J. Biochem. 144, 635–642 (2008)
doi:10.1093/jb/mvn110

Cell-free Synthesis of the Torque-Generating Membrane Proteins,
PomA and PomB, of the Na+-driven Flagellar Motor in Vibrio
alginolyticus
Hiroyuki Terashima, Rei Abe-Yoshizumi, Seiji Kojima and Michio Homma*
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya
464-8602, Japan
Received August 18, 2008; accepted August 29, 2008; published online September 6, 2008

Flagellar motor proteins, PomA and PomB, are essential for converting the sodium
motive force into rotational energy in the Na+-driven flagella motor of Vibrio
alginolyticus. PomA and PomB, which are cytoplasmic membrane proteins, together
comprise the stator complex of the motor and form a Na+ channel. We tried tosynthesize PomA and PomB by using the cell-free protein synthesis system,
PURESYSTEM. We succeeded in doing so in the presence of liposomes, and showed
an interaction between them using the pull-down assay. It seems likely that theproteins are inserted into liposomes and assembled spontaneously. The N-terminal
region of in vitro synthesized PomB appeared to be lost, but this problem was
suppressed by fusing GFP to the N-terminus of PomB or by mutagenesis at Pro-11 orPro-12. A structural change of the N-terminal region of PomB by these modifications
may prevent cleavage during protein synthesis in PURESYSTEM. The mutations did
not affect the functioning of the motor. Using this system, biochemical analysis
of PomA and PomB can be performed easily and efficiently.
Key words: bacterial flagellum, in vitro protein synthesis, ion-driven motor,
membrane protein, stator.
Abbreviations: CHAPS, 3-[(3-cholamidopropyl) dimetylammonio] propanesulfonate; DTT, Dithiothreitol;
HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid.

interaction sites involved in torque generation in the
H+- and Na+-driven motor. A periplasmic region justabove the MotB TM region is proposed to form a plug inthe H+ channel (10, 15). When the MotA/B complex is
produced, no reduction in growth is observed with wildtype MotB, but this occurs if there are mutations in theplug region, suggesting that the opening/closing of the
H+ channel cannot be regulated in the plug mutants.
Protein purification from cells is essential for biochemical or structural analysis. However, some proteins,
especially membrane proteins, are difficult to isolate
from cells. A cell-free protein synthesis system, whichcontains all components required for transcription,translation and energy regeneration to synthesize thetarget proteins in vitro, has been developed (16, 17). Thissystem allows us to produce proteins that are either toxic
to the cell, or impair its growth when overexpressed.
Moreover, using cell-free protein synthesis allows us toskip several steps required for protein production or
purification, thereby save time. By using this method, it
is expected that comprehensive biochemical analysis ofmany mutant proteins can be carried out more easily.
There are several kinds of cell-free protein synthesis
system, which are based on, for example, cell extracts
derived from E. coli, wheat germ or rabbit reticulocyte(16, 18, 19). Alternatively, there is a system, named
PURESYSTEM, which contains all purified and reconstituted components required for protein synthesis (20,21). These cell-free protein synthesis systems haveusually been used for synthesizing soluble proteins.

The bacterial flagellar motor is a rotational molecularmachine, which converts the electrochemical potential
difference of a coupled ion, which is H+ for Escherichia
coli or Salmonella (1, 2) and Na+ for alkalophilic Bacillus
or Vibrio alginolyticus (3), into rotational energy. The
flagellar motor is composed of a stator part and a rotor
part. It is believed that 10–12 functional units of thestator complexes surround the rotor (4, 5). In the polar
flagellum of V. alginolyticus, the cytoplasmic membraneproteins, PomA and PomB, together comprise the stator
complex of the motor and function as a Na+ channel togenerate torque (6). The purified PomA/B complex was
shown in vitro to have sodium-conducting activity, and ispredicted to be in a 4A:2B stoichiometry complex (7).
PomA has four transmembrane (TM) segments and alarge cytoplasmic loop, while PomB has a single TM
domain and a peptidoglycan-binding motif, whichanchors the motor to the cell wall (6). An Asp residueof PomB in the TM region is speculated to be the Na+binding site during torque generation (8). Escherichia
coli has MotA and MotB which are orthologs of PomA
and PomB, respectively and the MotA/B complex functions as the H+ channel (9, 10). The cytoplasmic domainsof MotA and PomA are thought to interact with FliG, oneof the rotor components and to generate the rotary force(11–14). It is still unknown what happens at the*To whom correspondence should be addressed. Tel: +81-52-7892991, Fax: +81-52-789-3001, E-mail: g44416a@cc.nagoya-u.ac.jp
Vol. 144, No. 5, 2008635

