Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/12/16/M109.080739.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 9, pp. 6648 –6657, February 26, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

AAAⴙ Chaperone ClpX Regulates Dynamics of Prokaryotic
Cytoskeletal Protein FtsZ*□
S

Received for publication, October 29, 2009, and in revised form, December 4, 2009 Published, JBC Papers in Press, December 18, 2009, DOI 10.1074/jbc.M109.080739

Shinya Sugimoto‡1, Kunitoshi Yamanaka‡, Shingo Nishikori‡, Atsushi Miyagi§¶, Toshio Ando§, and Teru Ogura‡2
From the ‡Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University,
2-2-1 Honjo, Kumamoto 860-0811, the §Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192,
and the ¶Takara-Bio Endowed Laboratory, Institute for Protein Research, Osaka University, 6-2-3 Furuedai, Suita,
Osaka 565-0874, Japan

ClpX is a member of the Clp/Hsp100 class of AAA⫹
(ATPases associated with diverse cellular activities) family proteins (1). A hallmark of the AAA⫹ proteins is the formation of ahexameric ring structure with a central pore in an ATP-independent or -dependent manner. ClpX forms a complex with thetetradecameric protease component, ClpP, and the complex
ClpXP degrades various cellular proteins (2). ClpX consists ofan N-terminal domain and the following AAA⫹ domain. The
N-terminal domain recognizes specific substrate proteins,
whereas the AAA⫹ domain transmits energy of ATP tomechanical action like unfolding and translocation of substrate
proteins (3). The N-terminal domain of ClpX is essential for
degradation of some proteins, including MuA and ␭O, but notfor the degradation of other substrate proteins, such as SsrAtagged proteins (4). Furthermore, the N-terminal domain itself* This work was supported by a grant-in-aid for scientific research from theJapan Society for the Promotion of Science, by the Core Research for Evolutional Science and Technology program grant from the Japan Scienceand Technology Agency, and by grants from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S5 and Table S1.
1
Research Fellow of the Japan Society for the Promotion of Science.
2
To whom correspondence should be addressed. Tel.: 81-96-373-6578; Fax:
81-96-373-6582; E-mail: ogura@gpo.kumamoto-u.ac.jp.

6648 JOURNAL OF BIOLOGICAL CHEMISTRY

forms a dimer and has a potential role in quality control ofsubstrate proteins like general chaperones (4).
FtsZ, a structural homolog of the eukaryotic cytoskeleton
protein tubulin, is an essential protein and plays a key role inbacterial cell division (5–7). FtsZ polymerizes into filaments by
the head-to-tail association of individual protomers at mid-cell,which functions as the scaffold for assembly of at least a dozencell division proteins, and a ring-like large protein complex(Z-ring) is subsequently formed. As in the case of tubulin, polymerization of FtsZ is triggered by the GTP binding (8 –10). In
vitro, FtsZ assembles into several polymeric forms such as single straight or curved filaments, bundles, and sheets, dependingon the experimental conditions (8 –14). The observation thatthe expression level of FtsZ is constant in cells at various growthphases suggests that Z-ring formation is primarily regulated at
the level of FtsZ assembly. To date, several proteins regulating
FtsZ dynamics have been identified (7). For example, in Escherichia coli, inner membrane proteins FtsA and ZipA collaboratein the anchoring of FtsZ to membrane and thus stabilize theZ-ring (15, 16). In contrast, MinC and SulA inhibit FtsZ assembly (17, 18).
By a proteomic approach, ⬃50 proteins, including FtsZ, havebeen identified as candidates of the ClpX substrate, but how
ClpX recognizes FtsZ and the roles of ClpX in the fate of FtsZ
are unclear (2). Recently, it has been reported that in Bacillus
subtilis ClpX inhibits FtsZ polymerization in vivo and in vitro inan ATP-independent manner but does not degrade it (19, 20).
However, molecular mechanisms of regulating FtsZ monomerpolymer dynamics are still largely unknown. More recently, it
has been reported that in E. coli ClpX disassembles FtsZ polymers in cooperation with ClpP by degrading polymers preferentially rather than monomers and that ClpX revealed no significant activity to inhibit FtsZ polymerization (21). These
controversial results might be accounted for by the difference
in bacterial species and/or experimental conditions, but further
studies are required to evaluate these differences.
In this study, using E. coli systems, we examined the nature of
FtsZ polymers and roles of ClpX in regulation of FtsZ polymer
dynamics. We demonstrated that high speed atomic force
microscopy (AFM)3 is a superior method for visualizing FtsZ
filament dynamics. ClpX significantly shifts FtsZ dynamic equi3

The abbreviations used are: AFM, atomic force microscopy; IPTG, isopropyl
1-thio-␤-D-galactopyranoside; SEM, scanning electron microscopy; DAPI,
4,6-diamidine-2-phenylindole; ATP␥S, adenosine 5⬘-O-(thiotriphosphate).

