Mutations in the conserved proline 43 residue of the uncE protein (subunit c) of Escherichia coli F1F0-ATPase alter the coupling of F1 to F0.
The conserved Pro43 residue of the uncE protein (subunit c) of the Escherichia coli F1F0-ATPase was changed to Ser or Ala by oligonucleotide-directed mutagenesis, and the mutations were incorporated into the chromosome. The resultant mutant strains were capable of oxidative phosphorylation as indicated by their ability to grow on succinate and had growth yields on glucose that were 80-90% of wild type. Membrane vesicles from the mutants were slightly less efficient than wild type vesicles in ATP-driven proton pumping as indicated by ATP-dependent quenching of quinacrine fluorescence. The decreased quenching response was not due to increased H+ leakiness of the mutant membranes or to loss of F1-ATPase activity from the membrane. These results indicate that the mutant F1F0-ATPases are defective in coupling ATP hydrolysis to H+ translocation. The membrane ATPase activity of the mutants was inhibited less by dicyclohexylcarbodiimide than that of wild type. The decrease in sensitivity to inhibition by dicyclohexylcarbodiimide was caused primarily by dissociation of the F1-ATPase from the mutant F0 in the ATPase assay mixture. These results support the idea that Pro43, and neighboring conserved polar residues play an important role in the binding and functional coupling of F1 to F0. Although a Pro residue is found at position 43 in all species of subunit c studied, surprisingly, it is not absolutely essential to function.
Miller
M J
MJ
Department of Physiological Chemistry, University of Wisconsin Medical School, Madison 53706.
Fraga
D
D
Paule
C R
CR
Fillingame
R H
RH
D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005838:Genotype; D007700:Kinetics; D046911:Macromolecular Substances; D009154:Mutation; D015345:Oligonucleotide Probes; D011392:Proline; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D004024:Dicyclohexylcarbodiimide; D011392:Proline; D006180:Proton-Translocating ATPases