ß 2008 The Japanese Biochemical Society.

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H. Terashima et al.

Recently, however, membrane proteins, such as GPCRs(G-protein coupled receptors), EmrE (multidrug effluxpomp), cytochrome b5 and MtlA (mannitol permease) andso on, have been synthesized using this system andsuccessfully used in experiments (22–26).
In order to understand the flagellar motor function, it
has been a long-term aim to know how sodium ions flow
through the PomA/PomB complex and how the opening
and closing the PomA/B channel is regulated. As the
purification and functional reconstitution of membraneproteins of PomA and PomB is still not easy, we took
advantage of the cell-free protein synthesis system as
described above. In this study, we tried to produce theflagellar motor proteins, PomA and PomB, using a cellfree protein synthesis system.
MATERIALS AND METHODS

Bacteria and Media—The strains used are shownin Table 1. Vibrio alginolyticus was cultured at 308C in
VC medium [0.5% (w/v) polypeptone, 0.5% (w/v) yeast
extract, 0.4% (w/v) K2HPO4, 3% (w/v) NaCl, 0.2% (w/v)
Table 1. Bacterial strains and plasmids.
Strain or plasmid Genotype or description
Vibrio alginolyticus strains
NMB191
Rifr, Pof+, Laf pomAB

Reference(27)

Escherichia coli strains
JM109
recA1 endA1 gyrA96 thi
hsdR17 supE44
relA1 (lac-proAB)(F’ traD36
proAB lacIq lacZM15)
BL21(DE3)
T7 expression host(33)

Plasmids
pET22b(+)
pBAD33
pHFAB
pET-pomB
pET-GB
pHFGBA2

Novagen(34)(30)
In this study
In this study(30)

pYA303

T7 expression vector, Ampr
Cmr, PBAD
pomA and pomB in pBAD33
pomB in pET22b(+)
Gfp-pomB in pET22b(+)
his6-gfp-pomB and pomA
in pBAD33
pomA and pomB in pSU41

r

r

(32)

(35)

Rif , rifampicin resistant; Amp , ampicillin resistant; Cmr, chloramphenicol resistant; PBAD, araBAD promoter; Pof+, normal polar
flagellar formation; Laf, defective in lateral flagellar formation.

