THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 280, No. 27, Issue of July 8, pp. 25659 –25664, 2005
Printed in U.S.A.

Interactions of MotX with MotY and with the PomA/PomB Sodium
Ion Channel Complex of the Vibrio alginolyticus Polar Flagellum*
Received for publication, January 7, 2005, and in revised form, April 22, 2005
Published, JBC Papers in Press, May 2, 2005, DOI 10.1074/jbc.M500263200

Mayuko Okabe‡, Toshiharu Yakushi§, and Michio Homma
From the Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku,
Nagoya 464-8602, Japan

Rotation of the sodium ion-driven polar flagellum ofVibrio alginolyticus requires the inner membrane sodium ion channel complex PomA/PomB and the outermembrane components MotX and MotY. None of the detergents used in this study were able to solubilize MotX
when it was expressed alone. However, when co-expressed with MotY, MotX was solubilized by some detergents. The change in the solubility of MotX suggests that
MotY interacts with MotX. In agreement with this, apull-down assay showed the association of MotY with
MotX. Solubilized MotX and MotY eluted in the void
volume from a gel-filtration column, suggesting that
MotX and MotY form a large oligomeric structure(s). In
the absence of MotY, MotX affected membrane localization of the PomA/PomB complex and of PomB alone but
not of PomA alone, suggesting an interaction between
MotX and PomB. We propose that MotX exhibits multiple interactions with the other motor components, firstwith MotY for its localization to the outer membrane
and then with the PomA/PomB complex through PomBfor the motor rotation.

The bacterial flagellum is driven by a specific ion flow
depending on the electrochemical potential (1, 2). Two typesof coupling ions, protons and sodium ions, are known. The
motor is embedded in the cytoplasmic membrane at the baseof the flagellar structure and is composed of the stator androtor components. The rotor consists of a rod and two ring
structures termed the MS and C rings. Two additional rings,
the L and P rings that are embedded in the outer membrane
and the peptidoglycan layer, respectively, surround the rod
and act as a molecular bushing of the rotor in Gram-negative
bacteria. It has been proposed that the C ring, which ismounted onto the cytoplasmic face of the MS ring, is surrounded by the stator particles (3, 4). In the proton-driven
motors of Escherichia coli and Salmonella, the stator part of
the force-generating unit is composed of MotA and MotB (5,6). MotA has four transmembrane segments and one large
cytoplasmic region (7). MotB has a single N-terminal transmembrane segment, in which an important Asp is present to* This work was supported by grants-in-aid for scientific researchfrom the Ministry of Education, Science, and Culture of Japan, theJapan Science and Technology Corporation (to M. H. and T. Y.), and theSoft Nano-machine Project of Japan Science and Technology Agency (toT. Y. and M. H.). The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
‡ Present address: Dept. of Molecular Biophysics and Biochemistry,
Yale University, 266 Whitney Ave., Bass 225, New Haven, CT
06520-8114.
§ To whom correspondence should be addressed. Tel.: 81-52-7892992; Fax: 81-52-789-3001; E-mail: 4juji@cc.nagoya-u.ac.jp.
This paper is available on line at http://www.jbc.org

convey protons (8), and a peptidoglycan-binding motif at the
C terminus (9, 10). Several extensive lines of genetic andphysiological studies have shown that the transmembranesegments of MotA and MotB form a H⫹ channel complex(11–13).
The polar flagellum of Vibrio alginolyticus is driven by asodium ion motor (14 –16). In this motor, PomA, PomB, MotX,
and MotY are required for force generation (17–20). PomA
and PomB are orthologs of MotA and MotB, respectively. It
has been shown that the PomA/PomB complex catalyzes sodium ion influx when the purified PomA/PomB complex isreconstituted into proteoliposomes (21). A complex formed
from four PomA and two PomB molecules seems to act as a
sodium ion channel.
MotX and MotY have been identified in Vibrio sp. as specificmotor proteins for the sodium ion-driven motor (22, 23). No
homologues have been reported in E. coli or Salmonella. Recently, MotY homologues were reported in the lateral flagellar
system of Vibrio sp., in which the coupling ions are protons, andin the flagellum of Pseudomonas aeruginosa (24, 25). MotY has
a peptidoglycan-binding motif in the C-terminal region, whichis also found in MotB and PomB (20). MotX and MotY seem tointeract with each other, although direct evidence for this interaction has not yet been obtained (19, 23). Recently we haveshown that the N-terminal segments of MotX and MotY arecleaved, and the mature proteins are fractionated with theouter membrane, suggesting that they are outer membraneproteins (26).
The cytoplasmic region of MotA is postulated to interact withthe rotor component FliG. Electrostatic interactions between
conserved charges on FliG and those on MotA are important for
the rotation of the proton-driven motor of E. coli (27). It isbelieved that both proton- and sodium ion-driven motors rotate
by a similar mechanism because several chimeric motors composed of both types of components are functional (28 –30). However, amino acid substitutions in the conserved charged residues of PomA or FliG in the sodium ion-driven motor of V.
alginolyticus do not abolish motility (31, 32). These discrepancies between the proton- and sodium ion-driven motors suggestthat other charged residues are required for the flagellar rotation in the sodium ion-driven motor or that MotX and MotY
compensate for the effects of the amino acid substitutions in
PomA and FliG. If the latter is true, there may be some interactions among the motor proteins, although there is no evidence for interactions between MotX and/or MotY and the
PomA/PomB complex.
In this study, we have shown that MotX interacts with
MotY and affects membrane localization of the PomA/PomB
complex when MotY is absent. This is the first direct evidence
for interaction of the inner membrane ion channel complex with the outer membrane component in the flagellar
motor.

