Abstract
PomA and PomB form a complex that conducts sodium ions and generates the torque for the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. PomA has four transmembrane segments. One periplasmic loop (loop(1-2)) connects segments 1 and 2, and another (loop(3-4)), in which cysteine-scanning mutagenesis had been carried out, connects segments 3 and 4. When PomA with an introduced Cys residue (Cys-PomA) in the C-terminal periplasmic loop (loop(3-4)) was examined without exposure to a reducing reagent, a 43-kDa band was observed, whereas only a 25-kDa band, which corresponds to monomeric PomA, was observed under reducing conditions. The intensity of the 43-kDa band was enhanced in most mutants by the oxidizing reagent CuCl(2). The 43-kDa band was strongest in the P172C mutant. The motility of the P172C mutant was severely reduced, and P172C showed a dominant-negative effect, whereas substitution of Pro with Ala, Ile, or Ser at this position did not affect motility. In the presence of DTT, the ability to swim was partially restored, and the amount of 43-kDa protein was reduced. These results suggest that the disulfide cross-link disturbs the function of PomA. When the mutated Cys residue was modified with N-ethylmaleimide, only the 25-kDa PomA band was labeled, demonstrating that the 43-kDa form is a cross-linked homodimer and suggesting that the loops(3-4) of adjacent subunits of PomA are close to each other in the assembled motor. We propose that this loop region is important for dimer formation and motor function.
Highlights
Many bacteria rotate flagellar filaments to swim
In PomA, periplasmic loops are predicted for residues Val-21 through Leu-36 between transmembrane segments 1 and 2 and for residues Ser-167 through Ala-180 between transmembrane segments 3 and 4 (Fig. 1)
The accessibility of loop3–4 to the periplasm was confirmed by labeling with biotin maleimide; none of the Cys residues of loop1–2 were labeled by the reagent
Summary
Many bacteria rotate flagellar filaments to swim. The flagellar filament is attached via a flexible hook to a protein complex termed the basal body, which is embedded in the cell surface. Escherichia coli has Hϩ-driven flagellar motors whose torque-generating units consist of two proteins, MotA and MotB. These two proteins are believed to form a Hϩ channel to permit the Hϩ influx and to provide the energy for motor rotation (4 –7). It has been inferred that PomA and PomB form an ion channel [8] This inference was supported by the observation that mutations conferring resistance to phenamil, which is a known Naϩ-channel inhibitor and and strongly inhibits the Naϩ-driven motor, mapped to pomA and pomB [14, 16, 17]. The accessibility of loop to the periplasm was confirmed by labeling with biotin maleimide; none of the Cys residues of loop were labeled by the reagent These results suggest that the environments of the two loops are different [20].
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