glucose] or in VPG medium [1% (w/v) polypeptone,
0.4% (w/v) K2HPO4, 3% (w/v) NaCl, 0.5% (w/v)
glycerol]. Escherichia coli was cultured in LB broth [1%(w/v) polypeptone, 0.5% (w/v) yeast extract, 0.5% (w/v)
NaCl]. Ampicillin was added to a final concentration of
100 mg/ml for E. coli. Chloramphenicol was added to a
final concentration of 2.5 mg/ml for V. alginolyticus and25 mg/ml for E. coli.
Plasmid Construction—Plasmids and primers are listed
in Tables 1 and 2, respectively. Fragments containing
pomB with an NdeI site at the 50 and BamHI at the 30
were amplified using PCR, and these fragments wereinserted into the NdeI and BamHI sites of pET22b(+)(Novagen, Germany); this plasmid was named pETpomB. Site-directed mutagenesis in pomB was performedby using the QuickChange procedure (Promega). Fragments containing gfp-pomB were amplified by 1st PCR
using gfp(+) PURE forward and pomB-BamHI(), andthen by 2nd PCR using universal primer(+) containing
XbaI site and pomB-BamHI() and the 1st PCR products
as the template. These fragments were inserted into the
XbaI and BamHI sites of pET22b(+) and named pET-GB.
Preparation of Liposome—L-a-Phosphatidylcholine type
II-S from soybean (Sigma, USA) was dissolved inPA3-buffer [10 mM HEPES–KOH pH 7.5, 5 mM MgCl2,
10% (w/v) glycerol] to a final concentration of 44 mg/ml,
stirred at RT for 30 min and then sonicated. Liposomes
for the Na+-uptake assay were prepared using PA3buffer containing 200 mM KCl. The prepared samples
were frozen using liquid N2 and stored at 808C.
Protein Synthesis and Purification—PURESYSTEM
classic II mini (Post Genome Institute, Japan) was used
for the protein synthesis. PCR products of pomA were
amplified from pHFAB by using primers pomA(+) pureforward and pomA() pure reverse and added 750 ng as
template DNAs in the reaction solution. PCR products ofpomB or their mutant genes were also amplified frompHFAB, pET-pomB or its mutant genes by primers pomB(+)
pure forward pomB() pure reverse, his6-pomB(+) pureforward and pomB-his6() pure reverse and added 1500 ng
as template DNAs. The detergents were added at the critical micelle concentration (CMC) or the liposome to a final
concentration of 4.4 mg/ml. The protein synthesis reaction was incubated at 308C for 1.5 h. Then, the reaction
solution containing the detergent was diluted twice by
40 mM HEPES–KOH pH 7.5 containing the detergent
at CMC, 1/10 volume of Ni-NTA agarose was added

Table 2. Primers for PCR.
PrimerpomA(+) PURE forward
pomA() PURE reverse
pomB(+) PURE forward
pomB() PURE reverse
his6-pomB(+) PURE forward
pomB-his6() PURE reverse
gfp(+) PURE forward
universal primer(+)
NdeI-pomB(+)
pomB-BamHI() revevrse

Sequence
50 -AAGGAGATATACCAATGGATTTAGCAACCCTATTAGG-30
50 -TATTCATTACTCGTCAATCTCAAGGG-30
50 -AAGGAGATATACCAATGGATGATGAAGATAACAAATGCG-30
50 -TATTCATTATTGAATTACCGGCACTTCTTCGCTG-30
50 -AAGGAGATATACCAATGCATCACCATCACCATCACATGGATGATGAAGATAACAAATGCG-3 0
50 -TATTCATTAGTGATGGTGATGGTGATGTTGAATTACCGGCACTT-30
50 -AAGGAGATATACCAATGGTGAGCAAGGGCGAGG-30
50 -GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTT
TGTTTAACTTTAAGAAGGAGATATACCA-30
0
5 -GGAATTCCATATGGATGATGAAGATAACAAATGCG-30
50 -CGCGGATCCTTATTGAATTACCGGCAC
J. Biochem.

Cell-free Synthesis of PomA and PomB

Vol. 144, No. 5, 2008(w/v) glycerol and 0.25% (w/v) bactoagar] was used
for V. alginolyticus swarm assays. Chloramphenicol andarabinose were added at the final concentration of 2.5 mg/
ml and 0.02% (w/v), respectively. An aliquot (1 ml) ofovernight culture was spotted onto a plate, which wasthen incubated at 308C.
RESULTS

Synthesis of PomA and PomB in the Presence ofDetergent or Liposome—To synthesize PomA and PomB,
we used the cell-free synthesis system of PURESYSTEM(20, 21). We used the mixed pomA and pomB PCR
products as the template DNAs in the cell-free system. It
has been shown that PomA and PomB interact with eachother and that their interaction is important for thestability of the proteins (27). We could detect the proteinsin the reaction mixture even without detergent byimmunoblotting using anti-PomA or PomB antibodies(Fig. 1). The PomA signal was detected as doublet bands,
this may be a character of the very hydrophobic PomA
protein from V. alginolyticus and not because of thedegradation (27). After the reaction, the proteins forthe cell-free synthesis were removed by treatment with
Ni-NTA agarose and the ribosomes were removed using
an ultrafiltration device. Using this procedure, however,the synthesized proteins were lost completely. This isprobably because PomA and PomB aggregated. To
handle the membrane proteins, we added some detergents to the cell-free system at the critical micelleconcentration. PomA and PomB were synthesized in
the presence of 0.46% (w/v) CHAPS, 0.125% (w/v) sucrose

