Okamoto C T, Karam S M, Jeng Y Y, Forte J G, Goldenring J R
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles 90033, USA.
Am J Physiol. 1998 Apr;274(4):C1017-29. doi: 10.1152/ajpcell.1998.274.4.C1017.
gamma-Adaptin and clathrin heavy chain were identified on tubulovesicles of gastric oxyntic cells with the anti-gamma-adaptin monoclonal antibody (MAb) 100/3 and an anti-clathrin heavy chain MAb (MAb 23), respectively. In Western blots, crude gastric microsomes from rabbit and rat and density gradient-purified, H-K-ATPase-rich microsomes from these same species were immunoreactive for gamma-adaptin and clathrin. In immunofluorescent labeling of isolated rabbit gastric glands, anti-gamma-adaptin and anti-clathrin heavy chain immunoreactivity appeared to be concentrated in oxyntic cells. In primary cultures of rabbit oxyntic cells, the immunocytochemical distribution of gamma-adaptin immunoreactivity was similar to that of the tubulovesicular membrane marker in oxyntic cells, the H-K-ATPase. Further biochemical characterization of the tubulovesicular gamma-adaptin-containing complex suggested that it has a subunit composition that is typical of that for a clathrin adaptor: in addition to the gamma-adaptin subunit, it contains a beta-adaptin subunit and other subunits of apparent molecular masses of 50 kDa and 19 kDa. From solubilized gastric microsomes from rabbit, gamma-adaptin could be copurified with the major cargo protein of tubulovesicles, the H-K-ATPase. Thus this tubulovesicular coat may bind directly to the H-K-ATPase and may thereby mediate the regulated trafficking of the H-K-ATPase at the apical membrane of the oxyntic cell during the gastric acid secretory cycle. Given the similarities of the regulated trafficking of the H-K-ATPase with recycling of cargo through the apical recycling endosome of many epithelial cells, we propose that tubulovesicular clathrin and adaptors may regulate some part of an apical recycling pathway in other epithelial cells.
分别用抗γ-衔接蛋白单克隆抗体（MAb）100/3和抗网格蛋白重链单克隆抗体（MAb 23）在胃壁细胞的微管泡上鉴定出γ-衔接蛋白和网格蛋白重链。在蛋白质免疫印迹中，来自兔和大鼠的粗制胃微粒体以及从这些相同物种中经密度梯度纯化的富含H⁺-K⁺-ATP酶的微粒体对γ-衔接蛋白和网格蛋白呈免疫反应性。在分离的兔胃腺的免疫荧光标记中，抗γ-衔接蛋白和抗网格蛋白重链的免疫反应性似乎集中在壁细胞中。在兔壁细胞的原代培养中，γ-衔接蛋白免疫反应性的免疫细胞化学分布与壁细胞中微管泡膜标志物H⁺-K⁺-ATP酶的分布相似。对含γ-衔接蛋白的微管泡复合物的进一步生化特性分析表明，其亚基组成是网格蛋白衔接蛋白的典型组成：除了γ-衔接蛋白亚基外，它还包含一个β-衔接蛋白亚基以及表观分子量为50 kDa和19 kDa的其他亚基。从兔的可溶胃微粒体中，γ-衔接蛋白可与微管泡的主要货物蛋白H⁺-K⁺-ATP酶共纯化。因此，这种微管泡衣被可能直接与H⁺-K⁺-ATP酶结合，从而在胃酸分泌周期中介导壁细胞顶端膜上H⁺-K⁺-ATP酶的调节性转运。鉴于H⁺-K⁺-ATP酶的调节性转运与许多上皮细胞通过顶端回收内体进行货物回收的相似性，我们提出微管泡网格蛋白和衔接蛋白可能调节其他上皮细胞中顶端回收途径的某些部分。
