Suppression mutations in the defective beta subunit of F1-ATPase from Escherichia coli.
The Escherichia coli mutant of the proton-translocating ATPase KF11 (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., and Futai, M. (1980) J. Biochem. (Tokyo) 88, 695-703) has a defective beta subunit with serine being replaced by phenylalanine at codon 174. Four suppression mutants (RE10, RE17, RE18, and RE20) from this strain capable of growth on minimal plate agar supplemented by succinate were isolated. The original point mutation at codon 174 was intact in these strains. Additional point mutations, Ala-295 to Thr, Gly-149 to Ser, Leu-400 to Gln, Ala-295 to Pro, for RE10, RE17, RE18, and RE20, respectively, were identified by the polymerase chain reaction and sequencing. These mutations, except for RE10, were confirmed as a single mutation conferring a suppressive phenotype by genetic suppression assay using KF11 as the host cells. The results indicated that Ser-174 has functional interaction with Gly-149, Ala-295, and Leu-400. The residues are located within the previously estimated catalytic domain of the beta subunit, indicating that this domain is indeed folded for the active site of catalytic function. Growth rates of the revertants in the minimal medium with succinate increased compared with that of KF11, showing that ATP synthesis recovered to some extent. The ATP hydrolytic activity in the revertant membranes increased in RE17 and RE20 but did not in RE10 and RE18, suggesting that synthesis and hydrolysis are not necessarily reversible in the proton-translocating ATPase (F1F0).
Miki
J
J
Department of Biotechnology, Faculty of Engineering Sciences, Okayama University, Japan.
Fujiwara
K
K
Tsuda
M
M
Tsuchiya
T
T
Kanazawa
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D016153:Genes, Suppressor; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D006180:Proton-Translocating ATPases / Q000235:genetics*; D015183:Restriction Mapping
D006180:Proton-Translocating ATPases