Advani R J, Yang B, Prekeris R, Lee K C, Klumperman J, Scheller R H
Medical School, University of Utrecht, Institute for Biomembranes, 3584CX Utrecht, The Netherlands.
J Cell Biol. 1999 Aug 23;146(4):765-76. doi: 10.1083/jcb.146.4.765.
A more complete picture of the molecules that are critical for the organization of membrane compartments is beginning to emerge through the characterization of proteins in the vesicle-associated membrane protein (also called synaptobrevin) family of membrane trafficking proteins. To better understand the mechanisms of membrane trafficking within the endocytic pathway, we generated a series of monoclonal and polyclonal antibodies against the cytoplasmic domain of vesicle-associated membrane protein 7 (VAMP-7). The antibodies recognize a 25-kD membrane-associated protein in multiple tissues and cell lines. Immunohistochemical analysis reveals colocalization with a marker of late endosomes and lysosomes, lysosome-associated membrane protein 1 (LAMP-1), but not with other membrane markers, including p115 and transferrin receptor. Treatment with nocodozole or brefeldin A does not disrupt the colocalization of VAMP-7 and LAMP-1. Immunoelectron microscopy analysis shows that VAMP-7 is most concentrated in the trans-Golgi network region of the cell as well as late endosomes and transport vesicles that do not contain the mannose-6 phosphate receptor. In streptolysin- O-permeabilized cells, antibodies against VAMP-7 inhibit the breakdown of epidermal growth factor but not the recycling of transferrin. These data are consistent with a role for VAMP-7 in the vesicular transport of proteins from the early endosome to the lysosome.
通过对膜运输蛋白囊泡相关膜蛋白（也称为突触小泡蛋白）家族中的蛋白质进行表征，一幅关于对膜区室组织至关重要的分子的更完整图景正开始浮现。为了更好地理解内吞途径中的膜运输机制，我们针对囊泡相关膜蛋白7（VAMP - 7）的胞质结构域制备了一系列单克隆抗体和多克隆抗体。这些抗体在多种组织和细胞系中识别一种25kD的膜相关蛋白。免疫组织化学分析显示其与晚期内体和溶酶体的标志物溶酶体相关膜蛋白1（LAMP - 1）共定位，但与其他膜标志物，包括p115和转铁蛋白受体不共定位。用诺考达唑或布雷菲德菌素A处理不会破坏VAMP - 7和LAMP - 1的共定位。免疫电子显微镜分析表明，VAMP - 7最集中在细胞的反式高尔基体网络区域以及不包含甘露糖 - 6 - 磷酸受体的晚期内体和运输小泡中。在链球菌溶血素O通透的细胞中，针对VAMP - 7的抗体抑制表皮生长因子的降解，但不抑制转铁蛋白的循环利用。这些数据与VAMP - 7在蛋白质从早期内体到溶酶体的囊泡运输中所起的作用一致。