C
H
AP
0.
12
S
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SM
0.
00
9%
D
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0.
58
%
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0.
46
%

-d

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ge

nt

cell-free

Sy
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F
Sy
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U
F
Sy
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U
F
Sy
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U
F
Sy
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F
ce
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(Qiagen, Germany), and was shaken at 48C for 1 h.
The resin-bound proteins and ribosomes were removed
by an ultrafiltration device (Microcon YM-100, Millipore,
USA). The reaction solution containing liposomes was
diluted twice by 40 mM HEPES–KOH pH 7.5 and sonicatedfor 30 s in a bath. The samples were then diluted twiceby 100 mM MgCl2 and stood on ice for 10 min. Liposome
was precipitated by centrifugation at 10,000g for 10 min.
The pellet was re-suspended in SDS loading buffer.
Detection of Proteins—The samples were mixed with
SDS loading buffer and boiled at 958C for 5 min. SDS–PAGE
and immunoblotting, using the antibodies against PomA,
PomB, His-probe (Santa Cruz, USA) and GFP (BD living
colors, USA), were performed as described previously (27).
Solubilization of Proteins—The reaction solution forthe protein synthesis was mixed at the final concentration of 1% (w/v) CHAPS and incubated on ice for 10 min.
The solubilized samples were centrifuged at 10,000g for
10 min at 48C and then the supernatants were centrifuged at 200,000g (TLA-100.3, Beckman, USA) for 1 h at
48C to remove insoluble material.
Pull-down Assay—The protein synthesis was carriedout as above with slight modifications. PURESYSTEM
advance (Post Genome Institute, Japan) was usedinstead of PURESYSTEM classic II mini (Post Genome
Institute, Japan), the PCR products of pomA templateand liposome were changed to 150 ng and the final
concentration of 2.2 mg/ml, respectively. His6-tag wasadded to the C terminus of PomB protein. The reaction
solution for the protein synthesis was mixed at the final
concentration of 1% (w/v) CHAPS and incubated for
10 min on ice. The solubilized samples were centrifuged
at 10,000g for 10 min at 48C. Ni-NTA agarose was added
to the supernatant, and the mixture was incubated at
48C for 30 min. The Ni-NTA agarose was washedby centrifugation three times with wash buffer [50 mM
HEPES–KOH pH 7.5, 100 mM KCl, 10 mM MgCl2, 1%(w/v) CHAPS and 50 mM imidazole] at 48C. The
associated proteins were eluted by elution buffer[50 mM HEPES–KOH pH 7.5, 100 mM KCl, 10 mM
MgCl2, 1% (w/v) CHAPS and 300 mM imidazole] at 48C.
The proteins were detected by immunoblotting as
described above.
Size Exclusion Chromatograph Assay—A Superdex 200
10/300 GL size exclusion column (GE Healthcare, Sweden)
was equilibrated with size exclusion buffer [50 mM HEPES–
KOH pH 7.5, 100 mM KCl, 10 mM MgCl2, 1% (w/v) CHAPS
and 1mM DTT]. Solubilized samples, as prepared above,were applied to the Superdex 200 10/300 GL column andeluted with the size exclusion buffer.
22
Na+-uptake Assay—A total of 630 ml of the assay buffer
containing 20 mM HEPES/KOH pH 7.5, 200 mM choline
chloride, 5 mM MgCl2 and 1 mM 22NaCl (14.8 kBq/ml) and
70 ml of the cell-free reaction mixture were mixed andincubated at 308C for 10 min. Valinomycin was added at the
final concentration of 25 mM to form a diffusion membranepotential. At intervals, 90 ml of the reaction mixture was
sampled out and passed through Dowex 50WX8-400 (Sigma,
USA) to trap the unincorporated 22Na+. The radioactivity of
the flow-through fraction was measured using a g-counter.
Swarm Assay—VPG-0.25% agar plate [1% (w/v)
bactotrypton, 0.4% (w/v) K2HPO4, 3% (w/v) NaCl, 0.5%637

