Le Borgne R, Hoflack B
European Molecular Biology Laboratory, Heidelberg, Germany.
J Cell Biol. 1997 Apr 21;137(2):335-45. doi: 10.1083/jcb.137.2.335.
The transport of the two mannose 6-phosphate receptors (MPRs) from the secretory pathway to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi-specific assembly protein and clathrin. Using an in vitro assay that reconstitutes the ARF-1-dependent translocation of cytosolic AP-1 onto membranes of the TGN, we have previously reported that the MPRs are key components for the efficient recruitment of AP-1 (Le Borgne, R., G. Griffiths, and B. Hoflack. 1996. J. Biol. Chem. 271:2162-2170). Using a polyclonal antibody against the mouse gamma-adaptin, we have now examined the steady state distribution of AP-1 after subcellular fractionation of mouse fibroblasts lacking both MPRs or reexpressing physiological levels of either MPR. We report that the amount of AP-1 bound to membranes and associated with clathrin-coated vesicles depends on the expression level of the MPRs and on the integrity of their cytoplasmic domains. Thus, these results indicate that the concentration of the MPRs, i.e., the major transmembrane proteins sorted toward the endosomes, determines the number of clathrin-coated vesicles formed in the TGN.
两种甘露糖6-磷酸受体（MPRs）从分泌途径向胞吞途径的转运是由包被有AP-1高尔基体特异性组装蛋白和网格蛋白的载体囊泡介导的。利用一种体外检测方法，该方法可重建胞质AP-1依赖于ARF-1向反式高尔基体网络（TGN）膜上的转运，我们先前曾报道MPRs是有效招募AP-1的关键组分（勒博尔涅，R.，G. 格里菲思，和B. 霍夫拉克。1996。《生物化学杂志》271:2162 - 2170）。现在，我们使用一种针对小鼠γ衔接蛋白的多克隆抗体，在对缺乏两种MPRs或重新表达生理水平的任一MPR的小鼠成纤维细胞进行亚细胞分级分离后，检测了AP-1的稳态分布。我们报道，与膜结合并与网格蛋白包被囊泡相关的AP-1量取决于MPRs的表达水平及其胞质结构域的完整性。因此，这些结果表明，MPRs（即主要被分选至内体的跨膜蛋白）的浓度决定了在TGN中形成的网格蛋白包被囊泡的数量。