VOLUME 285 • NUMBER 9 • FEBRUARY 26, 2010

Downloaded from www.jbc.org at KUMAMOTO UNIV LIB, on February 24, 2010

AAAⴙ chaperone ClpX has been suggested to be a modulatorof prokaryotic cytoskeletal protein FtsZ, but the details of recognition and remodeling of FtsZ by ClpX are largely unknown.
In this study, we have extensively investigated the nature of FtsZ
polymers and mechanisms of ClpX-regulated FtsZ polymer
dynamics. We found that FtsZ polymerization is inhibited by
ClpX in an ATP-independent manner and that the N-terminal
domain of ClpX plays a crucial role for the inhibition of FtsZ
polymerization. Single molecule analysis with high speed
atomic force microscopy directly revealed that FtsZ polymer isin a dynamic equilibrium between polymerization and depolymerization on a time scale of several seconds. ClpX disassembles
FtsZ polymers presumably by blocking reassembly of FtsZ. Furthermore, Escherichia coli cells overproducing ClpX and N-terminal domain of ClpX show filamentous morphology withabnormal localization of FtsZ. These data together suggest that
ClpX modulates FtsZ polymer dynamics in an ATP-independent fashion, which is achieved by interaction between the
N-terminal domain of ClpX and FtsZ monomers or oligomers.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/12/16/M109.080739.DC1.html

ClpX-mediated Inhibition of FtsZ Polymerization
librium from polymer to monomer, and the N-terminal domainof ClpX has the potential activity to inhibit FtsZ polymerization. Based on our biochemical and cell biological evidence, we
propose novel molecular mechanisms for remodeling of FtsZ
polymer dynamics by ClpX.

FEBRUARY 26, 2010 • VOLUME 285 • NUMBER 9

JOURNAL OF BIOLOGICAL CHEMISTRY

6649

Downloaded from www.jbc.org at KUMAMOTO UNIV LIB, on February 24, 2010

EXPERIMENTAL PROCEDURES
Bacterial Strains and Plasmids—E. coli strains used in thisstudy (supplemental Table S1) were grown at 37 °C in LB
medium containing appropriate antibiotics (40 ␮g/ml kanamycin and 50 ␮g/ml ampicillin). The plasmids used in this study
are also listed in supplemental Table S1. The ftsZ gene wasamplified from E. coli JM109 and cloned into pET-15b (Novagen) and pGFPuv (Clontech) plasmids using NdeI and BamHI
restriction sites and SphI and XbaI restriction sites, respectively. The ftsZ⌬C encoding FtsZ residues 1–316, clpX encoding ClpX residues 1– 424, clpX⌬N encoding ClpX residues
61– 424, clpX-N encoding ClpX residues 1– 60 or sspB genes
was also amplified from E. coli JM109 and cloned into pET-28b
plasmid using NdeI and BamHI restriction sites. DNA fragment
encoding the SsrA tag was cloned downstream of gfpuv in pGFPuv. DNA sequences of the cloned genes were confirmed.
Purification of Proteins—E. coli BL21(DE3) was transformed
with pET15-FtsZ, pET28-FtsZ⌬C, pET28-ClpX, pET28ClpX⌬N, pET28-ClpX-N, pET28-SspB, and pGEX-ClpP (provided by Dr. S. Hishida) or pUHE-ClpB (provided by Dr. B.
Bukau). E. coli KY2266 (22) was transformed with
pGFPuv-SsrA.
E. coli cells overproducing FtsZ or FtsZ⌬C were grown at
37 °C in LB medium containing 50 ␮g/ml ampicillin or 40
␮g/ml kanamycin, respectively, and expression of the FtsZ variants was induced by the addition of isopropyl 1-thio-␤-D-galactopyranoside (IPTG) (1 mM), followed by incubation at 37 °C
for 3 h. Cells from 2 liters of culture were harvested by centrifugation and suspended in 100 ml of buffer A (20 mM Tris-HCl(pH 8.0), 400 mM NaCl, 20 mM imidazole, 10% (w/v) glycerol)
supplemented with protease inhibitor mixture (Nakalai
Tesque). After sonication on ice, cell lysates were centrifuged at
15,000 ⫻ g for 1 h at 4 °C, and the supernatant was loaded ontoa HisTrap HP column (GE Healthcare). The column waswashed with buffer A containing 50 mM imidazole, and recombinant proteins were then eluted using 50 –500 mM imidazole
gradient in buffer A. To remove contaminated GTP or GDP,
eluted fractions were further purified by 35% ammonium sulfate precipitation and HiTrap DEAE column (GE Healthcare)chromatography using 0 –500 mM NaCl gradient in buffer B (50mM Tris-HCl (pH 8.0), 1 mM EDTA, 10% (w/v) glycerol). Eachfraction containing FtsZ or FtsZ⌬C was stored separately at⫺80 °C.
E. coli cells overproducing ClpX, ClpX⌬N, ClpX-N, or SspB
were grown at 30 °C in LB medium containing 40 ␮g/ml kanamycin to an A600 ⫽ 0.5, and then IPTG was added to a final
concentration of 0.1 mM. After an additional incubation for 2 h
at 30 °C, cells were harvested and suspended in buffer C (50 mM
Tris-HCl (pH 7.5), 100 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol, 20 mM imidazole, 0.01% Triton X-100, 10% (w/v) glycerol)
supplemented with protease inhibitor mixture. After sonication on ice, cell lysates were centrifuged at 15,000 ⫻ g for 1 h at

