The synthesis of ATP during oxidative phosphorylation is catalyzed by a reversible H+-translocating ATPase. The enzyme has an ATP hydrolyzing sector (F1) and a proton-translocating sector (FO), which can be disassociated from each other and studied as individual entities. We are styding the FO-sector of the H+-ATPase in Escherichia coli, which is composed of 3 types of subunits. The long-term goal is to define in chemical detail how the FO moiety translocates protons across the membrane, and how proton-flux is coupled to ATP synthesis in F1. The research proposed will provide much needed chemical information on the constitutents of the H+-conducting pathway through FO, and on the functional interaction between FO and F1. Detailed chemical information on the protein components will be required before detailed mechanistic models can be proposed and tested. We will analyze E. coli mutants in FO to determine the functional role of each subunit. We will assay for loss of the following functions: (1) FO mediated H+-translocation, (2) Binding of F1 to FO (3) coupling of H+-translocation from FO to ATP synthesis in F1. The mutants will be further analyzed to determine the site of mutation so that specific amino acid residues can be related to function. This will be done by DNA sequencing. We will also test the role of specific amino acids by site-specific mutagenesis. Information on the organization and structure of subunit in FO will be obtained by chemical approaches (including cross-linking, chemical modification, limited proteolysis, and peptide directed antibodies). This information will be related to folding models predicted from the primary amino sequence. The information provided by these studies should contribute to elucidation of the mechanism of oxidative phosphorylation. Study of this membrane enzyme, which may have an unprecedented structural complexity, should also contribute to our general knowledge of membrane structure. Study of the FO sector as an H+-transporter should increase our presently vague knowledge of membrane transporters. A detailed understanding of membrane transport carriers and pumps may ultimately prove important to many areas of medical science, extending from renal and cardiac physiology to cancer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Wisconsin Madison
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Fillingame, Robert H; Steed, P Ryan (2014) Half channels mediating H(+) transport and the mechanism of gating in the Fo sector of Escherichia coli F1Fo ATP synthase. Biochim Biophys Acta 1837:1063-8
Steed, P Ryan; Kraft, Kaitlin A; Fillingame, Robert H (2014) Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm. Proc Natl Acad Sci U S A 111:16730-5
Steed, P Ryan; Fillingame, Robert H (2014) Residues in the polar loop of subunit c in Escherichia coli ATP synthase function in gating proton transport to the cytoplasm. J Biol Chem 289:2127-38
Moore, Kyle J; Fillingame, Robert H (2013) Obstruction of transmembrane helical movements in subunit a blocks proton pumping by F1Fo ATP synthase. J Biol Chem 288:25535-41
DeLeon-Rangel, Jessica; Ishmukhametov, Robert R; Jiang, Warren et al. (2013) Interactions between subunits a and b in the rotary ATP synthase as determined by cross-linking. FEBS Lett 587:892-7
Uhlemann, Eva-Maria E; Pierson, Hannah E; Fillingame, Robert H et al. (2012) Cell-free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane. Protein Sci 21:279-88
Dong, Hui; Fillingame, Robert H (2010) Chemical reactivities of cysteine substitutions in subunit a of ATP synthase define residues gating H+ transport from each side of the membrane. J Biol Chem 285:39811-8
Steed, P Ryan; Fillingame, Robert H (2009) Aqueous accessibility to the transmembrane regions of subunit c of the Escherichia coli F1F0 ATP synthase. J Biol Chem 284:23243-50
Moore, Kyle J; Angevine, Christine M; Vincent, Owen D et al. (2008) The cytoplasmic loops of subunit a of Escherichia coli ATP synthase may participate in the proton translocating mechanism. J Biol Chem 283:13044-52
Moore, Kyle J; Fillingame, Robert H (2008) Structural interactions between transmembrane helices 4 and 5 of subunit a and the subunit c ring of Escherichia coli ATP synthase. J Biol Chem 283:31726-35

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