The synthesis of ATP during oxidAtive phosphorylation is catalyzed by a reversible H+-translocating ATP synthase complex. The H+ ATP synthase, or H+ ATPase, is composed of two sectors: F1 is the ATPase moiety and is easily dissociated from the membrane, whereas F0 extends through the membrane and functions as the H+- translocase. The long term challenge before us is to define how the F0 moiety translocates H+, and how H+ flux through F0F1 is coupled to ATP synthesis. In Escherichia coli, F0 is composed of 3 types of subunits (a, b, and c), and F1 of 5 types of subunits. These subunits are coded by the unc operon which has been cloned and sequenced. The F1F0 complexes of E. coli and mitochondria are very similar, so conclusions from one should be applicable to the other. This grant is aimed at providing information on the functional role of subunits c and a through analysis of mutants, as well as more information on structure. Subunit c is small (79 amino acids) and will be analyzed in greatest detail. We will determine the regions and specific residues that are essential to function by saturation mutagenesis, the optimal position and critical interactions of an essential carboxyl by moving it around, and regions of F1 that are """"""""coupled"""""""" with subunit c by analysis of second site revertants. Subunit c is predicted to fold like a hairpin in the membrane, and must also do so in organic solvent as judged by 1H NMR, and retention of inhibitor binding properties. The structural and functional properties is organic solvent will be correlated with the properties in fully assembled F0, in wild-type and mutant membranes, and mutant proteins will be examined for predicted changes in structure. Functional domains of the larger subunit a will be defined by random mutagenesis, and interesting hot spot areas further characterized by localized mutagenesis. The folding of subunit a in the membrane will be evaluated using antibodies directed against putatively external peptide sequences. The combined genetic and NMR structural approaches with subunit c potentially could provide a high resolution picture of the central component of a membrane H+-translocator. These studies should therefore provide insight into no only the mechanism of oxidative phosphorylation, but also of membrane transport. A detailed understanding of membrane transport proteins, ATPase-pumps, etc. is fundamental to many areas of medical science, including the pathology of many diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023105-15
Application #
3271505
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1976-05-01
Project End
1994-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
15
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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
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
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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