4 °C, and the supernatant was loaded onto a HisTrap HP column. The column was washed with buffer C containing 50 mM
imidazole, and recombinant proteins were then eluted using
50 –500 mM imidazole gradient in buffer C. The eluted fractions
were diluted more than 10-fold into buffer D (50 mM Tris-HCl(pH 7.5), 100 mM NaCl, 2 mM dithiothreitol, 20% (w/v) glycerol)and loaded onto a HiTrap Q column (GE Healthcare). The column was washed with buffer D, and recombinant proteins were
eluted using 100 –1,000 mM NaCl in buffer D. Each fraction
containing recombinant proteins was stored separately at⫺80 °C.
E. coli cells overproducing glutathione S-transferase-fused
ClpP (GST-ClpP) were grown at 37 °C in LB medium containing 50 ␮g/ml ampicillin, and expression of GST-ClpP was
induced by the addition of 1 mM IPTG, followed by incubation
at 30 °C for 3 h. Cells were harvested and suspended in buffer E(50 mM Tris-HCl (pH 7.4), 10 mM KCl, 10 mM MgCl2, 1 mM
dithiothreitol, 10% (w/v) glycerol). After sonication on ice, celllysates were centrifuged at 15,000 ⫻ g for 1 h at 4 °C, and thesupernatant was loaded onto a GSTrap HP column (GE Healthcare). The column was washed with buffer E, and recombinant
proteins were then eluted with buffer E containing 10 mM
reduced glutathione. The eluted fractions containing GSTClpP were treated with PreScission protease (BD Biosciences)
at 4 °C for 12 h to remove a glutathione S-transferase moiety.
ClpP was further purified by a HiTrap Q column chromatography using 0 –500 mM KCl gradient in buffer F (50 mM Tris-HCl(pH 7.5), 2 mM EDTA, 2 mM dithiothreitol, 10% glycerol). Fractions containing ClpP were concentrated with Amicon
Ultra-30 (Millipore) and stored at ⫺80 °C.
GFP-SsrA was overexpressed in E. coli KY2266 (22). Cells
were suspended in buffer G (20 mM Tris-HCl (pH 7.4), 1 mM
EDTA) and disrupted by sonication. Subsequently, GFP-SsrA
was purified by HiTrap Q column chromatography using 0 –1 M
NaCl gradient in buffer G. Fractions containing GFP-SsrA werepooled and stored at ⫺80 °C. ClpB was purified as described
previously (23).
Protein concentrations were determined by measuring band
intensities in SDS-polyacrylamide gels. All protein concentrations are given for protomers.
FtsZ Polymerization Assays—In light scattering assay, FtsZ
(12 ␮M) was preincubated in buffer H (40 mM HEPES-KOH (pH7.5), 5 mM MgCl2, 175 mM NaCl, 0.02% Triton X-100, 10% glycerol) at 30 °C for several minutes to correct a base line. Polymerization was started by the addition of 5 mM GTP. Light
scattering was continuously recorded every 10 s using a Jasco
FP-750 fluorescence spectrophotometer (Jasco Corp.) withboth excitation and emission wavelengths set to 500 nm with
5-nm slit widths. Light scattering of ClpX (indicated concentrations) was also monitored with time after the addition of
ClpX.
In sedimentation assay, polymerization of FtsZ (6 ␮M) or
FtsZ⌬C (6 ␮M) was initiated in buffer H by adding 5 mM GTP. If
required, ClpX (6 ␮M), ClpX⌬N (6 ␮M), ClpX-N (1.5–12 ␮M),
ClpB (6 ␮M), ClpP (14 ␮M), SspB (6 ␮M), and ATP (5 mM) wereadded. After incubation at 30 °C for 15 min, the reaction was
immediately ultracentrifuged at 4 °C in a Beckman TLA 100
rotor (Beckman-Coulter) at 100,000 rpm for 30 min. Processing