α-PomA

α-PomB

Fig. 1. Cell-free synthesis of PomA and PomB in theabsence or presence of detergents. The cell-free synthesis
was performed using PCR products from the pomA and pomB
genes as template DNA. The detergent, 0.46% (w/v) CHAPS,
0.125% (w/v) sucrose monocaprate, 0.009% (w/v) n-dodecyl-bD-maltoside or 0.58% (w/v) n-octyl-b-D-thioglucoside, which are
the CMC, was added in PURESYSTEM. Cell, Vibrio whole-cell
producing PomA and PomB from pYA303; cell-free, cell-freeprotein synthesis; Syn, a whole fraction of the protein synthesis;
UF, a soluble fraction after chelating Ni-NTA agarose binding
proteins and ribosomes by an ultrafiltation device filtrating themolecular mass of >100 kDa. Vibrio cells (cell) were cultured at
308C for 3 h in VPG medium. The cells collected by centrifugation were suspended in SDS loading buffer at a concentration
corresponding to an A660 of 50. The 5 ml preparations of thesynthesized proteins were mixed with 5 ml of SDS loading buffer.
Immunoblotting to detect PomA and PomB was performed using
anti-PomA antibody (PomA1312) and anti-PomB antibody(PomB93), which were previouly prepared (27).

A

α-PomAnot solubilized

solubilized by 1% CHAPScellcell-free
syn sup ppt cell

Sy
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LP
LS
U
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U
-S

H. Terashima et al.

Sy
n
LP
LS
U
-P
U
-S

638

α-PomA

α-PomB

α-PomB
with liposome(L-α-phosphatidylcholine)
Fig. 2. Cell-free synthesis of PomA and PomB in thepresence of liposome made of L-a-phosphatidylcholine.
The cell-free synthesis was performed using PCR products fromthe pomA and pomB genes as the template DNA. Cell, Vibrio
whole-cell producing PomA and PomB from pYA303; cell-free,cell-free protein synthesis; syn, a whole fraction of the protein
synthesis; ppt, a precipitation fraction when liposome was
precipitated by adding magnesium at a final concentration of
50 mM; sup, the supernatant fraction. PomA and PomB weredetected as Fig. 1.

monocaprate or 0.009% (w/v) n-dodecyl-b-D-maltoside,
but not in the presence of n-octyl-b-D-thioglucoside(Fig. 1). Only in the presence of CHAPS, were PomA
and PomB recovered as soluble forms after ultrafiltration. Under all conditions, synthesized PomB was
detected as a protein with smaller molecular mass thanthat of the control PomB produced in vivo.
Next, we carried out the protein synthesis by adding
liposomes made of L-a-phosphatidylcholine. PomA
and PomB were produced and were detected in thefraction containing lipids precipitated by adding magnesium at a final concentration of 50 mM (Fig. 2, ppt) as
reported previously (28). Therefore, PomA and PomB
seem to be inserted into the liposomes as previously
suggested (25, 26). Synthesized PomB also had a smallermolecular mass in this condition.
Interaction Between PomA and PomB—Liposomes
containing the synthesized PomA and PomB weresolubilized by 1% CHAPS, and fractionated. They weredetected in the supernatant, after ultracentrifugation, as
the soluble fraction (Fig. 3A). Without CHAPS they weredetected in the precipitate after ultracentrifugation(Fig. 3A). Next, we examined the interaction between
PomA and PomB in the liposomes. The His6-tag fused to
PomB at the C-terminus was used to pull-down PomA.
After PomA and PomB were synthesized by the cell-freesystem in the presence of liposomes, the proteins(solubilized by CHAPS) were applied to Ni-NTA agarose
and the bound proteins were detected by immunoblotting
using anti-PomA and PomB antibodies (Fig. 3B). As aresult, PomA from the cell-free system was detected
together with the His6-tag fused-PomB. This suggests aninteraction between PomA and PomB in the cell-freesystem.
To examine molecular sizes of PomA and PomB fromthe cell-free system, we solubilized the synthesized PomA
and PomB using CHAPS and fractionated them by size
exclusion column. When only PomA was synthesized andB

