Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
Correspondence
Michio Homma
g44416a{at}cc.nagoya-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Escherichia coli and Salmonella typhimurium have proton-driven motors that have been extensively studied (Berry & Armitage, 1999; Blair, 1995
; Derosier, 1998
). The stator complex consists of the cytoplasmic membrane proteins MotA and MotB, which are essential for torque generation. MotA has four transmembrane segments and a large cytoplasmic loop (Zhou et al., 1995
). MotB has a single transmembrane domain and a peptidoglycan-binding motif (Chun & Parkinson, 1988
); there is an Asp residue in the putative transmembrane region, which is speculated to be protonated during torque generation. The protonation of the Asp residue induces a conformational change of the motor protein (Kojima & Blair, 2001
). MotA and MotB together have been suggested to form a proton channel, based on extensive genetic and physiological evidence (Blair & Berg, 1990
; Garza et al., 1995
, 1996
; Sharp et al., 1995a
; Stolz & Berg, 1991
).
Vibrio alginolyticus has two types of flagella in one cell: a lateral flagellum with a proton-driven motor and a polar flagellum with a sodium-driven motor (Atsumi et al., 1992; Kawagishi et al., 1995
). In the polar flagellar motor, the following four proteins have been identified and found to be essential for torque generation: PomA, PomB, MotX and MotY (Asai et al., 1997
; Furuno et al., 1999
; Okabe et al., 2001
; Okunishi et al., 1996
). PomA and PomB are homologous to MotA and MotB, respectively, of the proton-driven motor. Thus it is thought that PomA and PomB form a complex and have similar function to MotA and MotB. The direct interaction between PomA and PomB has been demonstrated (Yorimitsu et al., 1999
). PomA was required for the stability of PomB. PomB also has an essential charged residue, Asp, which is highly conserved in the transmembrane region of the motor protein. MotX and MotY were first identified in Vibrio parahaemolyticus (McCarter, 1994a
, b
), and homologous genes have been identified only in Vibrio species. We suggest that MotX and MotY mutually stabilize each other and that MotX is more directly involved in motor function than MotY (Okabe et al., 2001
). MotY has a peptidoglycan-binding motif at the C-terminal region. The motif is also observed in MotB and PomB. The roles of MotX and MotY have not always been clear. However, these proteins are located in the outer membrane (Okabe et al., 2002
) and may be essential for sodium recognition in the motor and/or for the fast rotation speed of flagella in Vibrio cells (Asai et al., 2003
). The maximal rotational speed of the sodium-driven motor is 1700 revolutions per second, whereas the maximal rotational speed of the proton-driven E. coli motor is 300 revolutions per second (Yorimitsu & Homma, 2001
). The sodium-driven motor has advantages for the study of motor function because sodium-motive force can be easily manipulated. Moreover, the specific sodium channel inhibitors, amiloride and phenamil, can be used to study the mechanism of torque generation in this system (Atsumi et al., 1990
; Sugiyama et al., 1988
). Phenamil-resistant mutations were mapped near the cytoplasmic ends of the putative transmembrane segment of PomA and PomB (Jaques et al., 1999
; Kojima et al., 1999
). It is inferred that the high-affinity phenamil-binding site is located around the interface of the cytoplasmic ends of PomA and PomB.
We previously purified the components of the torque-generating unit, PomA and PomB, which were solubilized by the non-ionic detergent -octylglucoside. We showed that the PomA/PomB complex exhibits significant sodium uptake activity (Sato & Homma, 2000a
). From further experiments, however, we found that the complex dissociates easily with
-octylglucoside. In the present study, the membrane-associated motor proteins were solubilized and isolated by using sucrose monocaprate, another non-ionic detergent. We partly characterized the motor complex composed of PomA and PomB, which appears to be much larger than previously believed.
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METHODS |
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Association of PomA and PomB.
Cells were harvested and washed with V-buffer (25 mM Tris/HCl, pH 7·5, 10 mM MgSO4, 300 mM NaCl). The washed cells were suspended in 400 ml 20TMPD (20 mM Tris/HCl, pH 8·0, 5 mM MgSO4, 0·5 mM PMSF and 1 mM DTT) and mixed with a homogenizer (Polytron, type PT3000), with the rotation speed set at 10 000 r.p.m. for 2 min. After adding DNase I to 20 µg ml1, membrane vesicles were prepared by subjecting the suspension to a single passage through a French press (5501-M, Ohtake Works) at 500 kg cm2 at 4 °C. Undisrupted cells were removed by low-speed centrifugation (10 000 g for 20 min at 4 °C) and the membrane fraction was recovered from the supernatant by centrifugation at 200 000 g for 2 h at 4 °C. The membrane pellet was suspended in 20TNPDG (20 mM Tris/HCl, pH 8·0, 0·15 M NaCl, 0·5 mM PMSF, 1 mM DTT and 10 %, w/v, glycerol). Sucrose monocaprate or -octylglucoside was added to the membrane fraction suspended in 20TNPDG to give final concentrations of 2·5 % and 10 mg membrane protein ml1. Two millilitres of the suspension was mixed for 30 min at 4 °C and centrifuged for 30 min at 100 000 g. Imidazole was added to each supernatant at 5 mM final concentration, then the solubilized membrane preparation was mixed with 25 µl Ni-NTA agarose (QIAGEN) for 1 h at 4 °C. The loaded resin was extensively washed with 20TNPDG containing 5 mM imidazole and 0·5 % sucrose monocaprate or 1·25 %
-octylglucoside. Bound proteins were eluted with 100 µl 20TNPDG containing 400 mM imidazole and 0·5 % sucrose monocaprate or 1·25 %
-octylglucoside.
