CHARACTERIZATION OF THE COATED VESICLE PROTON PUMP

这项研究的长期目标是确定结构， ATP依赖性质子泵的分布及其调节机制 网格蛋白包被的囊泡。 最初的努力将集中在亲和力上 该泵的标记、纯化和重构。 酸化已被认为是加工过程中的一个重要步骤 受体介导的内吞作用后的配体。 因此，许多配体 （包括胰岛素、EGF 和脱唾液酸糖蛋白）与其分离 受体暴露于低 pH (5.0-6.0) 且受体循环受阻 通过消散 pH 梯度的试剂。 这些药物还可以阻止感染 某些病毒和毒素通过内吞作用进入细胞。 这些 结果表明，低 pH 值会激活配体-受体解离 受体回收所必需的，其他研究表明这 酸化发生在前溶酶体区室中。 我们已经确定了一种能够酸化的 ATP 依赖性质子泵 网格蛋白包被的囊泡，内吞细胞中已知的最早的区室 途径。 该泵的部分特征在于其 离子传输特性、周转期间的磷酸化和抑制剂 敏感性。 亲和标记研究将使用 ATP 保护抑制剂 NBD-C1 用于识别 ATP 结合亚基 泵。 洗涤剂溶解的蛋白质的纯化和重构 进入磷脂囊泡将提供关于 该泵的结构和活性及其与其他阳离子的关系 转运ATP酶。 最后，将产生针对纯化的抗体 蛋白质并用于免疫细胞化学研究以确定 该泵的细胞内分布。 这些研究对于 我们对这款泵的了解，除了它的作用之外 受体介导的内吞作用，可能参与调节 细胞内 pH 值以及某些病毒和毒素的进入。

The long term goal of this research is to determine the structure, distribution and mechanism of regulation of the ATP-dependent proton pump of clathrin-coated vesicles. Initial efforts will be focused on affinity labeling, purification and reconstitution of this pump. Acidification has been identified as an essential step in the processing of ligands following receptor-mediated endocytosis. Thus, a number of ligands (including insulin, EGF and asialoglycoproteins) dissociate from their receptors on exposure to low pH (5.0-6.0) and receptor recycling is blocked by agents which dissipate pH gradients. These agents also block infection by certain viruses and toxins which enter the cell by endocytosis. These results suggest that low pH activates the ligand-receptor dissociation necessary for receptor recycling, and other studies suggest that this acidification occurs in a prelysosomal compartment. We have identified an ATP-dependent proton pump capable of acidifying clathrin-coated vesicles, the earliest known compartment in the endocytic pathway. This pump has been partially characterized with respect to its ion transport properties, phosphorylation during turnover and inhibitor sensitivity. Affinity labeling studies will be carried out using the ATP-protectable inhibitor NBD-C1 to identify the ATP binding subunit of the pump. Purification of the detergent solubilized protein and reconstitution into phospholipid vesicles will provide definitive information on the structure and activity of this pump and on its relation to other cation transport ATPases. Finally, antibodies will be raised against the purified protein and used in immunocytochemical studies to determine the intracellular distribution of this pump. These studies are essential to our understanding of this pump which, in addition to its role in receptor-mediated endocytosis, may be involved in regulation of intracellular pH and the entry of certain viruses and toxins.