Mutagenic analysis of the a subunit of the F1F0 ATP synthase in Escherichia coli: Gln-252 through Tyr-263.
The a subunit of F1F0 ATP synthase contains a highly conserved region near its carboxyl terminus which is thought to be important in proton translocation. Cassette site-directed mutagenesis was used to study the roles of four conserved amino acids Gln-252, Phe-256, Leu-259, and Tyr-263. Substitution of basic amino acids at each of these four sites resulted in marked decreases in enzyme function. Cells carrying a subunit mutations Gln-252-->Lys, Phe-256-->Arg, Leu-259-->Arg, and Tyr-263-->Arg all displayed growth characteristics suggesting substantial loss of ATP synthase function. Studies of both ATP-driven proton pumping and proton permeability of stripped membranes indicated that proton translocation through F0 was affected by the mutations. Other mutations, such as the Phe-256-->Asp mutation, also resulted in reduced enzyme activity. However, more conservative amino acid substitutions generated at these same four positions produced minimal losses of F1F0 ATP synthase. The effects of mutations and, hence, the relative importance of the amino acids for enzyme function appeared to decrease with proximity to the carboxyl terminus of the a subunit. The data are most consistent with the hypothesis that the region between Gln-252 and Tyr-263 of the a subunit has an important structural role in F1F0 ATP synthase.
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610.
P E
PE
Hartzog
B D
BD
Cain
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D025243:Bacterial Proton-Translocating ATPases; D001483:Base Sequence; D002463:Cell Membrane Permeability; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D011522:Protons
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011522:Protons; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C074428:atpB protein, E coli