pomA + pomB
Syn FTW E1 E2

pomA + pomB-his6
Syn FTW E1 E2

α-PomA

α-PomB

Fig. 3. Biochemical profiles of PomA and PomB.
(A) Solubilization of proteins and liposomes by CHAPS. Aftersynthesizing PomA and PomB in the presence of liposome,CHAPS was added at the final consentration of 1% (w/v). Afterremoving the insoluble materials by low-speed centrifugation,
the supernatant was ultracentrifuged. Syn, a whole fraction of
the protein synthesis; L-P, ppt of low-speed centrifugation; L-S,
sup of low-speed centrifugation; U-P, ppt of ultracentrifugation;
U-S, sup of ultracentrifugation; cell, Vibrio whole-cell producing
PomA and PomB from pYA303. (B) Interaction between PomA
and PomB detected by pull-down assay. Ni-NTA agarose wasadded into the solubilized solution by CHAPS. PomB-His6 boundto the resin was eluted by 300 mM imidazole. Syn, a wholefraction of the protein synthesis; FT, the flow-through fraction;
W, the wash fraction; E1, the first elution fraction; E2, thesecond elution fraction. The cell-free synthesis was performed
using PCR products from the pomA and pomB genes or the
pomA and pomB-his6 genes as template DNA. PomA and PomB
were detected as Fig. 1.

applied to the size exclusion column, it was eluted in awide range of the fractions with the peak around fraction
22 (Fig. 4A). When only PomB was synthesized, it was
eluted in the fraction of molecular size of 75 kDa,
estimated using the molecular size standards (Fig. 4B).
When PomA and PomB were synthesized together,profiles of each protein were essentially same as that of
PomA or PomB only (Fig. 4C). On the other hand, when
they were incubated for 20 h at 308C following protein
synthesis, PomB was detected in fractions of 18–20,
whose molecular size are 440–158 kDa, and PomA wasshifted to be eluted in the same fractions and the higher
molecular fractions (Fig. 4D). It is likely that PomA and
PomB oligomerized in the liposomes. We carried out
measurement of Na+-uptake activity of PomA and PomB
using the cell-free system and radioisotope, 22Na+.
However, we could not detect a significant Na+-uptake
into the liposomes (data not shown).
Molecular Mass of in vitro Synthesized PomB—PomB
synthesized by the cell-free system had a smaller molecular mass than that produced in the cells. To investigate
J. Biochem.

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Cell-free Synthesis of PomA and PomB
fraction
12

18

22

27

A
PomA only(α-PomA)

PomA

PomB only(α-PomB)

PomB

PomA + PomB

PomB

B

C
(α-PomA+α-PomB)
PomA

D
PomB

PomA + PomB
incubated at 30°C
for 20 hr(α-PomA+α-PomB)

PomA

e

CHAPS and applied to superdex 200 10/300 GL size exclusion
column. (D) PomA and PomB together were synthesized andincubated for 20 h at 308C, and then solubilized and applied asabove. PomA and PomB were detected as Fig. 1.

Bcell-free

ll
ce

ll

llce

ce

6

s
hi

B-

gfp-pomB

po

6

hi

m

om
-p

B
m
po

s

B
m
po

B

fre

cell

29 kDa

43

e

A

75

fre

Fig. 4. Size exclusion chromatography of PomA and/or
PomB from cell-free synthesis. (A) PomA only, (B) PomB onlyand (C) PomA and PomB together were synthesized, and thensolubilized immediately after the protein synthesis by using