Preparation of the motor complex.
A culture of bacteria was grown to stationary phase in 18 litres of VC medium. The membrane fraction was prepared as above and suspended in 20TNPDG. This suspension was homogenized with a Polytron homogenizer and sucrose monocaprate was added to 2 % final concentration. The suspension was mixed on ice and centrifuged for 20 min at 10 000 g. After addition of imidazole to 10 mM final concentration, the clarified extract was mixed with Ni-NTA agarose (QIAGEN), incubated at 4 °C for 1 h with gentle mixing, and then packed into the column. The loaded resin was washed with 20TNPDG containing 0·5 % sucrose monocaprate and 10 mM imidazole. Elution was conducted with 20TNPDG containing 0·5 % sucrose monocaprate and 400 mM imidazole. The eluate from the Ni-NTA column was diluted twofold with 20TPDG (20 mM Tris/HCl, pH 8·0, 0·5 mM PMSF, 1 mM DTT and 10 %, w/v, glycerol) and applied to a Q-Sepharose HiTrap Q HP column (Pharmacia) equilibrated with 20TPDG containing 75 mM NaCl. The column was washed with 20TPDG containing 75 mM NaCl and bound material was eluted with a 75700 mM linear gradient of NaCl in 20TPDG.
Gel-filtration chromatography.
A Superose 6 PC 3.2/30 gel-filtration column (Pharmacia) was equilibrated with 20TNPDG containing 0·5 % sucrose monocaprate. A 50 µl sample was applied to the Superose 6 column and eluted with the same buffer as used for the equilibration, at 30 µl min1 flow. The sample was recovered as 60 µl fractions 25 min after the sample injection.
Sucrose density-gradient centrifugation.
The suspension was layered on 530 % linear sucrose gradient in 20TNPDG containing 0·5 % sucrose monocaprate. Centrifugation was carried out in a Beckman SW41Ti rotor for 22 h at 24 000 r.p.m. and 6 °C.
SDS-PAGE and immunoblotting.
These were performed as previously described (Okabe et al., 2001). The antipeptide antibodies against PomA and PomB, which are referred to as PomA91 and PomB93, respectively, were prepared as previously described (Yorimitsu et al., 1999
).
Amino acid sequence of the N terminus.
Proteins separated by SDS-PAGE were transferred to a PVDF membrane and stained by Coomassie blue R250. The protein band corresponding to PomB was excised and analysed with a peptide sequencer (analysis done by APRO Science Co., Tokushima, Japan).
22Na+ uptake by proteoliposomes.
Proteoliposomes were reconstituted by the detergent dilution method as follows. A sample was mixed with 5·0 mg E. coli phospholipids (Avanti Polar Lipids) in 20 mM Tris/HCl, pH 8·0, 200 mM KCl, 0·5 %, w/v, sucrose monocaprate, 10 %, w/v, glycerol. The mixture (100 µl) was sonicated briefly and incubated on ice for 20 min and rapidly diluted (40-fold) into the dilution buffer (20 mM Tris/HCl, pH 8·0, 200 mM KCl). After 15 min at 4 °C with gentle shaking, proteoliposomes formed were recovered by centrifugation at 200 000 g for 1 h, resuspended in 100 µl dilution buffer, frozen in dry ice/ethanol and stored at 80 °C. For assays, the suspension was thawed at room temperature.
The standard incubation mixtures contained the following, in 500 µl, at 30 °C: (i) 20 mM Tris/HCl, pH 8·0, 200 mM choline chloride, 1 mM 22NaCl (0·4 µCi ml1; 14·8 kBq ml1), and (ii) proteoliposomes loaded with 200 mM KCl (50 µl). Components (i) and (ii) were separately incubated at 30 °C for 3 min; they were then mixed and valinomycin was added at the final concentration of 2 µM to form a diffusion membrane potential. At intervals, 90 µl of the reaction mixture was passed through Dowex 50WX8-100 (Sigma) prewashed with 20 mM Tris/HCl buffer, pH 8·0, containing 200 mM choline chloride, to trap the unincorporated 22Na+. The radioactivity of the flow-through fraction was determined by a -counter.
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RESULTS |
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The eluate from the Ni-NTA agarose resin was applied to an anion-exchange column and eluted with a NaCl gradient. Each fraction was analysed by SDS-PAGE followed by staining with Coomassie brilliant blue (Fig. 3a) and by immunoblots using antibodies generated against PomA and PomB (Fig. 3b
). PomB was efficiently recovered. PomA and PomB mostly co-eluted and the main peaks were at approximately 0·3 M NaCl. However, the elution profiles of PomA and PomB, at the peak fractions 11 and 10, respectively, did not completely correspond with each other.
