SUBUNIT STRUCTURE AND FUNCTION IN VACUOLAR H+/ATPASES

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The aim of the proposed research is to define the subunit structure and function relationships in vacuolar proton-translocating ATPases (H+- ATPases). Vacuolar H+-ATPases are found in all eukaryotic cells, where they may play both constitutive and specialized roles. Constitutive organelle acidification is involved in receptor-mediated endocytosis, targeting of proteins in the biosynthetic pathway, activation of zymogens, protein degradation, ion homeostasis, and uptake of basic compounds into regulated secretory granules. Specialized cells of the kidney and bone contain vacuolar H+-ATPases at the plasma membrane, where they pump protons out of the cell, resulting in urinary acidification and bone resorption. Constitutive organelle acidification is also exploited by viruses, such as influenza, toxins, including diphtheria toxin, and cellular pathogens, such as Salmonella,in invasion of the host cell, suggesting that the ability to regulate compartment acidification could have implications for a wide variety of human disease states. Although the cellular regulation of acidification is quite complex, it is clear that the central player is the vacuolar H+ATPase. Vacuolar H+-ATPases are remarkably similar in fungi, plants, and animals, both in overall structure and in the primary amino acid sequences of the subunits. This proposal focuses on the vacuolar H+-ATPase of the yeast Saccharomyces cerevisiae, with the goal of bridging the genetic information available about the enzyme in yeast and the biochemical characterization derived from work on other cell types. The experiments proposed will also extend the characterization of several of the subunits that have not been well- studied. A collection of subunit-specific antibodies will be generated and used as probes in biochemical studies aimed at defining subunit interactions in the native enzyme. In order to address structure- function relationships in the peripheral sector of the enzyme, which contains the catalytic sites for ATP hydrolysis, the structural genes for three of the peripheral subunits will be randomly mutagenized and mutations affecting assembly and function of the enzyme will be identified. The functional relationships in the membrane sector, containing the proton pore, will be elucidated by biochemicaIly defining interactions between the membrane subunits and the regions of these subunits important for binding of the peripheral sector and coupling of ATP hydrolysis and proton transport. Structural models generated by both the biochemical and genetic approaches will be genetically tested by directed mutagenesis of regions of the subunit genes believed to be functionally important.