25659

25660Interactions among Flagellar Motor Components

TABLE I
Bacterial strains and plasmids
Rif r, rifampicin-resistant; Laf ⫺, defective in lateral flagellar formation; Kmr, kanamycin-resistant
Strain or plasmid

V. alginolyticus strains
VIO542NMB94
NMB191
Plasmids
pSU41
pIO6
pKJ401
pKJ502
pKJ503
pKJ601
pKJ602
pKJ701
pMO401
pMO402
pMO711
pMO712
pMO721
pMO722
pSK603
pYA301
pYA303

Genotype or description

Rifr Laf ⫺ motY542
Laf ⫺ motX94
Rifr Laf⫺ ⌬pomAB

20
19
34

kan (Kmr) Plac lacZ␣
pSU38, 1.0-kb HindIII-XbaI fragment (motY⫹)
pSU41, 700-bp XbaI-BamHI fragment (motX⫹)
pSU41, 1.0-kb SalI-XbaI fragment (motY⫹)
pSU41, 1.0-kb SalI-XbaI fragment (motY-his6⫹)
pKJ502 (motY⫹), 700-bp XbaI-BamHI fragment (motX⫹)
pKJ502 (motY⫹), 700-bp XbaI-BamHI fragment (motX-his6⫹)
pKJ601 (motY⫹ motX⫹), 1.9-kb BamHI-SacI fragment (pomAB⫹)
pSU41, 700-bp ClaI-BamHI fragment (motX⫹)
pSU41, 700-bp ClaI-BamHI fragment (motX-his6⫹)
pKJ401 (motX⫹), 1.9-kb BamHI-SacI fragment (pomAB⫹)
pKJ502 (motY⫹), 1.9-kb BamHI-SacI fragment (pomAB⫹)
pKJ401 (motX⫹), 800-bp BamHI fragment (pomA⫹)
pKJ401 (motX⫹), 1.1-kb BamHI-SacI fragment (pomB⫹)
pSU41, 1.1-kb BamHI-SacI fragment (pomB⫹)
pSU41, 800-bp BamHI fragment (pomA⫹)
pSU41, 1.9-kb BamHI-SacI fragment (pomAB⫹)

