Genetic Complementation between Mutant b Subunits in F sub(1)F sub(0) ATP Synthase

• Published in: The Journal of biological chemistry Vol. 279; no. 30; pp. 31205 - 31211
• Main Authors: Grabar, Tammy Bohannon, Cain, Brian D
• Format: Journal Article
• Language: English
• Published: 23-07-2004
• Subjects: Escherichia coli
• ISSN: 0021-9258; 1083-351X

In Escherichia coli, a parallel homodimer of identical b subunits constitutes the peripheral stalk of F sub(1)F sub(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 sub(1)F sub(0) ATP synthase complexes containing two different b subunits. Two mutations already known to inactivate the F sub(1)F sub(0) ATP synthase complex have been studied using this experimental system. An evolutionarily conserved arginine, b sub(Arg-36), was known to be crucial for F sub(1)F sub(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 sub(1)F sub(0) ATP synthase complexes. Activity assays suggested that the heterodimeric F sub(1)F sub(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 sub(1)F sub(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.