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/12/16/M109.080739.DC1.html

ClpX-mediated Inhibition of FtsZ Polymerization

6650 JOURNAL OF BIOLOGICAL CHEMISTRY

FIGURE 1. GTP-dependent assembly of FtsZ into filaments. A, FtsZ (6 ␮M)
was polymerized in the absence (⫺) and presence (⫹) of 5 mM GTP and separated by ultracentrifugation into supernatant (Sup) and pellet (Pel) fractions.
FtsZ in each fraction was analyzed by SDS-PAGE and Coomassie Brilliant Blue
staining. B, SEM image of FtsZ filament is shown. Scale bar is given as 1 ␮m.

media containing 40 ␮g/ml kanamycin and cultured at 37 °C
overnight. Cell morphologies were observed by fluorescencemicroscopy with a phase-constant mode. Because leaky expression of ClpX variants was enough to show abnormal morphologies, no IPTG was supplemented into the cultures. To examine the localization of FtsZ, pFtsZ-GFPuv and either pET-28b or
pET28-ClpX were simultaneously introduced into E. coli
MC4100(DE3). These transformants were cultured in LB
media containing 40 ␮g/ml kanamycin and 50 ␮g/ml ampicillin
at 37 °C overnight. As high level FtsZ-GFPuv expression induces
filamentous cells, no IPTG was supplemented into the cultures.
Cells were then fixed with methanol and stained with 4,6-diamidine-2-phenylindole (DAPI) (28). Localization of FtsZ-GFPuv and nucleoids was observed under fluorescence microscopy
with green fluorescent protein and DAPI filters, respectively.

RESULTS
FtsZ Polymerizes into Fibers in the Presence of GTP—GTPdependent polymerization of purified FtsZ was confirmed by
sedimentation assay. In the absence of GTP, almost all FtsZ wasretained in the supernatant fraction, although, in the presenceof GTP, FtsZ was found predominantly in the pellet fraction(Fig. 1A). Kinetics of FtsZ polymerization revealed that FtsZ
polymerized immediately after the addition of GTP, and thepolymers were stable for at least 30 min under the conditions
used (supplemental Fig. S1). Rapid increase in the population of
FtsZ polymers is consistent with the result of light scattering
analysis (data not shown). We also observed the structure of
GTP-induced FtsZ polymers by SEM and detected networks offilaments (Fig. 1B) as demonstrated previously by transmission
electron microscopy (24).
FtsZ Polymers Undergo Dynamic Conformational Changes—
The AFM is a powerful tool to capture high resolution images of
biological samples in liquids without any fixation chemicals
unlike electron microscopy. However, the conventional AFM
takes at least 30 – 60 s to capture an image, whereas dynamic
biomolecular processes occur on a millisecond time scale or
less. FtsZ polymers are also known to be in a dynamic equilibrium, and subunits within the polymer are rapidly exchanged
on a time scale of 7 s (29). Therefore, it had been impossible tocapture the movements of FtsZ polymers by the conventional
AFM. Here, we applied a recently developed method, highspeed AFM, which enables us to visualize conformational
VOLUME 285 • NUMBER 9 • FEBRUARY 26, 2010

