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The gamma subunit of the Escherichia coli ATP synthase. Mutations in the carboxyl-terminal region restore energy coupling to the amino-terminal mutant gamma Met-23–>Lys.

1993; Elsevier BV; Volume: 268; Issue: 2 Linguagem: Inglês

10.1016/s0021-9258(18)54014-x

1083-351X

Robert K. Nakamoto, Masatomo Maeda, Masamitsu Futai,

Mitochondrial Function and Pathology

Abstract The gamma subunit mutations, gamma Met-23-->Lys or Arg, in the Escherichia coli ATP synthase were previously reported to cause dramatically inefficient energy coupling between ATPase catalysis and H+ translocation (Shin, K., Nakamoto, R.K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). In this paper, we report that second-site mutations in the gamma subunit can suppress the effects of gamma Met-23-->Lys. By screening randomly mutagenized uncG (gamma Met-23-->Lys), eight mutations in the carboxyl-terminal region were identified; strains carrying gamma Arg-242-->Cys, gamma Gln-269-->Arg, gamma Ala-270-->Val, gamma Ile-272-->Thr, gamma Thr-273-->Ser, gamma Glu-278-->Gly, gamma Ile-279-->Thr, or gamma Val-280-->Ala in combination with gamma Met-23-->Lys were able to grow by oxidative phosphorylation. H+ pumping assayed in membranes prepared from double mutation strains demonstrated that efficient ATP-dependent H+ transport was restored. Interestingly, the single mutations, gamma Gln-269-->Arg or gamma Thr-273-->Ser, caused reduced growth by oxidative phosphorylation; however, when these mutations were in combination with gamma Met-23-->Lys, growth was substantially increased. Furthermore, strains carrying gamma Met-23-->Lys, gamma Gln-269-->Arg, or gamma Thr-273-->Ser as single mutations were temperature sensitive, whereas, strains with the double mutations, gamma Met-23-->Lys/gamma Gln-269-->Arg or gamma Met-23-->Lys/gamma Thr-273-->Ser, were thermally stable. Taken together, these results strongly suggest that gamma Met-23, gamma Arg-242, and the region between gamma Gln-269 to gamma Val-280 are close to each other and interact to mediate efficient energy coupling.