MotX and MotY, specific components of the sodium-driven flagellar motor, colocalize to the outer membrane in Vibrio alginolyticus.

Abstract

Rotation of the sodium-driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium-driven motor of Vibrio, have been believed to be localized in the inner (cytoplasmic) membrane via their N-terminal hydrophobic segments. Here we show that MotX and MotY colocalize to the outer membrane. Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N-terminal hydrophobic segments, the N-termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX and MotY require each other for efficient localization to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that describes motor proteins function in the outer membrane for flagellar rotation.

Full text

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Molecular Microbiology (2002) 46(1), 125–134
© 2002 Blackwell Publishing Ltd
Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Science, 200245Original Article MotX and MotY colocalize to the outer membraneM. Okabe, T. Yakushi, M. Kojima and M. Homma
Accepted 5 July, 2002. *For correspondence. E-mail 4juji@
cc.nagoya-u.ac.jp; Tel. (+81) 52 789 2992; Fax (+81) 52 789 3001.
MotX and MotY, specific components of the
sodium-driven flagellar motor, colocalize to the outer
membrane in Vibrio alginolyticus
Mayuko Okabe, Toshiharu Yakushi,* Masaru Kojima
and Michio Homma
Division of Biological Science, Graduate School of
Science, Nagoya University, Chikusa-Ku, Nagoya, Japan.
Summary
Rotation of the sodium-driven polar flagella of Vibrio
alginolyticus requires four motor proteins: PomA,
PomB, MotX and MotY. MotX and MotY, which are
unique components of the sodium-driven motor of
Vibrio, have been believed to be localized in the inner
(cytoplasmic) membrane via their N-terminal hydro-
phobic segments. Here we show that MotX and MotY
colocalize to the outer membrane. Both proteins,
when expressed together, were detected in the outer
membrane fraction separated by sucrose density gra-
dient centrifugation. As mature MotX and MotY pro-
teins do not have N-terminal hydrophobic segments,
the N-termini of the primary translation products must
have signal sequences that are removed upon trans-
location across the inner membrane. Moreover, MotX
and MotY require each other for efficient localization
to the outer membrane. Based on these lines of evi-
dence, we propose that MotX and MotY form a com-
plex in the outer membrane. This is the first case that
describes motor proteins function in the outer mem-
brane for flagellar rotation.
Introduction
Bacterial flagellar motors are the locomotive organelles
that drive the cell by rotating their helical flagellar filament
(Berg, 1995; Blair, 1995; Macnab, 1996). In Gram-
negative bacteria, the rotor portion of the flagellar basal
body consists of an axial rod and four rings: L, P, MS and
C rings. The L, P and MS rings are thought to be embed-
ded in the outer membrane, the peptidoglycan layer, and
the inner (cytoplasmic) membrane respectively (Derosier,
1998). It is speculated that the cytoplasmic C ring is sur-
rounded by 6–16 stator particles (force-generating units)
in the inner membrane (Blair and Berg, 1988; Khan et al.,
1988; Muramoto et al., 1994). The motor is energized by
a transmembrane electrochemical potential using either
protons or sodium as the coupling ion (Manson et al.,
1977; Hirota et al., 1981).
Motility (mot) mutants of bacteria have paralysed fla-
gella and are unable to generate the torque required to
rotate the filament. Two motor genes, motA and motB,
have been identified in Escherichia coli and Salmonella,
whose motors use protons as the coupling ion for the
rotation of flagella (Dean et al., 1984; Stader et al., 1986).
MotA and MotB are inner membrane proteins and are
believed to convert the electrochemical potential to
mechanical energy in concert with the rotor component.
MotA has four transmembrane segments and one large
cytoplasmic segment, which has been proposed to elec-
trostatically interact with FliG (Zhou et al., 1995, 1998a;
Lloyd et al., 1999). MotB has a single N-terminal trans-
membrane segment where an Asp residue is essential
for proton flow as a part of the torque-generation process
(Zhou et al., 1998b). MotB also has a peptidoglycan-
binding motif at its C-terminus (Chun and Parkinson,
1988; De Mot and Vanderleyden, 1994). The transmem-
brane segments of MotA and MotB are thought to form
a proton channel (Blair and Berg, 1990; Sharp et al.,
1995a, b). MotA is thought to undergo a conformational
change that is coupled to proton flow to alter its interaction
with FliG, thereby driving rotation (Kojima and Blair,
2001).
Alkalophilic Bacillus firmus and Vibrio species use
sodium as the coupling ion for flagellar rotation (Imae
and Atsumi, 1989; Yorimitsu and Homma, 2001). Vibrio
alginolyticus and Vibrio parahaemolyticus have sodium-
driven polar flagella and proton-driven lateral flagella,
which are used for movement on a surface (Atsumi et al.,
1992; Kawagishi et al., 1995). The sodium-driven motor
of V. alginolyticus rotates stably and remarkably fast, up
to 1700 r.p.s., compared with rotation of the proton-driven
motors of E. coli and Salmonella, which has a speed of
around 300 and 200 r.p.s. respectively (Lowe et al., 1987;
Kudo et al., 1990; Magariyama et al., 1994; Muramoto
et al., 1995). Lateral flagellar motor genes, lafT and lafU

