J. Biochem. 135, 43–51 (2004) DOI: 10.1093/jb/mvh005 Multimeric Structure of the PomA/PomB Channel Complex in the Na+-Driven Flagellar Motor of Vibrio alginolyticus Tomohiro Yorimitsu*, Masaru Kojima, Toshiharu Yakushi and Michio Homma† Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602 Received October 3, 2003; accepted October 30, 2003 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 com- posed 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 pres- ence 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 PomA∼P172C and P172C∼P172C, but not P172C∼PomA. 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 com- plex consisting of two PomA and one PomB, and that they might interact with each other via not only PomA but also PomB. Key words: electrostatic interaction, energy transduction, flagellar motor, sodium type, Vibrio. Abbreviations: DTT, dithiothreitol; NEM, N-ethylmaleimide; PomBcl, PomB mutant with three Cys residues replaced with Ala. Many bacteria have filamentous organelles called flag- complex is thought to be changed by the H+ flux, and this ella that are used for motility. Bacterial flagella rotate by change is transmitted to FliG, which is a component of means of a motor, which is embedded in the membrane at the rotor or motor/switch complex. This complex is the base of the flagellar filament. The direction of motor formed by three proteins, FliG, FliM and FliN, and is rotation, clockwise or counterclockwise, is reversibly reg- required not only for torque generation, but also for flag- ulated by chemotactic signals. The torque of the motor is ellar assembly and for the regulation of the direction of generated by the electrochemical potential of specific ions rotation of the flagellar motor (17–19). across the cytoplasmic membrane (1, 2). Two types of Some bacteria, such as alkaliphilic Bacillus and Vibrio motors are known, H+-driven (3, 4) and Na+-driven (5, 6). species, use the electrochemical potential of Na+ ions to More than 40 proteins are needed to construct the motor. generate the torque of the flagellar motor (20, 21). Four Genetic and physiological studies of the H+-driven motors proteins—PomA, PomB, MotX, and MotY—have been of Escherichia coli and Salmonella typhimurium have shown to be necessary for the rotation of the polar flagel- identified two proteins, MotA and MotB, which have four lar motor of Vibrio spp., V. alginolyticus, V. parahaemo- and one transmembrane-spanning segments, respec- lyticus, and V. cholerae. V. alginolyticus and V. para- tively (7–10). MotA and MotB form a H+ channel complex haemolyticus also possess H+-driven lateral flagellar from their transmembrane segments (11–13). The MotA/ motors (22, 23). PomA and PomB are orthologous to MotA MotB complex surrounds the rotor part of the motor and and MotB (Fig. 1) and interact functionally with each functions as the stator part by being attached to the pep- other (24, 25). MotX and MotY, which were first charac- tidoglycan layer of the cell via a binding motif in the peri- terized as motor proteins in V. parahaemolyticus (26, 27), plasmic domain of MotB (14–16). To generate the torque are unique to the Na+-driven polar flagellar motor of of the flagellar motor, the conformation of the MotA/MotB Vibrio spp. (28, 29). Although their functions are not yet clear, a recent report showed that they co-fractionate with OmpA upon sucrose gradient centrifugation, which sug- *Present address: Department of Molecular, Cellular and Develop- gests that they are located in the outer membrane (30). + + mental Biology, University of Michigan, Ann Arbor, MI 48109, USA. The Na -type motor has advantages over the H -type †To whom correspondence should be addressed. Tel: +81-52-789- motor for studying the rotary mechanism in the driving 2991, Fax: +81-52-789-3001, E-mail: [email protected] force is easily manipulated by changing the Na+ concen- Vol. 135, No. 1, 2004 43 © 2004 The Japanese Biochemical Society. 