On the role of Arg-210 and Glu-219 of subunit a in proton translocation by the Escherichia coli F0F1-ATP synthase.
A strain of Escherichia coli was constructed which had a complete deletion of the chromosomal uncB gene encoding subunit a of the F0F1-ATP synthase. Gene replacement was facilitated by a selection protocol that utilized the sacB gene of Bacillus subtilis cloned in a kanamycin resistance cartridge (Ried, J. L., and Collmer, A. (1987) Gene (Amst.) 57, 239-246). F0 subunits b and c inserted normally into the membrane in the DeltauncB strain. This observation confirms a previous report (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 2815-2817) that subunit a is not required for the insertion of subunits b and c. The DeltauncB strain has been used to characterize mutations in Arg-210 and Glu-219 of subunit a, residues previously postulated to be essential in proton translocation. The aE219G and aE219K mutants grew on a succinate carbon source via oxidative phosphorylation and membranes from these mutants exhibited ATPase-coupled proton translocation (i.e. ATP driven 9-amino-6-chloromethoxyacridine quenching responses that were 60-80% of wild type membranes). We conclude that the aGlu-219 residue cannot play a critical role in proton translocation. The aR210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation. However, on removal of F1 from membrane, the aR210A mutant F0 was active in passive proton translocation, i.e. in dissipating the DeltapH normally established by NADH oxidation with these membrane vesicles. aR210A membranes with F1 bound were also proton permeable. Arg-210 of subunit a may play a critical role in active H+ transport that is coupled to ATP synthesis or hydrolysis, but is not essential for the translocation of protons across the membranes.
Valiyaveetil
F I
FI
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Fillingame
R H
RH
D001120:Arginine / Q000378:metabolism*; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid / Q000378:metabolism*; D017136:Ion Transport; D009154:Mutation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D011522:Protons
D011522:Protons; D018698:Glutamic Acid; D001120:Arginine; D006180:Proton-Translocating ATPases