title: Mutations in a helix-1 motif of the ATP synthase c-subunit of Bacillus pseudofirmus OF4 cause functional deficits and changes in the c-ring stability and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
abstract: The ATP synthase of the alkaliphile Bacillus pseudofirmus OF4 has a tridecameric c-subunit rotor ring. Each c-subunit has an AxAxAxA motif near the center of the inner helix, where neutralophilic bacteria generally have a GxGxGxG motif. Here, we studied the impact of four single and six multiple Ala-to-Gly chromosomal mutations in the A16xAxAxA22 motif on the capacity for nonfermentative growth and, for most of the mutants, on ATP synthesis by ADP- and P(i)-loaded membrane vesicles at pH 7.5 and 10.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of the holo-ATP synthases were used to probe stability of the mutant c-rotors and mobility properties of the c-rotors as well as the monomeric c-subunits that are released from them by trichloroacetic acid treatment. Mutants containing an Ala16-to-Gly mutation exhibited the most severe functional defects. Via SDS-PAGE, most of the mutant c-monomers exhibited increased mobility relative to the wild-type (WT) c-subunit, but among the intact c-rings, only Ala16-to-Gly mutants exhibited significantly increased mobility relative to that of the WT c-ring. The hypothesis that these c-rings have a decreased c-subunit stoichiometry is still untested, but the functional impact of an Ala16-to-Gly mutation clearly depended upon additional Ala-to-Gly mutation(s) and their positions. The A16/20G double mutant exhibited a larger functional deficit than both the A16G and A16/18G mutants. Most of the mutant c-rings showed in vitro instability relative to that of the WT c-ring. However, the functional deficits of mutants did not correlate well with the extent of c-ring stability loss, so this property is unlikely to be a major factor in vivo.
authors.affiliation: Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, New York 10029, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Oliver J
authors.initials: OJ
authors.lastname: Fackelmayer
authors.forename: David B
authors.initials: DB
authors.lastname: Hicks
authors.forename: Laura
authors.initials: L
authors.lastname: Preiss
authors.forename: Thomas
authors.initials: T
authors.lastname: Meier
authors.forename: Eric A
authors.initials: EA
authors.lastname: Sobie
authors.forename: Terry A
authors.initials: TA
authors.lastname: Krulwich
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005960:Glucosides / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005960:Glucosides; D021122:Protein Subunits; D011994:Recombinant Proteins; C018619:octyl-beta-D-glucoside; D025243:Bacterial Proton-Translocating ATPases