The generation of intracellular proton gradients across different intracellular membranes is necessary for the function of these membranes in a variety of cellular events. These diverse activities include the synthesis of ATP in mitochondria, disassembly of receptor ligand complexes within endosomal and lysosomal compartments in addition to the regulation of vesicular mediated membrane sorting within secretory elements. At the present time investigators are just beginning to define the structure and control of membrane bound ATPases which participate in the ATP dependent generaton of these proton gradients. In many cases these enzymes exhibit sensitivity to a common inhibitor. The ATPase complex of mitochondria is the best characterized of the intracellular proton translocating ATPases with regard to its structure and function. However these studies in the mitochondrial enzyme have been restricted for the most part to the sites of inhibitor binding analysis in vitro. The mitochondrial ATPase complex from yeast is genetically and biochemically the best characterized eukaryotic ATPase. In the proposed studies, the genes encoding the catalytic and associated subunits of this complex, isolated in previous and the proposed studies will be used to define at molecular detail the residues which directly participate in the binding hydrolysis and synthesis of ATP. These studies will also define the subunit-subunit, residue-residue contacts which are required to transduce an electrochemical gradient of protons to a high energy phosphate bond or its reversal and how this process may be controlled. These studies will utilize either localized or site directed in vitro mutagenesis of isolated genes encoding different ATPase subunits followed by the analysis of these mutations at the nucleotide level. The function and biochemistry of these modified subunits will be analyzed both in vivo by analysis of the mitochondrial ATPase from a host in which the gene encoding the wild type ATPase subunit has been replaced by mutant gene. The proposed in vitro analysis will extend our enzymatic, immunological and reconstitution techniques. Analysis of the F/1-ATPase at this detail will define for the first time the sites of subunit interplay in ATP-dependent H+ gradient formation and how this process is controlled.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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