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Comparative study of the ion flux pathway in stator units of proton- and sodium-driven flagellar motors

View Article: PubMed Central - PubMed

ABSTRACT

Flagellar motor proteins, MotA/B and PomA/B, are essential for the motility of Escherichia coli and Vibrio alginolyticus, respectively. Those complexes work as a H+ and a Na+ channel, respectively and play important roles in torque generation as the stators of the flagellar motors. Although Asp32 of MotB and Asp24 of PomB are believed to function as ion binding site(s), the ion flux pathway from the periplasm to the cytoplasm is still unclear. Conserved residues, Ala39 of MotB and Cys31 of PomB, are located on the same sides as Asp32 of MotB and Asp24 of PomB, respectively, in a helical wheel diagram. In this study, a series of mutations were introduced into the Ala39 residue of MotB and the Cys31 residue of PomB. The motility of mutant cells were markedly decreased as the volume of the side chain increased. The loss of function due to the MotB(A39V) and PomB(L28A/C31A) mutations was suppressed by mutations of MotA(M206S) and PomA(L183F), respectively, and the increase in the volume caused by the MotB(A39V) mutation was close to the decrease in the volume caused by the MotA(M206S) mutation. These results demonstrate that Ala39 of MotB and Cys31 of PomB form part of the ion flux pathway and pore with Met206 of MotA and Leu183 of PomA in the MotA/B and PomA/B stator units, respectively.

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Motility of MotB and PomB mutants on soft-agar plates (a)(c), and western blotting analysis of the proteins (b) (d). (a) Swarms of RP6894 (ΔmotAB) cells harboring a plasmid that express WT MotA/B, MotA/B(A39G) (G), MotA/B(A39V) (V), MotA/B(A39C) (C), MotA/B(A39S) (S), MotA/B(A39T) (T), MotA/B(A39Y) (Y) or MotA/B(A39W) (W). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing TG and 0.26% agar. Plates were incubated in the presence of 0.04% arabinose at 30°C for 10 hr. Vec. means a pBAD24 plasmid vector. (b) Western blotting analysis of MotA (left panel) and MotB (right panel) in whole cell extracts. Proteins were detected with an anti-MotA266 antibody and an anti-MotB2 antibody. (c) Swarms of NMB191 (ΔpomAB) cells harboring a plasmid that express WT PomA/B, PomA/B(C31G) (G), PomA/B(C31A) (A), PomA/B(C31S) (S), PomA/B(C31V) (V), PomA/B(C31T) (T), PomA/B(C31M) (M), PomA/B(C31I) (I), PomA/B(C31Y) (Y) or PomA/B(C31W) (W). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing VPG500 and 0.26% agar. Plates were incubated at 30°C for 6 hr. Vec. means a pSU41 plasmid vector. (b) Western blotting analysis of PomA (left panel) and PomB (right panel) in whole cell extracts. Proteins were detected with an anti-PomA1312 antibody and an anti-PomB93 antibody.
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f2-5_45: Motility of MotB and PomB mutants on soft-agar plates (a)(c), and western blotting analysis of the proteins (b) (d). (a) Swarms of RP6894 (ΔmotAB) cells harboring a plasmid that express WT MotA/B, MotA/B(A39G) (G), MotA/B(A39V) (V), MotA/B(A39C) (C), MotA/B(A39S) (S), MotA/B(A39T) (T), MotA/B(A39Y) (Y) or MotA/B(A39W) (W). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing TG and 0.26% agar. Plates were incubated in the presence of 0.04% arabinose at 30°C for 10 hr. Vec. means a pBAD24 plasmid vector. (b) Western blotting analysis of MotA (left panel) and MotB (right panel) in whole cell extracts. Proteins were detected with an anti-MotA266 antibody and an anti-MotB2 antibody. (c) Swarms of NMB191 (ΔpomAB) cells harboring a plasmid that express WT PomA/B, PomA/B(C31G) (G), PomA/B(C31A) (A), PomA/B(C31S) (S), PomA/B(C31V) (V), PomA/B(C31T) (T), PomA/B(C31M) (M), PomA/B(C31I) (I), PomA/B(C31Y) (Y) or PomA/B(C31W) (W). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing VPG500 and 0.26% agar. Plates were incubated at 30°C for 6 hr. Vec. means a pSU41 plasmid vector. (b) Western blotting analysis of PomA (left panel) and PomB (right panel) in whole cell extracts. Proteins were detected with an anti-PomA1312 antibody and an anti-PomB93 antibody.

