Cysteine-scanning mutagenesis of the periplasmic loop regions of PomA, a putative channel component of the sodium-driven flagellar motor in Vibrio alginolyticus.
The sodium-driven motor consists of the products of at least four genes, pomA, pomB, motX, and motY, in Vibrio alginolyticus. PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ion channel. In PomA, two periplasmic loops were predicted at positions 21 to 36 between membrane segments 1 and 2 (loop(1-2)) and at positions 167 to 180 between membrane segments 3 and 4 (loop(3-4)). To characterize the two periplasmic loop regions, which may have a role as an ion entrance for the channel, we carried out cysteine-scanning mutagenesis. The T186 residue in the fourth transmembrane segment and the D71, D148, and D202 residues in the predicted cytoplasmic portion of PomA were also replaced with Cys. Only two mutations, M179C and T186C, conferred a nonmotile phenotype. Many mutations in the periplasmic loops and all of the cytoplasmic mutations did not abolish motility, though the five successive substitutions from M169C to K173C of loop(3-4) impaired motility. In some mutants that retained substantial motility, motility was inhibited by the thiol-modifying reagents dithionitrobenzoic acid and N-ethylmaleimide. The profiles of inhibition by the reagents were consistent with the membrane topology predicted from the hydrophobicity profiles. Furthermore, from the profiles of labeling by biotin maleimide, we predicted more directly the membrane topology of loop(3-4). None of the loop(1-2) residues were labeled, suggesting that the environments around the two loops are very different. A few of the mutations were characterized further. The structure and function of the loop regions are discussed.
Asai
Y
Y
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan.
Shoji
T
T
Kawagishi
I
I
Homma
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D001710:Biotin / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004229:Dithiothreitol / Q000378:metabolism; D018274:Electroporation / Q000379:methods; D005407:Flagella / Q000502:physiology*; D008301:Maleimides / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D012964:Sodium / Q000494:pharmacology; D014733:Vibrio / Q000235:genetics / Q000502:physiology*
D001425:Bacterial Outer Membrane Proteins; D008301:Maleimides; C103352:PomA protein, bacteria; C043592:maleimide; D001710:Biotin; D012964:Sodium; D003545:Cysteine; D004229:Dithiothreitol