Structure and Function of the Plant Vacuolar H+-ATPase

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The purpose of this project is to characterize the structure and function of the plant vacuolar H+-ATPase. The specific goals of the project are (i) to determine the complete subunit composition by further purification and immunoprecipitation; (ii) to study the subunit organization by dissociating and identifying the peripheral subunits; (iii) to deduce the primary structure and later to define the functional domains of selected subunits by cloning and sequencing the gene encoding the 16 kDa proteolipid; (iv) to test the function of the 16 kDa proteolipid by reconstitution and molecular genetic methods; (v) to localize the subcellular distribution of the ATPase; and (vi) to begin preliminary studies on the biosynthesis of selected ATPase subunits. Plant cells contain a large central vacuole that occupies up to 80% of the cell volume. They are important for maintaining cell turgor and for the storage of ions and metabolites. Transport processes across the vacuolar membrane (tonoplast) are controlled by electrochemical gradients that are determined in part by the tonoplast localized vacuolar ATPase (V-ATPases) which is a proton-pumping ATPase (H+-ATPase). The V-ATPases represent a class of ATPase that is present in the endomembrane system of all eukaryotic cells. Thus, investigations on the structure and function of the plant vacuolar ATPase has relevance to the function of the endomembrane system in all higher organisms.