Mentions: Some critical residues of MotA, MotB, PomA and PomB that are involved in torque generation have been reported2,3. In particular, Asp32 of MotB and Asp24 of PomB (Figure 1), which are unique acidic residues in the transmembrane segment, are expected to function in conveying H+ and Na+, respectively3. Two conserved residues, Cys31 in PomB and Ala39 in MotB, are located in the membrane-spanning region and are on the same sides as Asp24 for PomB and Asp32 for MotB in a helical wheel diagram (Figure 1). Figure 2a shows the swarming abilities of wild-type (WT) MotA/B, MotA/B(A39G), MotA/B(A39V), MotA/B(A39C), MotA/B(A39S), MotA/B(A39T), MotA/B(A39Y) and MotA/B(A39W). MotA/B(A39C) (C) and MotA/B(A39S) (S) showed no significant reduction in swarm size compared with WT MotA/B, whereas MotA/B(A39G) (G) and MotA/B(A39T) (T) reduced the swarming ability, and MotA/B(A39V) (V), MotA/B(A39Y) (Y) and MotA/B(A39W) (W) completely disrupted the swarming ability. Previously, Blair and coworkers demonstrated that the A39V mutant of MotB was dominant and non-functional11, which is consistent with our results. The expression level of the proteins was examined by Western blotting analysis. As shown in Figure 2b, protein expression levels of MotB mutants were almost the same as those of the WT, except for MotB(A39G) and MotB(A39V) mutants where amounts of MotB were reduced, while the amounts of MotA were not affected by any of the MotB mutations. Figure 2c shows the swarming abilities of the WT PomA/B, PomA/B(C31G), PomA/B(C31A), PomA/B(C31S), PomA/B(C31V), PomA/B(C31T), PomA/B(C31M), PomA/B(C31I), PomA/B(C31Y) and PomA/B(C31W). PomA/B(C31S) (S) and PomA/B(C31T) (T) showed no significant reduction in swarm size compared with WT PomA/B, whereas PomA/B(C31G) (G), PomA/B(C31A) (A) and PomA/B(C31V) (V) reduced the swarming ability, and PomA/B(C31M) (M), PomA/B(C31Y) (Y) and PomA/B(C31W) (W) completely disrupted the swarming ability. The reduction of the swarming size caused by C31A has been reported previously by Yorimitsu and coworkers16. The expression level of the proteins was examined by Western blotting analysis. As shown in Figure 2d, protein expression levels of PomB C31G, C31A, A31T, C31V and C31M mutants were almost the same as those of the WT PomB, whereas levels of PomB(C31S) and PomB(C31I) were reduced by the mutations, and PomB(C31Y) and PomB(C31W) did not show any PomB expression. As for C31Y, PomA was also not expressed. These results suggest that the introduction of a large residue (e.g. Tyr or Trp) into the Cys31 position disrupts protein folding or its insertion into the membrane. In any case, the side chain volumes at positions 39 of MotB and 31 of PomB seem to be important for function. To confirm this, we plotted the swarming rate against the volume of the introduced amino acid side chain (Figure 3a).


