COATED VESICLE PROTON PUMP

本提案的长期目标是了解 （H+）-液泡家族的结构、机制和调控 ATP酶（或V-ATP酶）。 V-ATP酶负责酸化 在真核细胞中， 在许多细胞过程中起作用，包括受体介导的 内吞作用，细胞内膜运输，大分子降解 以及小分子的加工和耦合运输。 我们 以前的研究集中在V-ATPase的结构分析上 的网格蛋白包被囊泡，并提供了深入了解其 胞内分布 我们也开始着手解决 的亚基在V-ATP酶复合物和特定残基的作用， 催化和调节。 我们先前已经鉴定了73 kDa A亚基的Cys 254为 负责V-ATP酶对巯基敏感性的残基 试剂，并提出了证据，其参与，通过 二硫键的形成对V-ATP酶活性的调节。 进一步 定义位于A上的核苷酸结合位点的结构 亚基，将鉴定Cys 254的二硫键结合伴侣， 将确定与[32 P]-2-叠氮基-ATP的反应位点。 B 还将鉴定由[32 P]-Bz-ATP标记的亚基肽。 的 外周V1结构域中特定亚基的功能将被 通过使用蛋白质分离解离的V1多肽来解决 化学和免疫化学技术以及分离的 与完整V0结构域的不同组合的多肽， 这个实验室以前制定的方案。 重新组装 还将从其分离的组分中进行积分V0域 为了说明特定V0亚基在质子传导中的作用， 以及被动质子流的调节机制。 实验 以定位巴弗洛霉素结合的位点。 最后，将对MDBK细胞中V-ATP酶的组装途径进行研究。 通过用[35 S]甲硫氨酸代谢标记V-ATP酶测定 和免疫沉淀的标记泵使用两个可用的 制备的抗合成抗体的单克隆抗体和多克隆抗血清 缩氨酸 这些研究应该提供重要的新见解， 这类重要的（H+）-ATP酶。

The long-term objectives of this proposal are to understand the structure, mechanism and regulation of the vacuolar family of (H+)- ATPases (or V-ATPases). The V-ATPases are responsible for acidification of intracellular compartments in eukaryotic cells and serve an important function in a number of cellular processes, including receptor-mediated endocytosis, intracellular membrane traffic, macro-molecular degradation and processing and the coupled transport of small molecules. Our previous studies have focused on a structural analysis of the V-ATPase of clathrin-coated vesicles and have provided insight into its intracellular distribution. We have also begun to address the function of subunits in the V-ATPase complex and the role of specific residues in catalysis and regulation. We have previously identified Cys254 of the 73 kDa A subunit as the residue responsible for the sensitivity of the V-ATPases to sulfhydryl reagents and have presented evidence for its involvement through disulfide bond formation in regulation of V-ATPase activity. To further define the structure of the nucleotide binding site located on the A subunit, the disulfide bonded partner of Cys254 will be identified and the sites of reaction with [32P]-2-azido-ATP will be determined. B subunit peptides labeled by [32P]-Bz-ATP will also be identified. The function of specific subunits in the peripheral V1 domain will be addressed by separation of the dissociated V1 polypeptides using protein chemical and immunochemical techniques and reassembly of the separated polypeptides in different combinations with the integral V0 domain using the protocol previously worked out in this laboratory. Reassembly of the integral V0 domain from its separated components will also be carried out in order to address the role of specific V0 subunits in proton conduction and the mechanism by which passive proton flux is regulated. Experiments to localize the site of bafilomycin binding will also be performed. Finally, the assembly pathway of the V-ATPase in MDBK cells will be determined by metabolic labeling of the V-ATPase with [35S] methionine and immunoprecipitation of the labeled pumps using both available monoclonal antibodies and polyclonal antisera prepared against synthetic peptides. These studies should provide significant new insights into this important class of (H+)-ATPases.