Abstract
The mechanisms of energy coupling and catalytic co-operativity are not yet understood for H(+)-ATPase (ATP synthase). An Escherichia coli gamma subunit frameshift mutant (downstream of Thr-gamma 277) could not grow by oxidative phosphorylation because both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 5043-5048). The defect(s) of the gamma frameshift was obvious, because the mutant subunit had a carboxyl terminus comprising 16 residues different from those in the wild type. However, in this study, we surprisingly found that an Arg-beta 52-->Cys or Gly-beta 150-->Asp replacement could suppress the deleterious effects of the gamma frameshift. The membranes of the two mutants (gamma frameshift/Cys-beta 52 with or without a third mutation, Val-beta 77-->Ala) exhibited increased oxidative phosphorylation, together with 70-100% of the wild type ATPase activity. Similarly, the gamma frameshift/Asp-beta 150 mutant could grow by oxidative phosphorylation, although this mutant had low membrane ATPase activity. These results suggest that the beta subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the gamma mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme.
Highlights
The mechanisms of energy coupling and catalytic cooperativity are not yet understood for H؉-ATPase (ATP synthase)
These results suggest that the  subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the ␥ mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme
Similar to KF10rA/pBMG293fs, DK8 harboring pBMUG15fs could not grow by oxidative phosphorylation and exhibited about 23% of the membrane ATPase activity of the same strain with wild type plasmid pBWU15 (Table I)
Summary
E. coli and Growth Conditions—Strains DK8 (⌬uncB-C, ilv::Tn10, thi) [15] lacking the entire unc (ATP synthase) operon and KF10rA (thi, thy, recA1, uncG10 (Gln-␥14 3 end)) carrying pBMG293fs (frameshift in the uncG gene for the ␥ subunit) [8] were used. Construction of pBMUG15fs Carrying the Entire unc Operon with the ␥ Subunit Frameshift Mutation—The ␥ frameshift mutation was introduced into a recombinant plasmid, pBWU1 [17], carrying the entire wild type unc operon (see Fig. 1). A modified PCR [18] was employed to generate random mutations using universal primers (vector segments), and the SpeI–SacI fragment was isolated and ligated with pBMUG15fs treated with the same enzymes. The resulting plasmids were transformed into strain DK8, and cells showing positive growth on succinate by oxidative phosphorylation were isolated. These cells harbored recombinant plasmids carrying the ␥ frameshift mutation and  subunit mutation(s).
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