Comparative study of the ion flux pathway in stator units of proton- and sodium-driven flagellar motors
Motility of MotB and PomB mutants on soft-agar plates (a)(c), and western blotting analysis of the proteins (b) (d). (a) Swarms of RP6894 (ΔmotAB) cells harboring a plasmid that express WT MotA/B, MotA/B(A39G) (G), MotA/B(A39V) (V), MotA/B(A39C) (C), MotA/B(A39S) (S), MotA/B(A39T) (T), MotA/B(A39Y) (Y) or MotA/B(A39W) (W). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing TG and 0.26% agar. Plates were incubated in the presence of 0.04% arabinose at 30°C for 10 hr. Vec. means a pBAD24 plasmid vector. (b) Western blotting analysis of MotA (left panel) and MotB (right panel) in whole cell extracts. Proteins were detected with an anti-MotA266 antibody and an anti-MotB2 antibody. (c) Swarms of NMB191 (ΔpomAB) cells harboring a plasmid that express WT PomA/B, PomA/B(C31G) (G), PomA/B(C31A) (A), PomA/B(C31S) (S), PomA/B(C31V) (V), PomA/B(C31T) (T), PomA/B(C31M) (M), PomA/B(C31I) (I), PomA/B(C31Y) (Y) or PomA/B(C31W) (W). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing VPG500 and 0.26% agar. Plates were incubated at 30°C for 6 hr. Vec. means a pSU41 plasmid vector. (b) Western blotting analysis of PomA (left panel) and PomB (right panel) in whole cell extracts. Proteins were detected with an anti-PomA1312 antibody and an anti-PomB93 antibody.
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f2-5_45: Motility of MotB and PomB mutants on soft-agar plates (a)(c), and western blotting analysis of the proteins (b) (d). (a) Swarms of RP6894 (ΔmotAB) cells harboring a plasmid that express WT MotA/B, MotA/B(A39G) (G), MotA/B(A39V) (V), MotA/B(A39C) (C), MotA/B(A39S) (S), MotA/B(A39T) (T), MotA/B(A39Y) (Y) or MotA/B(A39W) (W). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing TG and 0.26% agar. Plates were incubated in the presence of 0.04% arabinose at 30°C for 10 hr. Vec. means a pBAD24 plasmid vector. (b) Western blotting analysis of MotA (left panel) and MotB (right panel) in whole cell extracts. Proteins were detected with an anti-MotA266 antibody and an anti-MotB2 antibody. (c) Swarms of NMB191 (ΔpomAB) cells harboring a plasmid that express WT PomA/B, PomA/B(C31G) (G), PomA/B(C31A) (A), PomA/B(C31S) (S), PomA/B(C31V) (V), PomA/B(C31T) (T), PomA/B(C31M) (M), PomA/B(C31I) (I), PomA/B(C31Y) (Y) or PomA/B(C31W) (W). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing VPG500 and 0.26% agar. Plates were incubated at 30°C for 6 hr. Vec. means a pSU41 plasmid vector. (b) Western blotting analysis of PomA (left panel) and PomB (right panel) in whole cell extracts. Proteins were detected with an anti-PomA1312 antibody and an anti-PomB93 antibody.
Mentions: Some critical residues of MotA, MotB, PomA and PomB that are involved in torque generation have been reported2,3. In particular, Asp32 of MotB and Asp24 of PomB (Figure 1), which are unique acidic residues in the transmembrane segment, are expected to function in conveying H+ and Na+, respectively3. Two conserved residues, Cys31 in PomB and Ala39 in MotB, are located in the membrane-spanning region and are on the same sides as Asp24 for PomB and Asp32 for MotB in a helical wheel diagram (Figure 1). Figure 2a shows the swarming abilities of wild-type (WT) MotA/B, MotA/B(A39G), MotA/B(A39V), MotA/B(A39C), MotA/B(A39S), MotA/B(A39T), MotA/B(A39Y) and MotA/B(A39W). MotA/B(A39C) (C) and MotA/B(A39S) (S) showed no significant reduction in swarm size compared with WT MotA/B, whereas MotA/B(A39G) (G) and MotA/B(A39T) (T) reduced the swarming ability, and MotA/B(A39V) (V), MotA/B(A39Y) (Y) and MotA/B(A39W) (W) completely disrupted the swarming ability. Previously, Blair and coworkers demonstrated that the A39V mutant of MotB was dominant and non-functional11, which is consistent with our results. The expression level of the proteins was examined by Western blotting analysis. As shown in Figure 2b, protein expression levels of MotB mutants were almost the same as those of the WT, except for MotB(A39G) and MotB(A39V) mutants where amounts of MotB were reduced, while the amounts of MotA were not affected by any of the MotB mutations. Figure 2c shows the swarming abilities of the WT PomA/B, PomA/B(C31G), PomA/B(C31A), PomA/B(C31S), PomA/B(C31V), PomA/B(C31T), PomA/B(C31M), PomA/B(C31I), PomA/B(C31Y) and PomA/B(C31W). PomA/B(C31S) (S) and PomA/B(C31T) (T) showed no significant reduction in swarm size compared with WT PomA/B, whereas PomA/B(C31G) (G), PomA/B(C31A) (A) and PomA/B(C31V) (V) reduced the swarming ability, and PomA/B(C31M) (M), PomA/B(C31Y) (Y) and PomA/B(C31W) (W) completely disrupted the swarming ability. The reduction of the swarming size caused by C31A has been reported previously by Yorimitsu and coworkers16. The expression level of the proteins was examined by Western blotting analysis. As shown in Figure 2d, protein expression levels of PomB C31G, C31A, A31T, C31V and C31M mutants were almost the same as those of the WT PomB, whereas levels of PomB(C31S) and PomB(C31I) were reduced by the mutations, and PomB(C31Y) and PomB(C31W) did not show any PomB expression. As for C31Y, PomA was also not expressed. These results suggest that the introduction of a large residue (e.g. Tyr or Trp) into the Cys31 position disrupts protein folding or its insertion into the membrane. In any case, the side chain volumes at positions 39 of MotB and 31 of PomB seem to be important for function. To confirm this, we plotted the swarming rate against the volume of the introduced amino acid side chain (Figure 3a).

View Article: PubMed Central - PubMed

ABSTRACT

Flagellar motor proteins, MotA/B and PomA/B, are essential for the motility of Escherichia coli and Vibrio alginolyticus, respectively. Those complexes work as a H+ and a Na+ channel, respectively and play important roles in torque generation as the stators of the flagellar motors. Although Asp32 of MotB and Asp24 of PomB are believed to function as ion binding site(s), the ion flux pathway from the periplasm to the cytoplasm is still unclear. Conserved residues, Ala39 of MotB and Cys31 of PomB, are located on the same sides as Asp32 of MotB and Asp24 of PomB, respectively, in a helical wheel diagram. In this study, a series of mutations were introduced into the Ala39 residue of MotB and the Cys31 residue of PomB. The motility of mutant cells were markedly decreased as the volume of the side chain increased. The loss of function due to the MotB(A39V) and PomB(L28A/C31A) mutations was suppressed by mutations of MotA(M206S) and PomA(L183F), respectively, and the increase in the volume caused by the MotB(A39V) mutation was close to the decrease in the volume caused by the MotA(M206S) mutation. These results demonstrate that Ala39 of MotB and Cys31 of PomB form part of the ion flux pathway and pore with Met206 of MotA and Leu183 of PomA in the MotA/B and PomA/B stator units, respectively.

No MeSH data available.


Related in: MedlinePlus