158

ll-

440

669

ce

molecular size marker

α-PomB

PomB

α-His

PomB

GFP-PomB

α-PomB

α-GFPFig. 5. The cell-free synthesis of His6-tag-fused PomB
proteins at the N- or C-terminus (A) or GFP-fused PomB
at the N-terminus (B). Cell, Vibrio whole-cell producing PomA
and PomB from pYA303 (A) or GFP-PomB from pET-GB (B); cellfree, cell-free protein synthesis. The cell-free synthesis was
performed using PCR products from the pomA and pomB

genes, the pomA and pomB-his6 genes, the pomA and his6pomB genes or the pomA and gfp-pomB genes as template DNA.
PomA and PomB were detected as Fig. 1. His6-tag and GFP weredetected by the antibodies against His-probe (a-His) and GFP(a-GFP), respectively.

the reason for this, we first synthesized PomB fromthe gene fused with his6-tag at the N-terminus or
C-terminus in the presence of liposome. PomB-His6
but not His6-PomB was detected using the antiHis-probe antibody (Fig. 5A). The molecular massof the in vitro synthesized His6-PomB was the same asthat of PomB. These results suggest that the N-terminusof PomB was lost in the cell-free system. Next, we

synthesized PomB from the gene fused with gfp at
the N-terminus. In vitro synthesized GFP-PomB had
the same molecular mass as in vivo synthesized GFPPomB (Fig. 5B). The additional GFP fused at the
N-terminus of PomB suppressed the reduction of PomB
molecular mass.
Change of PomB Molecular Mass by the N-terminal
Mutations—To investigate further the reduction of theVol. 144, No. 5, 2008

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H. Terashima et al.
PomB-P12G or PomB-P12N with PomA from plasmids
were inoculated in the soft agar plates, their swarming
abilities were the same as the wild-type strain (Fig. 7A).
In the cells, PomB mutants were detected at the samelevel as wild-type PomB and their molecular masses weresame as that of wild-type PomB (Fig. 7B). These results
suggest that the mutations of PomB Pro-11 and Pro-12
are not likely to affect the stator function.

PomB molecular mass, we carried out site-directedmutagenesis to alter the stability of the N-terminal
region of PomB or to disrupt a potential initiation site in
the coding region of pomB mRNA. We made the Ala
substituted mutant of PomB at Cys-10, Pro-11, Pro-12,
Gly-14, Leu-15, Met-19 or Gly-20. PomB-P11A and P12A
showed the native molecular mass. We made moremutants substituted to Gly and Asn at Pro-11 and Pro12. These substitutions also suppressed the reduction of
the molecular mass in vitro (Fig. 6A). The substitution at
Pro-12 seems to have the strongest effect in preventing achange in molecular mass, as most of PomB had thesame molecular mass as that produced in vivo and theremainder was smeared at the smaller molecular weight.
We suspected that the Met-19 might become an initiation
codon of pomB in the cell-free system, but the PomBM19A mutant still had a smaller molecular mass
(Fig. 6B). There seem to be no other potential initiation
codons around the PomB N-terminal region. It is
unlikely that the translation is initiated at another point.
We examined the in vivo function of the Pro mutantsin PomB using swarming assay. When the pomAB
Vibrio cells producing PomB-P11/12A, PomB-P12A,

cell

w

t
P1
1
P1 A
1
P1 G
1N
P1
1,
P1 12A
2
P1 A
2
P1 G
2
w N
t

B

cell-freecell

α-PomBPomB

α-PomA

PomA

Fig. 6. The cell-free synthesis of mutant PomB proteins.
The mutations in PomB are introduced into Pro-11 and/or Pro12 (A) or Met-19 (B). Cell, Vibrio whole-cell producing PomA
and PomB from pYA303; cell-free, cell-free protein synthesis.
The cell-free synthesis was performed using PCR products fromthe pomA and pomB genes, the pomA and mutant pomB genes.
PomA and PomB were detected as Fig. 1.