EXPERIMENTAL PROCEDURES

Bacterial Strains and Growth Conditions—The E. coli strain JM109
was used for DNA manipulations (33). The V. alginolyticus strains usedin this study are listed in Table I. V. alginolyticus cells were cultured at
30 °C in VC medium (0.5% Tryptone, 0.5% yeast extract, 0.4% K2HPO4,
3% NaCl, 0.2% glucose) or in VPG medium (1% Tryptone, 0.4%
K2HPO4, 3% NaCl, 0.5% glycerol). The E. coli strains used for DNA
manipulations and membrane localization experiments were cultured
at 37 °C in LB medium (1% Tryptone, 0.5% yeast extract, 0.5% NaCl).
When necessary, kanamycin was added to a final concentration of 100
␮g/ml (for V. alginolyticus) or 50 ␮g/ml (for E. coli).
Plasmid Construction—DNA manipulations were carried out according to standard procedures (35). Plasmids used in this study are listed
in Table I. Plasmid pKJ402, which was constructed by PCR usingsynthetic oligonucleotide primers, encodes the C-terminal hexahistidine-tagged MotX (MotX-His6). The nucleotide sequence of 5⬘-CATCACCATCACCATCACTAAGGATCC-3⬘ (the BamHI site is underlined) was
inserted between the 3⬘ end of the motX open reading frame and the
BamHI site of plasmid pKJ401. Because this plasmid complemented a
motX mutant of V. alginolyticus, the C-terminal His6 tag does notappear to perturb MotX function (data not shown). The 700-bp XbaIBamHI fragment (containing motX-his6) of pKJ402 was inserted into
pKJ502 (carrying motY) to construct a plasmid, pKJ602, that carries anartificial motY-motX-his6 operon. The 1.9-kb BamHI-SacI fragment(containing pomAB) of pYA303 was inserted into pKJ401 (carryingmotX), constructing a plasmid, pMO711, that carries an artificial motXpomAB operon. This BamHI-SacI fragment was inserted into pKJ502(carrying motY) to construct a plasmid, pMO712, which carries anartificial motY-pomAB operon. The 0.8-bp BamHI fragment (containing
pomA) of pYA301 was inserted into pKJ401 to construct a plasmid,
pMO721, which carries an artificial motX-pomA operon. The 1.1-kb
BamHI-SacI fragment (containing pomB) of pSK603 was inserted into
pKJ401; the resultant plasmid, pMO722, carries an artificial motXpomB operon. The motor genes of the plasmids constructed here do nothave their native promoters and are under the control of the lac promoter operator. Transformation of Vibrio cells by electroporation was
carried out as described previously (38).
Membrane Preparation—Vibrio cells harboring plasmids were cultured at 30 °C in VPG medium. Cells were harvested, washed twicewith V-buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 300 mM NaCl),and stored at ⫺80 °C until use. The frozen cells were thawed andresuspended (0.2 g, wet weight/ml) in 20 mM Tris-HCl, pH 8.0, 5 mM
MgSO4, 0.5 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml DNase I. 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 5 min at 4 °C), and the membrane fraction was recoveredfrom the supernatant by ultracentrifugation at 100,000 ⫻ g for 2 h. The
membrane pellet was suspended in 20 mM Tris-HCl, pH 8.0, 0.5 mM

Reference

36
20
26
26
26
26
This work26
19This work
This work
This work
This work
This work
37
37
37

phenylmethylsulfonyl fluoride, 20% (w/v) glycerol and then homogenized and stored at ⫺80 °C until use.
Detection of Proteins—SDS-PAGE and immunoblotting were performed as described previously (34). The antibodies against MotX peptide and MotY peptide, which are referred to as MotX192 and MotY264,
respectively, were prepared as described previously (19). Protein concentrations were determined with the BCA protein assay kit (Pierce).
Membrane Solubilization by Detergents—Membrane preparations
were suspended in 20 mM Tris-HCl, pH 8.0, containing 0 or 450 mM
NaCl to yield final concentrations of 10 mg of protein/ml. After 72
different kinds of detergents from Detergent Screens 1, 2, and 3 (Hampton Research) were added at 2-fold the critical micelle concentration,
each suspension was mixed gently overnight at 4 °C and centrifuged for
10 min at 200,000 ⫻ g to separate the solubilized supernatant and theinsoluble precipitate. For solubilization of MotX or MotY, five detergents were selected: n-heptyl-␤-D-thioglucoside, n-octyl-␤-D-glucoside,n-decylsulfobetaine (ZWITTERGENT 3-10), octanoyl-N-methylglucamide (MEGA-8), and nonanoyl-N-methylglucamide (MEGA-9).
Pull-down Assay—Membranes from NMB94 harboring pKJ602 or
pKJ601 were suspended in 20TPG (20 mM Tris-HCl, pH 8.0, 0.5 mM
phenylmethylsulfonyl fluoride, 10% (w/v) glycerol) containing 5 mM
imidazole and 1.5% (w/v) n-octyl-␤-D-glucoside to yield final concentrations of 10 mg of protein/ml. The suspension was stirred overnight at
4 °C and centrifuged for 10 min at 200,000 ⫻ g. The clarified supernatant was passed through a Ni2⫹-NTA-agarose1 column (Qiagen), andthen the column was washed with the same buffer. Elution was conducted three times with 20TPG containing 800 mM imidazole and 1.5%(w/v) n-octyl-␤-D-glucoside.
Gel-filtration Chromatography—The membrane fractions from
NMB94 harboring pKJ601 were suspended in 20 mM Tris-HCl, pH 8.0,
1.5% (w/v) n-octyl-␤-D-glucoside to yield final concentrations of 10 mg ofprotein/ml. The suspension was stirred overnight at 4 °C and centrifuged for 10 min at 200,000 ⫻ g. The clarified supernatant (200 ␮l) was
separated on a SuperdexTM 200HR 10/30 column (Amersham Biosciences) equilibrated with the same buffer at a flow rate of 0.5 ml/min.
The eluates were collected in 500-␮l fractions starting 14 min after
sample injection.
Membrane Localization of Motor Proteins—E. coli strain JM109 harboring plasmid pYA303, pMO711, pMO712, pSK603, pMO721, or
pMO722 was grown on LB medium at 37 °C to mid-exponential phase,and expression of motor proteins was induced for 1 h by the addition of
1 mM isopropyl ␤-D-thiogalactopyranoside prior to harvest. The cells
were harvested and converted to spheroplasts. The spheroplast suspension was sonicated, and the total membrane fraction was obtained bycentrifugation (200,000 ⫻ g, 10 min) after the removal of unbroken
spheroplasts. The total membranes were resuspended in 50 mM Tris-