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DISCUSSION |
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The purification procedure of the PomA/PomB motor complex was improved in this study. The detergent sucrose monocaprate allowed better solubilization of membrane fractions associated with the PomA/PomB complex; however, MotX or MotY was still not associated with this complex. We changed the position of the attached hexahistidine tag to motor components. Previously, the tag was fused to the N terminus of PomA. The binding efficiency of His6-PomA to Ni-NTA agarose was not adequate. Therefore, we made a His-tagged PomB protein. The hexahistidine sequence was inserted at the C terminus of PomB, where it was predicted to localize in the periplasmic space or bind to peptidoglycans. PomB-His6 functions like the wild-type PomB. Interaction with the rotor has been shown to be necessary to adjust the structure to a functional form of the stator, which is composed of A and B subunits (Garza et al., 1996). We speculate that a small fraction of the motor complex in the overproduced condition was activated by interaction with the rotor because the functional rotors were not overproduced. Therefore, cells can survive in the overproduced condition. Similarly, the overproduction of MotA and MotB did not severely affect cell growth (Wilson & Macnab, 1990
)
A complex composed of MotA and MotB is thought to be the force-generating unit. There is no direct evidence of this; however, the molar ratio of MotA and MotB is thought to be 1 : 1 and an ion-conducting pore forms with the transmembrane regions of each protein (Sharp et al., 1995a, b
). In E. coli, a systematic Cys-substitution against a single transmembrane segment of MotB was recently performed. Periodic formation of cross-links on one face of its
-helix with or without the co-expression of MotA was found (Braun & Blair, 2001
). It has been proposed that the two copies of MotB form a dimer in the MotA/MotB complex and the two Asp residues are displayed on separate faces. A new topological model was presented from the assumption that the stoichiometry of the force-generating unit is a 4 : 2 complex of MotA and MotB (Blair, 2003
). Furthermore, this stoichiometry of PomA and PomB was reported in one of our recent papers (Yorimitsu et al., 2004
). This model is consistent with the biochemical evidence that the ratio of PomA and PomB is 2 : 1. This has been determined from the intensity of the Coomassie blue staining. Also, the size of the complex estimated by gel filtration assay is 175 kDa (Sato & Homma, 2000a
). This size corresponds to the complex of four PomA and two PomB proteins. It has been shown that PomA forms a stable homodimer and both halves of the dimer seem to function together to conduct sodium ions (Sato & Homma, 2000a
, b
).
The molecular size of the PomA/PomB-His6 complex has been estimated to be greater than 900 kDa when solubilized and separated by the detergent sucrose monocaprate. When gel-filtration chromatography was used, PomB was eluted as an approximately 260 kDa complex, which is based on the analysis of fraction 9 by anion-exchange chromatography. The size of 260 kDa is much larger than the monomer size of PomB (37 kDa). In the presence of 50 mM DTT, the position of the main PomB eluted fraction was shifted to one corresponding to about half the size. Since PomB has three cysteine residues, the most probable explanation is that PomB forms a disulfide bridge between PomB molecules (Yorimitsu et al., 2004). MotB is predicted to form a dimer with an estimated size of 74 kDa based on cross-linking experiments (Braun & Blair, 2001
). However, the size of approximately 260 kDa is still much larger than the dimer size of PomB. The reliability of the estimated size of the membrane protein in the presence of the detergent is unknown. However, the size of the huge complex of PomA and PomB was confirmed by sucrose density-gradient centrifugation. On the other hand, the structure of MotB or PomB is expected to be very extended. Therefore, estimation of the molecular mass may be very difficult when globular proteins are used as standard proteins. Even with treatments of SDS and DTT, PomB was eluted at a size greater than 100 kDa as a globular protein. Currently, we cannot confirm that PomB still forms a complex.
Apparently, most of PomB does not associate with PomA. PomA and PomB proteins were co-purified in chromatography fractions with the highest molecular size (approx. 900 kDa), which suggests that they form the 900 kDa complex. This might not be an intact force-generating unit because the amount of PomA is smaller than that of PomB in the high molecular size complex. Specifically, the PomB band was clearly detected by Coomassie blue R250 while the PomA band was not. This may suggest that PomA interacts with multiple PomB proteins or PomB complexes. In this condition, the association of PomA and PomB was probably destroyed. Even though the amount recovered improved, the sodium uptake activity was lost in this procedure. We suspect that the native PomA/PomB complex does not have ion-conducting activity, rather the disordered complex treated with certain detergents such as -octyoglucoside does. We need to further clarify the biochemical character of the membrane components, PomA and PomB, or the complex, and to investigate improved conditions or detergents that maintain both association and activity.
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ACKNOWLEDGEMENTS |
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Received 18 June 2003;
revised 17 November 2003;
accepted 18 December 2003.