CHARACTERIZATION OF THE COATED VESICLE PROTON PUMP

涂层囊泡质子泵的表征

• 批准号: 3285548
• 负责人: MICHAEL D FORGAC
• 金额: $ 12.8万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-30 至 1988-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3285548
• 关键词: Golgi apparatus; acid base balance; adenosine triphosphate; adenosinetriphosphatase; affinity labeling; antibody; fluorescence spectrometry; ligands; lysosomes; membrane model; membrane reconstitution /synthesis; membrane structure; phospholipids; receptor mediated endocytosis

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.

这项研究的长期目标是确定其结构，