1

The abbreviation used is: Ni2⫹-NTA, nickel-nitrilotriacetic acid.

Interactions among Flagellar Motor Components

25661

FIG. 2. Pull-down assay of MotY with MotX-His6. Membranes of
NMB94 (motX) harboring pKJ602 (motYmotX-his6) (A) or pKJ601 (motYmotX) (B) were solubilized overnight with 1.5% n-octyl-␤-D-glucosidein the absence of NaCl. The supernatant was passed through a Ni2⫹NTA column. The bound materials were eluted with high imidazole
buffer after extensive washing of the column with low imidazole buffer
as described under “Experimental Procedures.” Solubilized membranesupernatant (S), column flow-through (F), column wash (W), and eluate(E) fractions were analyzed by SDS-PAGE and immunoblotting using
anti-MotX and anti-MotY antisera.

FIG. 1. Solubilization of MotX and MotY by n-octyl-␤-D-glucoside (A) or ZWITTERGENT 3-10 (B). Membranes of NMB94 harboring pKJ402 (motX) or pKJ601 (motYmotX) were treated overnight witha 2-fold concentration of the critical micelle concentration of the detergent in the presence or absence of NaCl. The supernatant (S) and pellet(P) were separated by ultracentrifugation (200,000 ⫻ g, 10 min) andsubjected to SDS-PAGE and immunoblotting using anti-MotX and antiMotY antisera.
HCl, pH 7.5, containing 1 mM EDTA and then applied to a 35–55%(w/w) stepwise sucrose gradient containing 50 mM Tris-HCl, pH 7.5, and
1 mM EDTA. After centrifugation at 80,000 ⫻ g for 12 h at 4 °C, thegradient was divided into 20 –25 fractions from the bottom to the top.
OmpA, SecG, MotX, MotY, PomA, and PomB proteins in each fraction
were detected by SDS-PAGE followed by immunoblotting with therespective antibodies (19, 34, 39, 40).
RESULTS

Solubilization of MotX and MotY—Some detergents such as
Triton X-100 solubilized MotY. However, MotX was not solubilized by any detergents used so far (26). To carry out biochemical characterization of these proteins in more detail, we examined 72 different kinds of detergents to determine whether theysolubilize MotX and MotY. Because we have examined solubilization in the presence of 150 mM NaCl in a previous study(26), in this study we examined detergents in the presence orabsence of 450 mM NaCl. We found that eight detergents, six
nonionic (n-heptyl-␤-D-thioglucoside, n-octyl-␤-D-glucoside, CYMAL-3, MEGA-8, n-hexyl-␤-D-glucoside, and MEGA-9) and two
zwitterionic (ZWITTERGENT 3-10 and FOS-choline-8), solubilized MotX and MotY when co-expressed from a plasmid (Fig. 1
for the second and seventh detergents in the above list). MotX
and MotY were solubilized by these detergents only in theabsence, but not in the presence, of salt. From the detergentslisted above, we used n-heptyl-␤-D-thioglucoside, n-octyl-␤-Dglucoside, MEGA-8, MEGA-9, and ZWITTERGENT 3-10 toassess the solubility properties of MotX when it was expressedalone. None of the detergents solubilized MotX alone (Fig. 1 forthe second and fifth detergents in the above list), suggestingthat the solubility of MotX changed in the presence of coexpressed MotY, probably because of interactions between
MotX and MotY.

