Assembly & Conductance of Fo Sector of E coli H+-ATPase

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DESCRIPTION: (Applicant's Description) F-type ATPases synthesize most of the ATP in biological systems. These enzymes interconvert the energy stored in a transmembrane proton gradient with cellular ATP levels. All three specific aims in this proposal address questions about the structure and function of the F0 sector, which can exist in different physical and functional states, depending on assembly state, changes in gene expression, and/or response to metabolic circumstances. The first aim is to test the hypothesis that changes in stoichiometry of the C subunit changes the catalytic and energy-coupling properties of the enzyme. The C stoichiometry will be manipulated either genetically by altering expression of uncE (c subunit) or metabolically by growing wild type cells on different carbon sources, at different pH's, or under aerobic vs. anaerobic conditions. Isolated membranes will be assayed for membrane-bound ATPase and ATP synthase activities, and the relativtive stoichiometries of the different F0 sectors will be quantified by immunoprecipitation and immunoblotting with anti-c antibodies. The second aim is to test the hypothesis that a change in c stoichiometry can change the proton conductance of purified F0 sectors. F0 containing different numbers of c subunit will be purified and reconstituted into liposomes, and the single-channel F0 proton flux will by measured after the formation of a potassium/valinomycin-induced electrical gradient. An additional goal of these studies is to modify the F0 purification procedure to minimize or eliminate contaminating ion-channel activities. The third aim is to examine the assembly of the F0, first by characterizing the nature of the F0 synthesized and assembled in the absence of the F1 subunits, and then by determining which F1 subunits interact with that immature F0 to produce the mature, proton-conducting F0 sector. By combining standard membrane fluorescence quenching assays with an improved liposome proton-flux assay, these studies will test the ability of different F1 subunits to assist the insertion, assembly, or stability of the newly-synthesized F0.