Downloaded from www.jbc.org at KUMAMOTO UNIV LIB, on February 24, 2010

the pellet for protein estimation and SDS-PAGE was performed
as described earlier (24). Band intensity on the gel was estimated using ImageQuant (GE Healthcare).
Scanning Electron Microscopy (SEM)—FtsZ polymers wereprepared as mentioned above, fixed with 2.5% glutaraldehyde
and 2% formaldehyde, placed on a glass slide, and air-dried atroom temperature. The samples were coated with platinum
and observed using a Keyence VE-9800 scanning electron
microscope (Keyence Co.).
High Speed AFM—The laboratory-built tapping mode highspeed AFM apparatus, which has been developed by Dr. Andoand co-workers at Kanazawa University (25), was used in thisstudy. The cantilever (Olympus) has a resonant frequency of⬃1 MHz in water and a spring constant of 0.1– 0.2 newton/m.
An amorphous carbon tip was grown on the original cantilevertip by electron beam deposition (26). For high speed AFM
observation, FtsZ (6 ␮M) was incubated in buffer H at 30 °C for
5 min. If required, GTP (5 mM) and ClpX (6 ␮M) were supplemented. A droplet (2 ␮l) was deposited on a freshly cleaved
mica surface, incubated for 3 min at room temperature, andwashed with buffer H. AFM observation was then performed
under buffer H minus Triton X-100. ClpX (6 ␮M) was incubated in buffer I (50 mM Tris-HCl (pH 8.0), 500 mM KCl) at
30 °C for 5 min. A droplet (2 ␮l) was deposited on a freshly
cleaved mica surface, incubated for 3 min at room temperature, and washed with buffer I. AFM observation was thenperformed under buffer J (40 mM HEPES-KOH (pH 7.4), 150
mM KCl, 20 mM MgCl2).
Chemical Cross-linking—FtsZ (6 ␮M) and ClpX (6 ␮M) wereincubated at 30 °C for 10 min in buffer J in the absence andpresence of the indicated nucleotides. Proteins were then crosslinked with 0.1% glutaraldehyde at 30 °C for 20 min. The reactions were quenched by the addition of an equal volume of 2⫻
SDS sample buffer (150 mM Tris-HCl (pH 6.8), 4% SDS, 20%
glycerol, 10% 2-mercaptoethanol). Proteins were analyzed by
SDS-PAGE and visualized with Coomassie Brilliant Blue staining. Here, polyacrylamide gradient gels (SuperSep 3–10% gel,Wako) were used to separate proteins with a broad range ofmolecular masses.
FtsZ Depolymerization Assay—After polymerization of FtsZ
(12 ␮M) and FtsZ⌬C (12 ␮M) in buffer H supplemented with
GTP (5 mM) at 30 °C for 20 min, ClpX (6 ␮M) was added to the
mixture. The reactions were further incubated at 30 °C for 15
min and immediately ultracentrifuged at 4 °C in a Beckman
TLA 100 rotor at 100,000 rpm for 30 min. Both supernatant andpellet fractions were analyzed by SDS-PAGE with Coomassie
Brilliant Blue staining. The reactions without the addition of
ClpX were used as a negative control.
Protein Degradation—FtsZ (3 ␮M) and GFP-SsrA (3 ␮M)
were degraded by ClpXP (ClpX, 6 ␮M; ClpP, 14 ␮M) in buffer K(25 mM HEPES-KOH (pH 7.6), 100 mM KCl, 20 mM MgCl2, 1
mM EDTA) at 30 °C for 120 min. Protein degradation was analyzed by SDS-PAGE with Coomassie Brilliant Blue staining.
Estimation of substrate degradation was performed using
ImageQuant (GE Healthcare).
Fluorescence Microscopy—E. coli MC4100(DE3) (27) wastransformed with pET-28b, pET28-ClpX, pET28-ClpX⌬N, or
pET28-ClpX-N. These transformants were inoculated to LB