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M. Okabe, T. Yakushi, M. Kojima and M. Homma

© 2002 Blackwell Publishing Ltd,

Molecular Microbiology

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46

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of
lents
and Wright, 1993). In contrast, four motor genes (
pomB, motX and motY) have been identified as essential
for rotation of the sodium-driven polar flagellum in
(Asai et al., 1997; Gosink and Häse, 2000; McCarter,
2001; Okabe et al., 2001).
PomA and PomB are orthologues of MotA and MotB
respectively. Physical interaction between PomA and
PomB has been demonstrated (Yorimitsu
A purified PomA/PomB complex was shown to have
sodium-conducting activity in vitro
2000a). It appears that PomA forms a stable homodimer
and the two subunits function together (Sato and Homma,
2000b). A periplasmic loop region of PomA is considered
important for dimer formation (Yorimitsu
A hybrid motor that consists of PomB, MotX and MotY
from V. alginolyticus and MotA
motor of Rhodobacter spheroides
driven motor in Vibrio cells (Asai
MotB
RS
functions with neither MotA
Furthermore, the chimeric MomB protein, which contains
the N-terminal transmembrane region of MotB
C-terminus of PomB, serves in conjunction with MotA
as a sodium-driven motor in
2000).
MotX and MotY are unique to the sodium-driven flagel-
lar motor of Vibrio. The deduced amino acid sequences
of MotX and MotY show high identity among the
species. MotX and MotY have been characterized as
membrane proteins because their sequences predict a
single transmembrane segment. Both proteins are recov-
ered from the membrane fraction when expressed in
either E. coli or Vibrio (McCarter, 1994a; Okabe
2001). The membrane localization of MotY in
depends on MotX, and MotY remains in the soluble frac-
tion unless MotX is coexpressed (McCarter, 1994a). MotX
and MotY mutually stabilize by interaction within the mem-
brane (Okabe
et al., 2001). MotY also has a putative
peptidoglycan-binding motif in its C-terminal region, as
does MotB and PomB (McCarter, 1994b; Okunishi
1996). MotX and MotY each have two cysteine residues,
one of which is found in the conserved tetrapeptide CQLV
that is in a similar position from the N-terminus.
In E. coli, there are no homologues of MotX and MotY.
Overproduction of MotX in E. coli
is suppressed by amiloride, a sodium channel blocker
(McCarter, 1994a). This harmful effect of MotX does not
require MotY, nor is the production of MotY alone detri-
mental to E. coli. Therefore, it was suggested that MotX
participates somehow in sodium translocation across the
inner membrane. A corollary to this proposed function is
that MotX and/or MotY may modulate the ion specificity
of the PomA/PomB complex.

V. parahaemolyticus
motA and

, are quite similar to their equiva-
in other proton motors (McCarter

motB

pomA

,

Vibrio



et al

., 1999).

