J. Biochem, 2004, Vol. 135, No. 1 43-51
© 2004 The Japanese Biochemical Society
BIOCHEMISTRY |
Multimeric Structure of the PomA/PomB Channel Complex in the Na+-Driven Flagellar Motor of Vibrio alginolyticus
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602
It is known that PomA and PomB form a complex that functions as a Na+ channel and generates the torque of the Na+-driven flagellar motor of Vibrio alginolyticus. It has been suggested that PomA works as a dimer and that the PomA/PomB complex is composed of four PomA and two PomB molecules. PomA does not have any Cys residues and PomB has three Cys residues. Therefore, a mutant PomB (PomBcl) whose three Cys residues were replaced by Ala was constructed and found to be motile as well. We carried out gel filtration analysis and examined the effect of cross-linking between the Cys residues of PomB on the formation of the PomA/PomB complex. In the presence of dithiothreitol (DTT), the elution profile of the PomA/PomB complex was shifted to a lower apparent molecular mass fraction similar to that of the complex of the wild-type PomA and PomBcl mutant. Next, to analyze the arrangement of PomA molecules in the complex, we introduced the mutation P172C, which has been shown to cross-link PomA molecules, into tandem PomA dimers (PomA
PomA). These mutant dimers showed a dominant-negative effect. DTT could restore the function of PomAP172C and P172CP172C, but not P172CPomA. Interdimer and intradimer cross-linked products were observed; the interdimer cross-linked products could be assembled with PomB. The formation of the interdimer cross-link suggests that the channel complex of the Na+-driven flagellar motor is composed of two units of a complex consisting of two PomA and one PomB, and that they might interact with each other via not only PomA but also PomB.
* Present address: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
To whom correspondence should be addressed. Tel: +81-52-789-2991, Fax: +81-52-789-3001, E-mail: g44416a{at}cc.nagoya-u.ac.jp
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