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/12/16/M109.080739.DC1.html

ClpX-mediated Inhibition of FtsZ Polymerization
with a fast scan rate (⬃1 frame/s). As shown in Fig. 3 and supplemental Fig. S2, we succeeded to detect directly the conformational changes of FtsZ polymers. Some straight filaments
gradually curved, and others laterally associated into bundles.
Partial dissociation and reassembly of filaments were also
detected (Fig. 3 and supplemental Fig. S2). To our knowledge,
this is the first vivid visualization of conformational changes of
FtsZ polymers on a second time scale using a high speed AFM.
ClpX Forms Heterogeneous Oligomers—Prior to investigating
the effect of ClpX on FtsZ polymerization in vitro, we analyzed
the oligomeric state of purified ClpX. In the absence of nucleotides, light scattering signal of ClpX increased in a dose-dependent manner (Fig. 4A), indicating that ClpX forms large
oligomers under the conditions used.
To investigate the oligomeric state of ClpX in detail, we conducted a glutaraldehyde cross-linking assay. As shown in Fig.
4B, smear bands around the position of dimers and hexamers
were predominantly detected in the absence of nucleotides.
Interestingly, hexamers were obviously detected in the presence of the nonhydrolyzable analog of ATP, ATP␥S, rather
than in the presence of ATP or ADP. In addition, in the presence of nucleotides, the amount of dimer was reduced compared with that in the absence of nucleotides, and large oligomers were concomitantly detected around the position ofwells (Fig. 4B). These observations are consistent with a previous report showing that ClpX showed heterogeneously sizedparticles with no obvious regularity and that the addition of
ATP␥S to ClpX produced a more uniform particle with a size
corresponding to a hexamer (30).
Structure and size distribution of ClpX molecules were also
evaluated by AFM. ClpX was observed as heterogeneous globular particles with a size distribution ranging from 3 to 50 nm inheight and 2 to 33 nm in width (Fig. 4C), indicating that ClpX
exists as heterogeneously sized particles ranging from a monomer to a large oligomer. The result of chemical cross-linking
also showed similar heterogeneous oligomeric states of ClpX
(Fig. 4B).
FIGURE 2. AFM images of FtsZ monomers and polymers. A, AFM image of
ClpX Inhibits FtsZ Polymerization—Light scattering assay is
FtsZ in the absence of GTP is shown. B, height of FtsZ at the white-lined crosssection in A was measured. C, AFM image of GTP-triggered FtsZ polymers is a useful method to analyze the time course of FtsZ polymerizagiven. D, height of FtsZ at the white-lined cross-section in C was measured. tion in vitro. However, because ClpX itself shows a large light
Scale bars in A and C are given as 100 nm.
scattering (Fig. 4A), this method was
not employed to investigate theability of ClpX to modulate the FtsZ
assembly. Therefore, we conducted
a sedimentation assay and evaluatedband intensities of the sedimented
FtsZ to quantify the formation ofpolymers. After polymerization at
30 °C for 15 min, the reaction wascentrifuged at 100,000 rpm for 30
min at 4 °C, and the pellet fraction
was analyzed by SDS-PAGE. Whenthe amount of polymerized FtsZ in
the presence of GTP is defined as
FIGURE 3. High speed AFM images of FtsZ polymers. A, raw AFM images of FtsZ polymers taken at the 100%, the addition of ClpX to theindicated times. There are many single filaments, bundles, and curved filaments. B, to emphasize the dynamic reaction reduced it to ⬃20% (Fig. 5,
nature of filaments, raw images in A are shown in gray scale, and selected filaments are colored in red. A straight
filament was curved, disassembled, reassembled, and bundled under the observation. Scale bar is given as 100 A and B). Surprisingly, ATP slightly
but significantly inhibited the activnm.
changes of native proteins directly in solution on much smaller
time scales (30 – 60 ms) (25).
First, we compared AFM images of FtsZ between the presence and absence of GTP. In the absence of GTP, small particles
with different shapes were detected (Fig. 2A). Based on the
results of chemical cross-linking that FtsZ predominantly
formed monomers (data not shown), FtsZ monomers seemed
to be adsorbed on mica. In contrast, in the presence of GTP,
networks of long filaments containing single straight, curved,
and bundled filaments were detected (Fig. 2C). The heights of
FtsZ filaments (average 5.2 nm, Fig. 2D) were similar to those ofmonomers and dimers (average 4.4 nm, Fig. 2B), suggestingthat FtsZ monomers and dimers assemble into a single filament
by longitudinal interaction.
Next, to ask whether FtsZ polymers undergo dynamic conformational changes, we continuously captured AFM images

JOURNAL OF BIOLOGICAL CHEMISTRY

6651

Downloaded from www.jbc.org at KUMAMOTO UNIV LIB, on February 24, 2010

FEBRUARY 26, 2010 • VOLUME 285 • NUMBER 9

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/12/16/M109.080739.DC1.html

ClpX-mediated Inhibition of FtsZ Polymerization

6652 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 285 • NUMBER 9 • FEBRUARY 26, 2010