(Sato and Homma,

et al

., 2000).


RS

from the proton-driven
functions as a sodium-
et al., 1999). In contrast,
nor PomA in





RS

Vibrio

.

RS

and the

RS
.,

V. alginolyticus

(Asai

et al



Vibrio


et al
E. coli

.,



et al

.,


is lethal and its lethality

In this paper, we present evidence that MotX and MotY
localize to the outer membrane. Our data require that an
alternative model for the function of MotX and MotY be
developed.

Results

Membrane localization of MotX and MotY

MotX and MotY were classified as membrane proteins
because they both possess a single hydrophobic domain
of sufficient length to span the inner membrane (McCarter,
1994a, b). Both proteins are predicted to have a large
C-terminal periplasmic domain, topology that makes
them type II membrane proteins (McCarter, 2001). It was
also known that MotY can be recovered in the soluble
fraction when expressed in
E. coli
(McCarter, 1994a). This result could be explained by the
relatively low hydrophobicity of the MotY transmembrane
region (Okabe et al., 2001). On the other hand, the
sequence alignment of MotX and MotY of
reveals that the putative transmembrane regions are
not highly conserved (Okabe
could indicate that the transmembrane domain of the
proteins is not particularly important. A prediction of sub-
cellular localization of MotX and MotY using an on-line
programme (http://psort.ims.u-tokyo.ac.jp/) (Nakai and
Kanehisa, 1991) showed that their most probable des-
tination is the periplasmic space. Also, this predicted that
their putative transmembrane segment may be cleaved
during translocation across the inner membrane.
To determine the subcellular localization of MotY, we
carried out subcellular fractionation such as spheroplast-
ing and membrane separation using sucrose density
gradient centrifugation. E. coli
combinations of the motor proteins were converted into
spheroplasts and then fractionated to periplasmic, cyto-
plasmic and membrane fractions. The
sor, LacI, maltose-binding protein, MBP, and OmpA were
used as the diagnostic marker proteins for the cytoplasm,
periplasm and membranes respectively. As shown in
Fig. 1, we compared the lanes designated as W and
others, and a significant portion of MotY seemed to be
degraded during the fractionation, even if coexpressed
with MotX from pKJ601 and pKJ701. When MotY was
expressed by itself, however, a relative amount of MotY
was highest in the periplasmic fraction (lanes 10–12).
Even under these conditions, a little MotY could be
detected in the membrane fraction if high amounts of the
sample were loaded on SDS-PAGE (data not shown).
When coexpressed with MotX, MotY was detected exclu-
sively in the membrane fraction (Fig. 1, lanes 14–16). The
plasmid pKJ601 ( motX
motY
motY mutants of Vibrio (data not shown), which means

in the absence of MotX


Vibrio

species

et al

., 2001). That fact

cells expressing either or

lac

operon repres-


+


+

) complemented

motX

and

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MotX and MotY colocalize to the outer membrane

127

© 2002 Blackwell Publishing Ltd,

Molecular Microbiology

,

46

, 125–134

MotX and MotY produced from the plasmid are functional.
The localization of MotX and MotY were not changed
upon coexpression with PomA/PomB (lanes18–20).
Figure 1 suggests that the previously observed soluble
form of MotY (McCarter, 1994a) resides in the periplasm,
not in the cytoplasm of
E. coli
is found in the membrane fraction despite the presence
or absence of the other motor proteins.
As coexpressed MotX and MotY were both detected in
the membrane fraction, the inner versus outer membrane
localization of the two proteins was determined by density-
gradient centrifugation (Fig. 2).
(
D
pomAB) harbouring plasmid pKJ701 (
pomAB ) was analysed for localization of MotX, MotY and
PomA/PomB. The plasmid pKJ701 complemented the
motX , motY or
D
pomAB mutation of
functional MotX, MotY and PomA/PomB proteins are
synthesized from the artificial
(data not shown). As shown in Fig. 2A, MotX and MotY
colocalized in the higher density fraction, in which an
OmpA-like protein could also be detected. In contrast,
PomA and PomB were detected in the lower density frac-
tion. This result clearly indicates that MotX and MotY
localize to a different membrane than PomA/PomB in
alginolyticus.
The inner versus outer membrane localization of MotX
and MotY was also determined in
ods for separation of the inner and outer membranes of
V. alginolyticus are not as well established as in
Also, the antisera against diagnostic inner and outer mem-
brane proteins are not available. Membranes of strain
JM109 harbouring plasmid pKJ601 (
fractionated by sucrose density gradient centrifugation
(Fig. 2B). The relative amounts of the inner membrane
protein SecG (Nishiyama et al