Interaction between MotX and MotY—To investigate
whether MotX actually interacts with MotY, we performed apull-down assay with MotX-His6. The binding efficiency of
MotX-His6 to a Ni2⫹-NTA resin was very poor, and almost thesame amount of MotX-His6 was recovered in the flow-throughfraction as was in the applied materials (Fig. 2A, compare lanes
1 and 2). This may be because of sequestration of the C-terminal His6 tag by oligomerization of MotX (see below). However,a small but significant amount of MotX-His6 appeared in theelution fractions. Untagged MotY was also found in the elution
fractions (Fig. 2A). On the other hand, neither MotX nor MotY
was detected when untagged MotX and MotY were used, confirming that no nonspecific binding to the column occurred.
Therefore, we conclude that MotX binds to MotY.
Analytical Gel-filtration Chromatography of MotX and
MotY—To estimate the molecular size of the MotX/MotY complex, the proteins solubilized by n-octyl-␤-D-glucoside were analyzed by a SuperdexTM 200 gel-filtration column. Both MotX
and MotY eluted in the void volume (Fig. 3). Thus, it is likelythat MotX and MotY form an oligomer(s) too large to analyze.
In agreement with this result, such large oligomers were also
detected in the high molecular weight fractions of a sucrose
density gradient centrifugation experiment (data not shown).
To rule out the possibility of artifactual oligomerization because of their overexpression, MotX solubilized from wild-type
cells expressing only chromosomal levels of motX was also
shown to elute in the void volume (data not shown).
The biochemical properties of MotY seemed to be drastically
influenced by salt. Because most of MotY was present in the
periplasmic space in soluble form in the absence of MotX (26),both membrane-bound (Fig. 4A) and soluble (Fig. 4B) MotY
were analyzed by gel-filtration chromatography. Both forms
eluted in the void volume in the absence of NaCl (Fig. 4, upper
panels). On the other hand, in the presence of 300 mM NaCl,both membrane-bound and soluble MotY appeared in the fractions between albumin (67 kDa) and chymotrypsinogen A (25kDa), which corresponds to the monomeric size of MotY (Fig. 4,
lower panels). Similarly, the monomeric form of MotY wasobserved at the same concentration of KCl (data not shown).
Alteration in Membrane Localization of PomA and PomB by
Co-expression with MotX—When co-expressed, MotX and MotY

25662

Interactions among Flagellar Motor Components

FIG. 3. Analytical gel-filtration chromatography of MotX andMotY with a SuperdexTM 200HR 10/30 column. The membrane
fractions of VIO542 (motY) harboring pKJ601 (motYmotX) were solubilized overnight with 1.5% n-octyl-␤-D-glucoside, and the solubilized
supernatant was separated and fractionated by the column as describedunder “Experimental Procedures.” MotX and MotY in each elution
fraction were detected by immunoblotting with anti-MotX and antiMotY antisera after SDS-PAGE. The positions of the molecular mass
markers, blue dextran (void volume) and catalase (232 kDa), are shown
below the panel.

FIG. 4. Analytical gel-filtration chromatography of MotY withor without salt. After VIO542 (motY) cells harboring pIO6 (motY)
were disrupted by sonication, the soluble and membrane fractions wereprepared in the presence or absence of NaCl. A, the membrane fractionsof VIO542 (motY) cells harboring pIO6 (motY) were solubilized with
1.5% n-octyl-␤-D-glucoside overnight without NaCl or with 300 mM
NaCl. The detergent-solubilized fractions were separated and fractionated by gel-filtration chromatography with a SuperdexTM 200HR 10/30
column as described under “Experimental Procedures.” B, the soluble
fractions were analyzed. MotY in each of the elution fractions was
detected by immunoblotting with anti-MotY antiserum after SDSPAGE. The positions of the molecular mass markers, blue dextran (voidvolume), ferritin (440 kDa), catalase (232 kDa), albumin (67 kDa), andchymotrypsinogen A (25 kDa), are shown below the panels.