Downloaded from www.jbc.org at KUMAMOTO UNIV LIB, on February 24, 2010that no obvious filament was
detected, ClpX inhibits the longitudinal assembly of FtsZ at the initialstep, in which binding of FtsZ protomers occurs.
N-terminal Domain of ClpX Playsa Crucial Role in the Inhibition of
FtsZ Polymerization—N-terminal
domains of several AAA⫹ chaperones are known to function in thesubstrate recognition and to act as a
primary binding site. The N-terminal domain of ClpX also plays a crucial role in the recognition of specific target proteins, including MuAand ␭O (4). In contrast, it does notplay a role for the recognition ofsome other substrates such as GFPSsrA (4). To address whether the
N-terminal domain of ClpX is
involved in the recognition of FtsZ,
we generated a ClpX mutant,
ClpX⌬N, lacking the N-terminal
domain and examined its effect on
FtsZ polymerization (Fig. 5C).
Compared with wild type ClpX,
ClpX⌬N showed very weak inhibiFIGURE 4. Oligomeric state of ClpX. A, light scattering of ClpX (indicated concentrations) in the absence of tion, suggesting that the N-terminal
nucleotide was measured as described under “Experimental Procedures.” RU, response units. B, ClpX (6 ␮M) was
incubated in the absence and presence of the indicated nucleotides (5 mM) at 30 °C for 10 min and then domain is responsible for the recogcross-linked with 0.1% glutaraldehyde at 30 °C for 20 min. After quenching the reaction by the addition of nition of FtsZ. A recent report
Tris-HCl-containing buffer, proteins were analyzed by SDS-3–10% gradient polyacrylamide gel. The molecular revealed that ClpX⌬N, together
sizes in kilodaltons are given at the left. The positioning of monomer, dimer, hexamer, and large oligomers isgiven. C, AFM images at two magnifications are shown. Scale bars are given as 100 nm. D, width (x axis) and with ClpP, showed a reduced butheight (y axis) of ClpX molecules (n ⫽ 120) in C and other frames were measured and plotted.
still significant ability to degrade
FtsZ (21), suggesting that the AAA⫹
ity of ClpX to prevent FtsZ polymerization (Fig. 5, A and B). The domain of ClpX has a weak ability to recognize and unfold FtsZ.
addition of ATP did not affect FtsZ polymerization itself. In
To examine the possibility that the N-terminal domain ofaddition, ATP␥S also reduced the activity of ClpX, although ClpX has the capacity to bind to FtsZ and to inhibit FtsZ polyADP did minimally, indicating that an ATP-bound form of merization, we constructed and purified a ClpX mutant,
ClpX is ineffective for inhibition of FtsZ polymerization. Thus, ClpX-N, composed of the N-terminal domain. ClpX-N critihenceforth ATP was not added to reactions. To ask whether the cally inhibited FtsZ polymerization in a dose-dependent maneffect on FtsZ polymerization is unique for ClpX, the effects of ner (Fig. 5E), suggesting that the N-terminal domain of ClpX
ClpB (another AAA⫹ chaperone), ClpP (protease component plays a primary role in the regulation of FtsZ dynamics and thatof ClpXP), and SspB (adaptor protein of ClpXP) were tested. the AAA⫹ domain is not essential for this function. It is interClpB, ClpP, and SspB slightly inhibited FtsZ polymerization, esting to mention that when the reactions were conducted with
but their effects were at negligible levels (Fig. 5C and supple- the same concentrations of ClpX-N and ClpX (6 ␮M as monomental Fig. S3). These results indicate that FtsZ polymerization mers), ClpX-N showed a stronger inhibitory effect on FtsZ pois specifically inhibited by ClpX. As SspB, which stimulates deg- lymerization than did ClpX (Fig. 5, B and E). This is likely due toradation of SsrA-tagged substrates by ClpXP (31), did not show differences in the number of N-terminal domains accessible forsignificant inhibition of FtsZ assembly, an FtsZ recognition FtsZ, which might be limited by formation of hexamers andmode of ClpX is distinct from that of SsrA-tagged substrates by oligomers in the case of ClpX (Fig. 4B).
ClpX and SspB.
ClpX Recognizes a C-terminal Moiety and Other Parts of FtsZ—
To examine how ClpX prevents FtsZ polymerization, struc- The C-terminal unstructured region of FtsZ (residues 328 –tures of FtsZ were visualized by AFM. In the presence of GTP, 383) is recognized by several FtsZ modulators, such as FtsA,
many filamentous structures were detected as shown in Figs. MinC, and ZipA (15–17). Furthermore, it has recently been
2C, 3, and 5D. In the simultaneous presence of GTP and ClpX, reported that this region is important for degradation of FtsZ by
no obvious long filament was detected, but small particles were ClpXP (21). To ask whether the C-terminal region of FtsZ isobserved (Fig. 5D). Here, ClpX dimers and FtsZ monomers/ essential for the inhibition of FtsZ polymerization by ClpX, we
dimers were not distinguishable. Taking into consideration generated an FtsZ mutant, FtsZ⌬C, lacking the C-terminal res-

