Genetic complementation between mutant b subunits in F1F0 ATP synthase.
In Escherichia coli, a parallel homodimer of identical b subunits constitutes the peripheral stalk of F(1)F(0) ATP synthase. Although the two b subunits have long been viewed as a single functional unit, the asymmetric nature of the enzyme complex suggested that the functional roles of each b subunit should not necessarily be considered equivalent. Previous mutagenesis studies of the peripheral stalk suffered from the fact that mutations in the uncF(b) gene affected both of the b subunits. We developed a system to express and study F(1)F(0) ATP synthase complexes containing two different b subunits. Two mutations already known to inactivate the F(1)F(0) ATP synthase complex have been studied using this experimental system. An evolutionarily conserved arginine, b(Arg-36), was known to be crucial for F(1)F(0) ATP synthase function, and the last four C-terminal amino acids had been shown to be important for enzyme assembly. Experiments expressing one of the mutants with a wild type b subunit demonstrated the presence of heterodimers in F(1)F(0) ATP synthase complexes. Activity assays suggested that the heterodimeric F(1)F(0) complexes were functional. When the two defective b subunits were expressed together and in the absence of any wild type b subunit, an active F(1)F(0) ATP synthase complex was assembled. This mutual complementation between fully defective b subunits indicated that each of the two b subunits makes a unique contribution to the functions of the peripheral stalk, such that one mutant b subunit is making up for what the other is lacking.
Grabar
Tammy Bohannon
TB
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
Cain
Brian D
BD
D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D021122:Protein Subunits; D011994:Recombinant Proteins; D025243:Bacterial Proton-Translocating ATPases