are detected in the outer membrane fraction after sucrose density
gradient ultracentrifugation. In contrast, in the absence of MotY,
MotX is detected in intermediate fractions between the inner andouter membrane fractions (26). When PomA, PomB, MotX, andMotY are expressed, MotX and MotY are mainly found in theouter membrane fractions, whereas PomA and PomB are present
in the inner membrane fractions (26).
To test whether MotX and/or MotY affect membrane localization of the PomA/PomB complex, we carried out sucrose
density gradient centrifugation experiments. The model organism E. coli was used because methods for the separation of theinner and outer membranes of V. alginolyticus are not as wellestablished. OmpA and SecG were used as the diagnostic
marker proteins for the outer and inner membranes, respectively. PomA and PomB were detected in the inner membrane
fractions in the absence of both MotX and MotY (Fig. 5A).
Next, E. coli cells expressing PomA, PomB, and either MotX
or MotY were fractionated (Fig. 5, B and C). When PomA,
PomB, and MotX were co-expressed, they were all detected inintermediate fractions between the inner and outer membrane
fractions. On the other hand, when PomA, PomB, and MotY
were co-expressed, MotY was found in the outer membrane,
whereas PomA and PomB were detected in the inner membrane fractions. These results suggest that MotX, but not

FIG. 5. Membrane localization of PomA, PomB, MotX, andMotY in E. coli JM109 harboring pYA303 (pomAB) (A), pMO711(motXpomAB) (B), or pMO712 (motYpomAB) (C). OmpA and SecG
were used as the diagnostic marker proteins for the outer and inner
membranes, respectively. Membrane preparations were analyzed bysucrose density gradient centrifugation followed by fractionation into
25 or 20 fractions. PomA, PomB, MotX, MotY, OmpA, and SecG in eachfraction were analyzed by immunoblotting after SDS-PAGE. OmpA and
SecG were detected in fractions 7–12 and 13–18 (A), 2–7 and 9 –14 (B),and 2–7 and 8 –13 (C), respectively. PomA, PomB, MotX, and MotY aredemarcated in the fractions with double-headed arrows.

MotY, affects membrane localization of PomA and PomB.
PomB was detected in the inner membrane fractions when it
was expressed by itself (Fig. 6A). When PomB was co-expressedwith MotX, PomB was detected in the same intermediate fractions as MotX, even in the absence of PomA (Fig. 6B). Coexpressed MotX did not affect membrane localization of PomA
in the absence of PomB (data not shown), suggesting that in theabsence of MotY, MotX binds to PomB but not to PomA.
DISCUSSION

Several observations have suggested that MotX interacts
with MotY, although no direct evidence for such an interaction
has been obtained (19, 23, 26). In this study, we attempted tocharacterize the biochemical properties of MotX and MotY in
more detail. We searched for conditions under which detergents can solubilize these proteins. We tried 72 different kindsof detergents and found that co-expressed MotX and MotY weresolubilized by eight different detergents in the absence of salt.
In contrast, MotX alone was not solubilized by these detergentsat all, indicating that MotY strongly affects the solubility of
MotX. Pull-down assay showed that untagged MotY co-eluted
with MotX-His6, indicating a direct interaction between these
proteins. Because overexpression of MotX can suppress a series

Interactions among Flagellar Motor Components

25663

FIG. 7. Schematic illustration of motor assembly and rotation.
i, MotY binds to and delivers MotX to the outer membrane. ii, after
MotX localizes to the outer membrane, the stator complex can rotate therotor. iii, in the absence of MotY, MotX does not localize to the outermembrane but can still interact with PomB. The MotX/PomA/PomB
complex cannot efficiently assemble to the motor. A, PomA; B, PomB; X,
MotX; Y, MotY; OM, outer membrane; IM, inner membrane.

FIG. 6. Membrane localization of PomB and MotX in E. coli
JM109 harboring pSK603 (pomB) (A) or pMO722 (motXpomB)(B). OmpA and SecG were used as the diagnostic marker proteins forthe outer and inner membranes, respectively. Membrane preparations
were analyzed by sucrose density gradient centrifugation followed byfractionation into 23 or 21 fractions. PomB, MotX, OmpA, and SecG ineach fraction were analyzed by immunoblotting after SDS-PAGE.
OmpA and SecG were detected in fractions 3– 8 and 11–16 (A) and 3– 8
and 9 –14 (B), respectively. PomB and MotX are demarcated in thefractions with double-headed arrows.