. MotX, on the other hand,

V. alginolyticus

NMB191
motX




+

motY

+


+




Vibrio

, indicating that

motY-motX-pomAB

operon

V.


E. coli

because meth-

E. coli

.

motX

+



motY

+

) were

., 1993), the outer mem-
brane protein OmpA (Klose et al., 1988), and MotX and
MotY were determined by immunoblotting. MotX and
MotY were contained in the same membrane fraction as
OmpA (Fig. 2B). A negligible amount of MotX and MotY
was detected in the inner membrane fraction, where SecG
was present. When E. coli cells expressing PomA, PomB,
MotX and MotY were fractionated, most of MotX and
MotY were again found in the outer membrane fraction,
whereas PomA and PomB were present in the inner mem-
brane fraction (Fig. 2C). A very little part of MotX could be
detected in the inner membrane fraction, indicating the
Fig. 1. Subcellular localization of MotX and MotY. Escherichia coli
strain JM109 cells harbouring plasmid pSU41 (vector), pKJ401
(motX+), pKJ502 (motY+), pKJ601 (motX+ motY+) or pKJ701 (motX+
motY+ pomAB+) were converted to spheroplasts. The periplasmic
fraction was separated from spheroplasts by low speed centrifugation
(10 000 g, 5 min). The spheroplasts were disrupted by sonication.
Then, cytoplasmic and membrane fractions were prepared by ultra-
centrifugation. Equivalent amounts of the whole cell (W), periplasmic
(P), cytoplasmic (C) and membrane (M) fractions were subjected to
SDS-PAGE, followed by immunoblotting using anti-MotX, MotY,
OmpA, MBP and LacI antisera.
Fig. 2. Membrane localization of motor proteins. Membrane prepara-
tions were analysed by sucrose density gradient centrifugation. The
gradient was divided into 21 or 24 fractions from the bottom. MotX,
MotY, PomA, PomB, OmpA and SecG in each fraction were detected
by immunoblotting using the appropriate antiserum. The membrane
was prepared from Vibrio alginolyticus strain NMB191 (DpomAB)
harbouring plasmid pKJ701 (motX+ motY+ pomAB+) (A), E. coli
JM109 cells harbouring plasmid pKJ601 (motX+ motY+) (B) and E.
coli JM109 harbouring plasmid pKJ701 (motX+ motY+ pomAB+) (C).
In B and C, OmpA and SecG were detected in fractions 3–8 and
11–16 respectively.

