CHARACTERIZATION OF THE COATED VESICLE PROTON PUMP

这项研究的长期目标是确定结构， 依赖三磷酸腺苷的质子泵的分布及其调节机制 包裹着网状蛋白的囊泡。最初的努力将集中在亲和力上 该泵的标识、提纯和改装。 酸化已被确定为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、表皮生长因子和去唾液酸糖蛋白)从它们的 低pH值(5.0-6.0)下的受体和受体循环受阻 通过消散pH梯度的试剂。这些药剂还可以阻止感染。 由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值激活了配体-受体的解离 受体循环所必需的，而其他研究表明，这是 酸化发生在溶酶体前隔室。 我们已经确定了一种依赖于三磷酸腺苷的质子泵能够酸化 网状蛋白包裹的囊泡，已知的最早的内吞体内隔室 路径。这台泵的部分特点是它的 离子转运特性、周转过程中的磷酸化及抑制物 敏感度。亲和力标签研究将使用 三磷酸腺苷保护抑制剂NBD-C1用于识别三磷酸腺苷结合亚单位 打气筒。洗涤剂增溶蛋白的纯化及重组 将提供有关磷脂小泡的明确信息 该泵的结构、活性及其与其他阳离子的关系 运输ATPase。最后，将产生抗体来对抗纯化的 蛋白质，并用于免疫细胞化学研究以确定 这种泵在细胞内的分布。这些研究对于 我们对这种泵的理解，除了它在 受体介导的内吞作用，可能参与调节 细胞内的pH值和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.