of motY mutations (19), it is suggested that MotY is involved in
the proper targeting of MotX to the flagellar motor rather thanin torque generation. In fact, MotX fails to localize to the outermembrane in the absence of MotY (26).
Analytical gel-filtration chromatography showed that MotX
and MotY, either expressed from a plasmid or from the chromosome, eluted in the void volume, suggesting that MotX andMotY together form a large oligomeric structure(s). At the
moment, it is not clear whether the MotX/MotY oligomer(s)correspond to a homogeneous complex or just an aggregate(s).
However, it is interesting to note that the stator protein complex also exists as a large oligomer consisting of at least four
PomA (MotA) and two PomB (MotB) proteins (21, 41, 42).
It has been reported that overproduction of MotX is lethal to
E. coli cells and that cell death caused by overproduction of
MotX was dependent on the external NaCl or KCl concentration (23). The oligomeric state of MotY is also strongly influenced by salt concentration; it is monomeric in the presence of
300 mM NaCl or KCl, but it forms a large oligomeric structure(s) in the absence of salt and elutes in the void volume by
gel-filtration chromatography (Fig. 4). Detergents that wereable to solubilize the MotX/MotY complex worked only in theabsence of salt (Fig. 1). Thus, salt concentration appears to bea key factor for the conformation and/or the oligomeric state of
MotX, MotY, and the MotX/MotY complex.
We have shown previously that when expressed alone, MotX
is not fractionated with the inner or the outer membrane (26).
Thus, MotX exhibits different cellular locations depending onwhether or not MotY is present. In this study, we found that in
the absence of MotY, MotX altered the membrane localizationof the PomA/PomB complex and PomB, but not PomA, to fractions between the inner and outer membrane in sucrose density
gradient centrifugation (Figs. 5B and 6B). This result suggests
strongly that MotX binds to PomB. It is not clear whether the
PomA/PomB complex and PomB are detached from the membrane vesicles or whether membrane vesicles containing PomA/
PomB and PomB are isolated from those containing SecG (usedas an inner membrane marker protein) and those containing
OmpA (used as an outer membrane marker protein). It is
possible, however, that MotX binds tightly to PomA/PomB or

PomB, thereby pulling them toward the intermediate fraction
when they are separated by sucrose density gradient centrifugation. In our previous studies of the chimeric flagellar motor,
MotX and MotY have been shown to be required for the motor
function if the periplasmic region of the chimeric B subunits
derive from PomB (28 –30). Therefore, we suggest that MotX
probably binds to the periplasmic region of PomB to make afunctional stator. Thus, the interaction between PomB and
MotX is physiologically relevant to the torque generation process, and therefore, MotX probably forms a part of the stator of
the sodium ion-driven flagellar motor in Vibrio sp.
Based on the available information, we propose a mechanism
of assembly and a functional state of the stator complex of
sodium ion-driven motor (Fig. 7). (i) MotX forms a complex with
MotY, which is then targeted to the outer membrane. (ii) MotX
associated with the outer membrane interacts with the PomA/
PomB complex in the inner membrane through the periplasmic
region of PomB to produce torque. At the moment, we cannot
rule out the possibility that MotY is dissociated from the complex because even in the absence of MotY, a less functional
stator is formed by overproduction of MotX (19). It is likely,
however, that MotY remains with MotX in the complex because
MotX and MotY are fractionated together with the outer membrane when all of the four components are expressed (26). (iii)
If MotY is absent, the MotX/PomA/PomB complex does notefficiently localize to an appropriate position in relation to themotor; therefore, the motor does not rotate fully.
Acknowledgments—We thank Hiroshi Nikaido (University of California, Berkeley) and Ikuro Kawagishi (Nagoya University) for stimulating discussions and encouragement and Tohru Minamino (Osaka
University) and Hedda Ferris (Yale University) for careful reading of
the manuscript and suggestions.
REFERENCES
1. Blair, D. F. (1995) Annu. Rev. Microbiol. 49, 489 –522
2. Imae, Y., and Atsumi, T. (1989) J. Bioenerg. Biomembr. 21, 705–716
3. Macnab, R. (1996) in Escherichia coli and Salmonella (Neidhardt, F. C., ed) pp.
123–145, 2nd Ed., American Society for Microbiology, Washington, D. C.
4. Derosier, D. J. (1998) Cell 93, 17–20
5. Dean, G. D., Macnab, R. M., Stader, J., Matsumura, P., and Burks, C. (1984)
J. Bacteriol. 159, 991–999
6. Stader, J., Matsumura, P., Vacante, D., Dean, G. E., and Macnab, R. M. (1986)
J. Bacteriol. 166, 244 –252
7. Zhou, J. D., Fazzio, R. T., and Blair, D. F. (1995) J. Mol. Biol. 251, 237–242
8. Zhou, J., Sharp, L. L., Tang, H. L., Lloyd, S. A., Billings, S., Braun, T. F., andBlair, D. F. (1998) J. Bacteriol. 180, 2729 –2735
9. Chun, S. Y., and Parkinson, J. S. (1988) Science 239, 276 –278
10. De Mot, R., and Vanderleyden, J. (1994) Mol. Microbiol. 12, 333–334
11. Blair, D. F., and Berg, H. C. (1990) Cell 60, 439 – 449
12. Sharp, L. L., Zhou, J. D., and Blair, D. F. (1995) Biochemistry 34, 9166 –9171
13. Stolz, B., and Berg, H. C. (1991) J. Bacteriol. 173, 7033–7037
14. Atsumi, T., McCarter, L., and Imae, Y. (1992) Nature 355, 182–184
15. Kawagishi, I., Maekawa, Y., Atsumi, T., Homma, M., and Imae, Y. (1995) J.
Bacteriol. 177, 5158 –5160
16. Yorimitsu, T., and Homma, M. (2001) Biochim. Biophys. Acta 1505, 82–93
17. Asai, Y., Kojima, S., Kato, H., Nishioka, N., Kawagishi, I., and Homma, M.
(1997) J. Bacteriol. 179, 5104 –5110
18. Furuno, M., Nishioka, N., Kawagishi, I., and Homma, M. (1999) Microbiol.
Immunol. 43, 39 – 43
19. Okabe, M., Yakushi, T., Asai, Y., and Homma, M. (2001) J. Biochem. (Tokyo)
130, 879 – 884
20. Okunishi, I., Kawagishi, I., and Homma, M. (1996) J. Bacteriol. 178,