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128M. Okabe, T. Yakushi, M. Kojima and M. Homma
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134
MotX may slightly change its localization upon coexpres-
sion with PomA/PomB.
Signal peptide cleavage of MotX and MotY
If MotX and MotY localize to the outer membrane, they
must have an N-terminal signal sequence (Pugsley,
1993). MotX and MotY were partially purified from V. alg-
inolyticus as described in Experimental procedures. Their
N-terminal amino acid sequences were analysed by
Edman degradation. The N-terminal sequence of MotX
was determined to be NVADV, indicating that the precur-
sor was cleaved between Ala21 and Asn22. The N-terminus
of MotY was determined to be VMGKR, indicating that the
processed form of MotY begins with Val22. Additionally, N-
terminus of MotX protein prepared from E. coli expression
system had the identical sequence with that from V. algi-
nolyticus. This result confirmed that the same processing
reaction of MotX takes place within the two bacteria.
Altering the membrane localization of MotX and MotY
As described above, when expressed alone, most of MotY
seemed to be in the periplasm as a soluble protein. On
the other hand, when MotX and MotY were coexpressed,
the both localized to the outer membrane (Fig. 2). We
speculate that properties of MotY were altered by MotX.
Figure 1 shows that, without MotX, most of MotY is recov-
ered in the periplasmic fraction, however, some could be
in the membrane fraction (data not shown). Membrane
fraction of strain JM109 harbouring plasmid pKJ401
(motX+) or pKJ502 (motY+) was fractionated by sucrose
density gradient centrifugation (Fig. 3). Comparing
Figs. 2B and C, and 3A, MotX seems to be detected in a
strange fraction, in which it does not correspond with
either OmpA or SecG, in the absence of MotY. This result
leads to three possibilities that MotX is present in (i) nei-
ther membrane (ii) the both membranes (iii) an interme-
diate membrane other than the inner and the outer
membranes. In contrast, MotY was detected exclusively
in the outer membrane fraction even in the absence of
MotX (Fig. 3B).
MotX and MotY colocalize to the outer membrane
Previous studies and Fig. 1 showed that MotY is targeted
to the membrane in the presence of MotX (McCarter,
1994b; Okabe et al., 2001). As shown in Figs 1 and 3A,
MotX expressed by itself is in the membrane fraction,
but its distribution corresponded with neither SecG nor
OmpA. However, MotX coexpressed with MotY mostly
localizes to the outer membrane (Figs 2B and C). These
results indicate that both MotX and MotY are required for
localization of either one to the outer membrane.
To elucidate the biochemical properties of MotX and
MotY, we attempted to solubilize them with several deter-
gents. Membrane fractions of strain NMB94 (motX)
expressing MotX (Fig. 4A) or of strain VIO542 (motY)
expressing MotY (Fig. 4B) were used. Like E. coli, the
latter strain mostly produces soluble MotY, but a little
fraction of MotY is recovered in the membrane fraction,
and the membrane-bound MotY can be not released by
washing with buffer not containing any detergents (data
not shown). MotX could not be solubilized any of the
following four detergents: Triton X-100, b-octylglucoside,
sucrose monocaprate and CHAPS. On the other hand,
MotY was solubilized by all four detergents (Fig. 4). Mem-
brane fractions from VIO542 cells expressing both MotX
and MotY (Fig. 4C) and from NMB191 (DpomAB) express-
ing MotX, MotY, PomA and PomB (Fig. 4D) from a plasmid
were also treated with detergents. Under these conditions,
MotY was not solubilized. MotY did not become solubi-
lized upon coexpression with MotX. This property was not
changed by the simultaneous expression of PomA/PomB.
These results may indicate that the membrane-binding
manner of MotY would be different in strains having and
lacking MotX. Also, a physical interaction between MotX
and MotY exists.
Discussion
The sodium-driven polar flagellum of Vibrio is completely
paralysed by motX or motY mutation, suggesting that
MotX and MotY proteins are essential for the sodium-
driven motor. Mutations of pomA or pomB confer the same
Fig. 3. Membrane localization of MotX and MotY solely expressed.
Membranes were prepared from E. coli JM109 cells harbouring either
plasmid pKJ401 (motX+, A) or pKJ502 (motY+, B) and analysed by
sucrose density gradient centrifugation. The gradient was divided into
23 fractions from the bottom. MotX, MotY, OmpA and SecG in each
fraction, which was separated by SDS-PAGE, were detected by
immunoblotting. OmpA and SecG were detected in fractions 4–9 and
10–15 respectively.