A

G

2N
P1

P1
2

1A
/P
12
2A A

P12A

P1

P11A/P12A

ve
ct

vector

w

wt

t

or

B

P1

cell-free

We succeeded in the cell-free synthesis of the proteins
PomA and PomB in the presence of several detergents or
lipids (liposomes). Membrane proteins are generally
difficult to produce using cell-free synthesis systems.
Even without detergents PomA or PomB were synthesized by PURESYSTEM, but we were unable to handle
them without detergents. Among the detergents used inthis study, n-octyl-b-D-thioglucoside inhibits synthesis.
CHAPS do not inhibit synthesis and can solubilize PomA
and PomB.
The N-terminal region of in vitro synthesized PomB
seems to be missing. Fused GFP at the N-terminus of
PomB was not lost in the cell-free system. We speculatethat the structure of the N-terminal region of PomB wasaffected by the GFP fusion and that the cleavage of the
N-terminal region of PomB was difficult. This assumption was supported by the evidence that Pro-12 and Pro11 mutations suppressed reduction of the molecular massof PomB. Assuming that Pro residues greatly affect theprotein structure, the structural change in the
N-terminal region may prevent the cleavage. There
should be little contamination by protease in
PURESYSTEM because the proteins in PURESYSTEM
are all purified. Even if the protease inhibitor was added
to PURESYSTEM, the molecular mass was still small(data not shown). When the cell extracts from E. coli
were added in PURESYSTEM, a certain fraction ofsynthesized PomB was detected in the full-length form
(Terashima et al., unpublished results). This mightsuggest that some factors of E. coli affect the transcription of pomB mRNA, the translation of PomB or

w
t
M
19
M A
19
L
w
t

A

DISCUSSION

0.02%– + – + – + – + – + – + arabinose
α-PomB

P12G

P12N

host; NMB191 (∆pomAB)
Fig. 7. Function and expression of the pomB mutants in vivo.
(A) The swimming ability of pomAB cells producing PomB Pro-12
mutants with wild-type PomA from plasmid was examined on the
0.25% soft agar plate incubated at 308C for 6 h. (B) The pomAB

α-PomAcells producing PomB Pro-12 mutants with wild-type PomA fromplasmid were cultured in the absence or presence of 0.02% (w/v)
arabinose at 308C for 3 h. PomA and PomB were detected as Fig. 1.
The arrows indicate PomA and PomB.
J. Biochem.

Cell-free Synthesis of PomA and PomB
structural stability of the N-terminal region of PomB,
preventing reduction of its molecular mass.
Although it is unclear how, in vitro synthesized PomA
and PomB seem to be inserted spontaneously into theliposome and moreover, they interact with each other.
Such an insertion of membrane protein has been
observed and is independent on the Sec export system(25, 26). We have solubilized the PomA/B complex fromthe membrane of V. alginolyticus cells using thedetergents, n-octyl-b-D-thioglucoside (7), sucrose monocaprate (29) and CHAPS (30, 31). The molecular sizes of
the PomA/B complexes solubilized by their detergentswere estimated as 175 kDa, 900 kDa and 550 kDa,
respectively. The PomA/B oligomer, which was formed
when the synthesized PomA and PomB were incubatedfor 20 h at 308C, had similar high molecular sizes to thein vivo PomA/B complex solubilized by the detergents.
However, we could not detect significant Na+-uptake
using the proteoliposome reconstituted with in vitro
synthesized PomA and PomB. We had measured theNa+ uptake activity of PomA/B complex using theproteoliposome reconstituted with PomA and PomB
which were purified from Vibrio cells (7). In the present
experiment, the amount and complex formation of thereconstituted PomA and PomB, buffer organization, etc.
were, however, different from the previous condition.
Therefore, protein synthesis, complex formation andmeasurement conditions must be further optimized.
The synthesized PomA/B in this study might not befunctional for the Na+-conductive activity even though
the complex formation seems to be normal. We are goingto measure it using the mutant PomBs, which are notmissing the N-terminal region.
ACKNOWLEDGEMENTS

We thank Toshiharu Suzuki and Yuki Sudo for invaluableadvices and useful discussions.
FUNDING

Ministry of Education, Science, and Culture of Japan;
the Japan Science and Technology Corporation (to H.T.,
to M.H. and to S.K.) for scientific research partially;
Soft Nano-Machine Project of the Japan Science andTechnology Agency (to M.H.); Strategic International
Cooperative Program of JST (to M.H.).
CONFLICT OF INTEREST

None declared.
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