25664

Interactions among Flagellar Motor Components

2409 –2415
21. Sato, K., and Homma, M. (2000) J. Biol. Chem. 275, 5718 –5722
22. McCarter, L. L. (1994) J. Bacteriol. 176, 4219 – 4225
23. McCarter, L. L. (1994) J. Bacteriol. 176, 5988 –5998
24. Doyle, T. B., Hawkins, A. C., and McCarter, L. L. (2004) J. Bacteriol. 186,
6341– 6350
25. Stewart, B. J., and McCarter, L. L. (2003) J. Bacteriol. 185, 4508 – 4518
26. Okabe, M., Yakushi, T., Kojima, M., and Homma, M. (2002) Mol. Microbiol. 46,
125–134
27. Zhou, J. D., Lloyd, S. A., and Blair, D. F. (1998) Proc. Natl. Acad. Sci. U. S. A.
95, 6436 – 6441
28. Asai, Y., Kawagishi, I., Sockett, E., and Homma, M. (1999) J. Bacteriol. 181,
6322– 6338
29. Asai, Y., Sockett, R. E., Kawagishi, I., and Homma, M. (2000) EMBO J. 19,3639 –3648
30. Asai, Y., Yakushi, T., Kawagishi, I., and Homma, M. (2003) J. Mol. Biol. 327,
453– 463
31. Yorimitsu, T., Sowa, Y., Ishijima, A., Yakushi, T., and Homma, M. (2002) J.
Mol. Biol. 320, 403– 413
32. Yorimitsu, T., Mimaki, A., Yakushi, T., and Homma, M. (2003) J. Mol. Biol.

334, 567–583
33. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene (Amst.) 33, 103–119
34. Yorimitsu, T., Sato, K., Asai, Y., Kawagishi, I., and Homma, M. (1999) J.
Bacteriol. 181, 5103–5106
35. Sambrook, J., Fritsch, E. F., Maniatis, T. (eds) (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY
36. Bartolome, B., Jubete, Y., Martı́nez, E., and de la Cruz, F. (1991) Gene (Amst.)
102, 75–78
37. Kojima, S., Asai, Y., Atsumi, T., Kawagishi, I., and Homma, M. (1999) J. Mol.
Biol. 285, 1537–1547
38. Kawagishi, I., Okunishi, I., Homma, M., and Imae, Y. (1994) Microbiology(Read) 140, 2355–2361
39. Yamada, H., Matsuyama, S., Tokuda, H., and Mizushima, S. (1989) J. Biol.
Chem. 264, 18577–18581
40. Nishiyama, K., Mizushima, S., and Tokuda, H. (1993) EMBO J. 12, 3409 –3415
41. Braun, T. F., Al-Mawsawi, L. Q., Kojima, S., and Blair, D. F. (2004) Biochemistry 43, 35– 45
42. Yakushi, T., Kojima, M., and Homma, M. (2004) Microbiology (Read) 150,
911–920