat pancreatic exocrine cells the antibody reacted with the plasmalemma as well as elements in the Golgi region. By immunoelectron microscopy, the antigen recognized by anti-B2 IgG was found to be restricted to cis-Golgi elements in myeloma cells where it was concentrated in the fenestrated cis-most cisterna and in some of the tubules and vesicles located along the cis face of the Golgi complex. By immunoprecipitation and immunoblotting, the anti-B2 IgG exclusively recognized a 58-kD protein in myeloma cells. The anti-B2 IgG reacted with several proteins in solubilized pancreatic B2 membranes, including a 58-kD protein, but affinity-purified anti-58-kD IgG reacted exclusively with the 58-kD protein. These results suggest that the 58-kD protein is a specific component of cis-Golgi membranes.
通过生成针对重（顺式）高尔基体亚组分的多克隆抗体，已鉴定出一种58-kD的顺式高尔基体蛋白。从大鼠胰腺匀浆中分离出的总微粒体被亚分级分离，以产生富含顺式、中间和反式高尔基体成分的粗微粒体组分（B1）和三个光滑膜亚组分（B2 - B4）。重（顺式）亚组分B2（d = 1.17 g/ml）通过Triton X - 114相分离进行分级分离，去污剂相中回收的蛋白质用于免疫兔子。在正常大鼠肾细胞和小鼠RPC 5.4骨髓瘤细胞上通过免疫荧光筛选时，获得的一种抗B2抗体呈现出“高尔基体”染色模式。在大鼠胰腺外分泌细胞中，该抗体与质膜以及高尔基体区域的成分发生反应。通过免疫电子显微镜观察，抗B2 IgG识别的抗原在骨髓瘤细胞中仅限于顺式高尔基体成分，它集中在最靠近顺式面的有孔扁平囊泡以及沿着高尔基体复合体顺式面分布的一些小管和囊泡中。通过免疫沉淀和免疫印迹分析，抗B2 IgG在骨髓瘤细胞中仅识别一种58-kD的蛋白质。抗B2 IgG与溶解的胰腺B2膜中的几种蛋白质发生反应，包括一种58-kD的蛋白质，但亲和纯化的抗58-kD IgG仅与58-kD蛋白质发生反应。这些结果表明，58-kD蛋白质是顺式高尔基体膜的一种特异性成分。
