title: Mutagenesis studies of the F1F0 ATP synthase b subunit membrane domain.
abstract: A homodimer of b subunits constitutes the peripheral stalk linking the F1 and F0 sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F1F0 ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F1F0 ATPase and no detectable ATP-driven proton pumping activity. Expression of the b(N2A,T6A,Q10A) subunit was also oxidative phosphorylation deficient, but the b(N2A,T6A,Q10A) protein was incorporated into an F1F0 complex. Single amino acid substitutions had minimal reductions in F1F0 ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F0, and that these interactions are strongest on the periplasmic side of the bilayer.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Hardy
authors.forename: Tammy Bohannon
authors.initials: TB
authors.lastname: Grabar
authors.forename: Deepa
authors.initials: D
authors.lastname: Bhatt
authors.forename: Brian D
authors.initials: BD
authors.lastname: Cain
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D010085:Oxidative Phosphorylation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases