Intermolecular crosslinking of PomA, The Na^+-driven flagellar motor component of Vibrio alginolyticus

Yorimitsu, Tomohiro; Asai, Yukako; Sato, Ken; Homma, M.

doi:10.2142/biophys.39.s148_1

Cited by 30 publications

(51 citation statements)

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“…Membranes (60 l) were treated with 15 l of Proteinase K at the indicated concentrations on ice for 30 min. The samples were precipitated with trichloroacetic acid, washed with acetone, and then analyzed by SDS-PAGE, followed by immunoblotting with antiPomB antibody (31).…”

Section: Methodsmentioning

confidence: 99%

Functional Reconstitution of the Na+-driven Polar Flagellar Motor Component of Vibrio alginolyticus

Sato

Homma

2000

Journal of Biological Chemistry

144

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The bacterial flagellar motor is a molecular machine that couples the influx of specific ions to the generation of the force necessary to drive rotation of the flagellar filament. Four integral membrane proteins, PomA, PomB, MotX, and MotY, have been suggested to be directly involved in torque generation of the Na ؉ -driven polar flagellar motor of Vibrio alginolyticus. In the present study, we report the isolation of the functional component of the torque-generating unit. The purified protein complex appears to consist of PomA and PomB and contains neither MotX nor MotY. The PomA/B protein, reconstituted into proteoliposomes, catalyzed 22 Na؉ influx in response to a potassium diffusion potential. Sodium uptake was abolished by the presence of Li ؉ ions and phenamil, a sodium channel blocker. This is the first demonstration of a purification and functional reconstitution of the bacterial flagellar motor component involved in torque generation. In addition, this study demonstrates that the Na ؉ -driven motor component, PomA and PomB, forms the Na ؉ -conducting channel.

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Section: Methodsmentioning

confidence: 99%

Functional Reconstitution of the Na+-driven Polar Flagellar Motor Component of Vibrio alginolyticus

Sato

Homma

2000

Journal of Biological Chemistry

144

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“…The D148Y and P16S mutations combined to produce a synergistic increase in resistance to phenamil and impaired motor function much more severely than the individual mutations in the absence of inhibitor (17). We have also shown that PomA and PomB physically interact with each other (18), and that Na ϩ influx can be detected in reconstituted proteoliposomes containing purified PomA/PomB complex (19). The molar ratio of the isolated complex is calculated to be 2PomA/1PomB, and PomA seems to form a stable dimer.…”

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confidence: 70%

“…Next, an equal volume of 15% (w/v) trichloroacetic acid was added to the cell suspension, and the resulting precipitate was washed once with acetone, dried, and then dissolved in SDS sample buffer without reducing reagent. This sample was subjected to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with antibody against PomA, as described previously (18).…”

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confidence: 99%

“…The membrane fraction was recovered by centrifugation at 100,000 ϫ g, solubilized with TNET buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5 mM EDTA, and 1% (w/v) Triton X-100), and incubated for 60 min at 4°C. Immunoprecipitation with anti-PomA antibody was carried out as described previously (18). The resulting precipitates were subjected to SDS-PAGE, followed by fluorography.…”

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confidence: 99%

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Intermolecular Cross-linking between the Periplasmic Loop3–4 Regions of PomA, a Component of the Na+-driven Flagellar Motor of Vibrio alginolyticus

Yorimitsu

Asai

Sato

et al. 2000

Journal of Biological Chemistry

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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 Cterminal 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.

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“…Cells were resuspended in SDS loading buffer and boiled at 95 uC for 5 min, and then were subjected to SDS-PAGE and immunoblotting as described previously (Yorimitsu et al, 1999 antibody to PomA (PomA1312) has been reported previously (Yorimitsu et al, 1999) and the antibody to PomB (PomB C2 B0455) was prepared as follows: an N-terminally truncated PomB variant consisting of residues 59-315 (PomB C2 ) with a His 6 -tag fused at its C terminus was constructed, and was overexpressed in BL21(DE3) cells from plasmid pTSK36, a derivative of pET22b. Cells were disrupted and a soluble fraction was isolated by ultracentrifugation.…”

Section: Methodsmentioning

confidence: 99%

A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque

et al. 2011

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A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque Takashi Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, JapanBacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion (H + or Na + ) as their energy source, and are composed of stator and rotor proteins. Sodium-driven and proton-driven motors have the stator proteins PomA and PomB or MotA and MotB, respectively, which interact with each other in their transmembrane (TM) regions to form an ion channel. The single TM region of PomB or MotB, which forms the ion-conduction pathway together with TM3 and TM4 of PomA or MotA, respectively, has a highly conserved aspartate residue that is the ion binding site and is essential for rotation. To investigate the ion conductivity and selectivity of the Na + -driven PomA/PomB stator complex, we replaced conserved residues predicted to be near the conserved aspartate with H + -type residues, PomA-N194Y, PomB-F22Y and/or PomB-S27T. Motility analysis revealed that the ion specificity was not changed by either of the PomB mutations. PomB-F22Y required a higher concentration of Na + to exhibit swimming, but this effect was suppressed by additional mutations, PomA-N194Y or PomB-S27T. Moreover, the motility of the PomB-F22Y mutant was resistant to phenamil, a specific inhibitor for the Na + channel. When PomB-F22 was changed to other amino acids and the effects on swimming ability were investigated, replacement with a hydrophilic residue decreased the maximum swimming speed and conferred strong resistance to phenamil. From these results, we speculate that the Na + flux is reduced by the PomB-F22Y mutation, and that PomB-F22 is important for the effective release of Na + from PomB-D24. INTRODUCTIONSome ion channels and transporters that exist in cell membranes can selectively translocate only a particular ion. The high ion specificity of an ion channel or transporter is extremely important for signal transduction, membrane excitability and the homeostasis of organisms. Each selective ion transporter, which is coupled to a sodium ion, such as LeuT, NtpK or NhaA, possesses an ion binding pocket. A particular ion and/or a transport substrate interacts with the ion binding pocket, induces a conformational change, and is then translocated to the opposite side across the membrane (Gouaux & Mackinnon, 2005;Yamashita et al., 2005;Hunte et al., 2005;Murata et al., 2005Murata et al., , 2008. For example, in the Na + -driven V-type ATPase of Enterococcus hirae, the transmembrane complex V o , which conducts Na + (or Li + ), is composed of a membrane rotor ring (K-ring) comprising oligomers of NtpK and a single copy of the NtpI subunit. It has been suggested that the cavity size of the K-ring binding pocket contributes to the ion specificity, that five residues (L 61 , T 64 , Q 65 , Q 110 , E 139 ) of NtpK are involved in the Na + binding to the K-ri...

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Intermolecular crosslinking of PomA, The Na^+-driven flagellar motor component of Vibrio alginolyticus

Cited by 30 publications

References 0 publications

Functional Reconstitution of the Na+-driven Polar Flagellar Motor Component of Vibrio alginolyticus

Functional Reconstitution of the Na+-driven Polar Flagellar Motor Component of Vibrio alginolyticus

Intermolecular Cross-linking between the Periplasmic Loop3–4 Regions of PomA, a Component of the Na+-driven Flagellar Motor of Vibrio alginolyticus

A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque

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