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MotX and MotY colocalize to the outer membrane129
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 125–134
polar-flagellar-motility phenotype, as PomA and PomB
proteins form a complex to conduct sodium ions. On the
other hand, the function of MotX and MotY is obscure,
which have been believed to be located in the inner mem-
brane. In this paper, we concluded that MotX and MotY
colocalize to the outer membrane from the following
observations: (i) MotX and MotY did not have their
putative transmembrane segments, suggesting they are
cleaved during translocation across the inner membrane;
(ii) They localized to the outer membrane in E. coli,
whereas PomA/PomB were in the inner membrane. In the
Vibrio cell, they were detected in the fraction different from
that of PomA/PomB, which may suggest their location in
the outer membrane; (iii) The outer membrane localiza-
tions of MotX and MotY were dependent on each other.
Furthermore, MotY could not be solubilized in the pre-
sence of MotX, supporting their interaction in the outer
membrane. The L-ring component, FlgH, is a flagellar
protein located in the outer membrane (Macnab, 1996).
This protein is thought to participate in the structure of
flagellum, bushing for rotating rod in the outer membrane,
rather than in the motor rotation. Therefore, we suggest
that MotX and MotY are the first example of outer mem-
brane motor proteins.
The putative transmembrane region of each protein,
MotX or MotY, exhibits relatively low similarity among the
Vibrio species (Okabe et al., 2001). This implies that this
region may not have important roles in structure and func-
tion. Such assumptions are reasonable because we dem-
onstrated that the hydrophobic N-terminal sequence of
MotX and MotY was not present in the mature protein. It
can be speculated that the peptide was cut during trans-
location across the inner membrane, probably in a Sec
machinery-dependent manner (Pugsley, 1993). We con-
clude that the predicted transmembrane regions are
signal sequences for export. The protein translocation
mechanism in V. alginolyticus seems to be equivalent to
the E. coli Sec system, because several Sec factors of V.
alginolyticus show high similarity to those of E. coli and
also can function in E. coli (Tokuda et al., 1990; Kunioka
et al., 1998; Nishiyama et al., 1998). In this study, MotX
proteins from the two expression systems of E. coli and
V. alginolyticus had the identical N-terminus, suggesting
functionally homologous enzymes are relevant to the pro-
cessing of secretory proteins in the organisms. From
these lines of evidence, we speculate that primarily trans-
lated forms of MotX and MotY are translocated across the
inner membrane and signal sequences are cleaved to
convert the mature proteins and localize to the outer mem-
brane of V. alginolyticus. MotX and MotY homologues
have not been reported from any other bacteria than
Vibrio species, including alkalophilic Bacillus species that
have sodium-driven flagella. That is reasonable, because
Bacilli do not have an outer membrane. Rhizobium meliloti
requires a periplasmic motor protein MotC for the proton-
driven flagellar rotation (Platzer et al., 1997). This protein,
however, has significant sequence homology with neither
MotX nor MotY.
The secondary-structures of MotX and MotY from V.
alginolyticus were predicted by the method of Chou and
Fasman (Chou and Fasman, 1974). This method showed
that MotX is more a-helical than MotY, which exhibits
an extensive b-strand structure (data not shown). Outer
membrane proteins often form a b-barrel structure in
their membrane-spanning domains (Pugsley, 1993;
Koebnik et al., 2000). A three-dimensional structure
prediction program, 3D-PSSM based on the SCOP
(http://www.sbq.bio.ic.ac.uk/~3dpssm/)
2000), showed that MotY has similarities to OmpA trans-
(Kelley et al.,
Fig. 4. Solubilization of MotX and MotY with several detergents.
Membranes from V. alginolyticus NMB94 (motX94) cells harbouring
plasmid pKJ401 (motX+, A), VIO542 (motY542) cells harbouring plas-
mid pIO6 (motY+, B), plasmid pKJ601 (motX+ motY+, C), and
NMB191 (DpomAB) cells harbouring plasmid pKJ701 (motX+ motY+
pomAB+, D) were solubilized with 2.5% each of Triton X-100 (TX), b-
octylglucoside (OG), sucrose monocaprate (SC) or CHAPS (CH). The
supernatant (s) and precipitate (p) were prepared by ultracentrifuga-
tion (200 000 g, 10 min) and subjected to SDS-PAGE and immunob-
lotting using anti-MotX antiserum (A, and upper panels of C and D)
and anti-MotY antiserum (B, and lower panels of C and D).