44 T. Yorimitsu et al. tration. Furthermore, Na+-channel blockers such as ami- (recA1, endA1, gyrA96, thi–, hsdR17, relA1, supE44, λ–, loride and phenamil specifically inhibit the rotation of ∆(lac-proAB); [F′, traD36, proAB, lacIq, lacZ∆M15]) was the Na+-type motor (31, 32). Based on several lines of evi- used for DNA manipulations and cultured at 37°C in LB dence obtained using these techniques, a complex of medium. When necessary, kanamycin was added to a PomA and PomB is thought to function as the Na+ chan- final concentration of 100 µg/ml for Vibrio cells, or 50 µg/ nel. The PomA/PomB complex, solubilized by β-octylglu- ml for E. coli cells. Plasmid pKS101, a pSU41-based plas- coside and purified and reconstituted into proteolipo- mid, was constructed to carry his6-pomA under lac pro- + somes, has the ability to conduct Na ions upon the moter control (33). Plasmid pKS101-P172C (His6-P172C) generation of a transmembrane electrical gradient cre- was constructed by inserting a 0.6-kb DraI–EcoRI frag- ated by a diffusion potential of K+ using valinomycin (33). ment of pYA301-P172C (39) into the corresponding site of + The Na -conducting activity in proteoliposomes reconsti- pKS101. Plasmid pKS102 harboring his6-pomA and tuted with the PomA/PomB complex is specifically pomB under lac promoter control, was constructed by in- blocked by phenamil and no activity is detected in prote- serting a 1.0-kb HindIII fragment of pKS101 into the cor- oliposomes reconstituted with only PomA. From studies responding site of pSK602 (40). Plasmid pKS106 encodes of Na+ and H+ hybrid motors, we could convert the E. coli a tandem PomA dimer (PomA∼PomA) under lac promoter H+-driven motor into the Na+-driven motor by exchang- control, and was constructed by linking two pomA open ing the MotA/MotB stator with the PomA/PotB7E stator reading frames in frame with a 22-residue linker, MHY- E (34). PotB7 is a chimera joining the N-terminal trans- PYDVPDYAIEGRM-His6, which comprises a hemaggluti- membrane region of PomB to the extracellular C-termi- nin (HA)-tag and six histidines (His6) (35). Figure 4A nal region of MotB. Based on its apparent molecular shows a schematic diagram indicating the restriction mass as measured by gel filtration chromatography, the sites in the structure of PomA and PomA∼PomA. Plas- isolated PomA/PomB complex retaining Na+ uptake mids encoding mutant tandem PomA dimers were con- activity seems to be composed of four PomA and two structed by inserting fragments into the corresponding PomB molecules (33). However, purified PomA proteins sites of pYA301-P172C as follows: pKS106 (PomA∼P172C), form a stable homodimer when expressed without PomB, 0.8-kb DraI fragment of pKS106 (PomA∼PomA); pKS106 which suggests that PomA may also function as a dimer (P172C∼PomA), 1.0-kb HindIII fragment of pKS106 ∼ within a (PomA)4(PomB)2 complex. This idea is supported (PomA PomA); and pKS106 (P172C~P172C), 0.8-kb DraI by the fact that when a Cys residue is introduced into fragment of pKS106 (P172C∼PomA). Likewise, plasmids PomA, which has no native Cys residues, a cross-link is encoding tandem PomA dimers along with PomB were con- formed at the motor site (25). Also, a tandem PomA structed by inserting fragments into the corresponding dimer, in which two pomA genes are genetically fused sites of pYA303 (40) as follows: pAAB101 (PomA∼PomA/ and produce a single polypeptide when expressed, is PomB), 0.8-kb DraI fragment of pKS106 (PomA∼PomA); functional as a torque generator (35). The introduction of pAAB101 (P172C∼PomA/PomB), 0.8-kb DraI fragment of a mutation into either or both halves of the tandem PomA pKS106 (P172C∼PomA); pAAB101 (PomA∼P172C/PomB), dimer inhibits motor function. In a recent study, Braun 1.0-kb HindIII fragment of pKS106 (PomA∼P172C). and Blair (36) showed that MotB with a Cys substitution Gel Filtration Assay—The PomA/PomB complex was in its sole transmembrane segment forms a specifically purified as previously described (33); except that DTT cross-linked dimer when expressed with MotA, suggest- was not used. Membrane vesicles were prepared from ing that MotB also functions as a dimer in the H+-driven NMB191 cells harboring pKS102 disrupted by a French flagellar motor. press and solubilized by 2.5% β-octylglucoside. From the Here, to obtain topological information about the solubilized extracts, the PomA/PomB complex was puri- PomA/PomB complex, we have carried out gel-filtration fied using a Ni-NTA column (QIAGEN) followed by a analysis of the PomA/PomB complex in the presence and MiniQ column (Amersham Pharmacia Biotech).