Interaction of PomB with the third transmembrane segment of PomA in the Na+-driven polar flagellum of Vibrio alginolyticus.
The marine bacterium Vibrio alginolyticus has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na(+)-driven flagellar rotation. PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it. Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent. The strain with this combination showed a lower affinity for Na(+) than the wild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na(+) conduction to flagellar rotation.
Yakushi
Toshiharu
T
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan. 4juji@cc.nagoya-u.ac.jp
Maki
Shingo
S
Homma
Michio
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D005407:Flagella / Q000737:chemistry / Q000502:physiology; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009068:Movement; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D044149:Vibrio alginolyticus / Q000737:chemistry* / Q000502:physiology*
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; C107540:PomA protein, Vibrio; C107541:PomB protein, Vibrio; D015222:Sodium Channels; D012964:Sodium