Structure, Function and Evolution of the Vacuolar H+ ATPASE

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The vacuolar H+ -ATPase is ubiquitous in eukaryotic cells and plays a central role in vacuolar solute accumulation in plants. Like FoF1-ATPases it is insensitive to vanadate and does not have a phosphoenzyme catalytic intermediate. We and other laboratories have shown that the vacuolar H+-ATPase is a large, multimeric enzyme, 400-500 kDA, containing three major subunits an up to five minor ones. The primary structures of the 70 kDa, 60 kDa, and 16 kDa subunits are now known. The 70 and 60 kDa kDa subunits contain numerous highly conserved regions thought to be involved in catalysis and regulation in the F1 beta and alpha subunits, respectively. The PI wishes to characterize the functional domains of the 70 and 60 kDa subunits directly using a combination of chemical probes and site-directed mutagenesis. Site-directed mutagenesis studies will be carried out both in E. coli and in carrot. He has already obtained overexpression of the 70 kDa subunit in E. coli. He plans to generate vacuolar H+-ATPase null mutants in carrot by transforming with antisense DNA to the 5'-untranslated region of 70 kDa cDNA. Site-directed mutagenesis will be carried out by retransforming the null mutants. Phylogenetic comparisons will determine whether or not the vacuolar H+ATPase is related to other archaebacterial H+-ATPases besides that of Sulfolobus acidocaldarius, its closest relative. Finally, electron microscope studies aimed at characterizing the surface features and quaternary structure of the V-ATPase are described.