Concerted effects of amino acid substitutions in conserved charged residues and other residues in the cytoplasmic domain of PomA, a stator component of Na+-driven flagella.
PomA is a membrane protein that is one of the essential components of the sodium-driven flagellar motor in Vibrio species. The cytoplasmic charged residues of Escherichia coli MotA, which is a PomA homolog, are believed to be required for the interaction of MotA with the C-terminal region of FliG. It was previously shown that a PomA variant with neutral substitutions in the conserved charged residues (R88A, K89A, E96Q, E97Q, and E99Q; AAQQQ) was functional. In the present study, five other conserved charged residues were replaced with neutral amino acids in the AAQQQ PomA protein. These additional substitutions did not affect the function of PomA. However, strains expressing the AAQQQ PomA variant with either an L131F or a T132M substitution, neither of which affected motor function alone, exhibited a temperature-sensitive (TS) motility phenotype. The double substitutions R88A or E96Q together with L131F were sufficient for the TS phenotype. The motility of the PomA TS mutants immediately ceased upon a temperature shift from 20 to 42 degrees C and was restored to the original level approximately 10 min after the temperature was returned to 20 degrees C. It is believed that PomA forms a channel complex with PomB. The complex formation of TS PomA and PomB did not seem to be affected by temperature. Suppressor mutations of the TS phenotype were mapped in the cytoplasmic boundaries of the transmembrane segments of PomA. We suggest that the cytoplasmic surface of PomA is changed by the amino acid substitutions and that the interaction of this surface with the FliG C-terminal region is temperature sensitive.
Fukuoka
Hajime
H
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan.
Yakushi
Toshiharu
T
Homma
Michio
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism*; D005407:Flagella / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D020409:Molecular Motor Proteins; D008969:Molecular Sequence Data; D009068:Movement; D016297:Mutagenesis, Site-Directed; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology*; D013696:Temperature; D014733:Vibrio / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D001425:Bacterial Outer Membrane Proteins; D020409:Molecular Motor Proteins; C103352:PomA protein, bacteria; D012964:Sodium