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/12/16/M109.080739.DC1.html

ClpX-mediated Inhibition of FtsZ Polymerization

FEBRUARY 26, 2010 • VOLUME 285 • NUMBER 9

JOURNAL OF BIOLOGICAL CHEMISTRY

6653

Downloaded from www.jbc.org at KUMAMOTO UNIV LIB, on February 24, 2010

idues 317–383. FtsZ⌬C polymerized in the presence of GTP (Fig.
6A) similarly to wild type FtsZ (Fig.
1A). Polymerization of FtsZ⌬C wasinhibited by ClpX (Fig. 6B). Compared with the case of wild type FtsZ
(Fig. 5, A and B), the level of ClpXmediated inhibition of FtsZ⌬C polymerization was markedly low (Fig.
6B), indicating that the C-terminal
region of FtsZ has a substantial role
for the recognition by ClpX, but it is
not essential for the inhibition of
FtsZ polymerization.
ClpX Blocks Reassembly of FtsZ
Polymers—Because FtsZ is in a
dynamic equilibrium between polymerization and depolymerization asshown by high speed AFM (Fig. 3and supplemental Fig. S2), we
assumed that ClpX can affect theoligomeric state of preformed FtsZ
polymers. The FtsZ polymers wereformed in the presence of GTP at
30 °C and then incubated with ClpX
as described under “Experimental
Procedures.” As expected, ClpX significantly reduced the amount of
FtsZ polymers as well as FtsZ⌬C
FIGURE 5. Inhibition of FtsZ polymerization by ClpX. A, FtsZ (6 ␮M) was polymerized in the presence of GTP
with or without ClpX and the indicated nucleotides. FtsZ polymers were collected by ultracentrifugation and polymers (Fig. 7), showing again
analyzed by SDS-PAGE. B, band intensities of FtsZ polymers in A were quantified as described under “Experi- that the C-terminal region of FtsZ ismental Procedures.” C, degrees of GTP-induced FtsZ polymers formed in the absence and presence of the
not required for the ClpX-mediatedindicated chaperones (6 ␮M per monomer) are given. D, inhibition of FtsZ polymerization by ClpX is visualized
by AFM. AFM images of FtsZ, in the absence and presence of GTP, and ClpX are also shown. All scale bars are inhibition of FtsZ reassembly as ingiven as 100 nm. E, dose dependence of ClpX-N in inhibition of FtsZ assembly is examined. The indicated the case of the prevention of the inimonomer concentrations of ClpX-N were added to the FtsZ polymerization reaction. B, C, and E, level of FtsZ
polymers formed in the presence of GTP was defined 100%. All polymerization reactions were started by the tial step of FtsZ assembly (Fig. 5). It
addition of 5 mM GTP. Data from at least three replicates are presented as means ⫾ S.E.
should be emphasized that noenergy of ATP hydrolysis was
required for this reaction, indicating that binding to FtsZ isenough to block the assembly and reassembly of FtsZ and that
this activity does not contain an energy-consuming process
such as protein unfolding and translocation.
ClpX Slightly Unfolds FtsZ—Recently, it has been reported
that E. coli ClpXP degraded FtsZ more rapidly in the presence
than in the absence of GTP, suggesting that ClpXP preferentially degrades FtsZ polymers rather than monomers (21). In
addition, ClpX-mediated inhibition of FtsZ polymerization was
not observed (21). Based on these results, they proposed that
ClpXP may participate in cell division by modulating theequilibrium between monomeric and polymeric FtsZ via
degradation of FtsZ polymers. In contrast, it has beenreported that in B. subtilis ClpXP does not degrade FtsZ andthat ClpX regulates FtsZ polymer dynamics by preventing
FtsZ polymerization (19). Our present results mentioned
above are consistent with the latter result, but contribution
FIGURE 6. Inhibition of GTP-dependent polymerization of FtsZ⌬C. A, GTPdependent polymerization of FtsZ⌬C was confirmed as shown in Fig. 1A. Sup, of FtsZ degradation (or unfolding) by ClpXP (or ClpX) to thesupernatant; Pel, pellet. B, inhibition of FtsZ⌬C polymerization by ClpX was regulation of FtsZ polymer dynamics remains to be eluciexamined as shown in Fig. 5A. The quantified values of FtsZ⌬C polymers in
the absence and presence of ClpX are indicated at the bottom. Data from at dated. Therefore, we reassessed FtsZ degradation by ClpXP
using E. coli proteins.
least three replicates are presented as means ⫾ S.E.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/12/16/M109.080739.DC1.html

ClpX-mediated Inhibition of FtsZ Polymerization

FIGURE 7. Depolymerization of FtsZ polymers mediated by ClpX. FtsZ and
FtsZ⌬C (12 ␮M each) were polymerized by the addition of GTP and thenunsupplemented (⫺) or supplemented (⫹) with ClpX. After the depolymerization reaction, depolymerized (and/or unpolymerized) and polymerized
FtsZ was separated by ultracentrifugation and analyzed by SDS-PAGE as
described under “Experimental Procedures.” The positioning of FtsZ and
FtsZ⌬C is given. Sup, supernatant; Pel, pellet.

As shown in Fig. 8, ClpXP showed a weak activity to degrade
FtsZ. The degradation rate of FtsZ was ⬃2-fold faster than thatof FtsZ⌬C, indicating that the C-terminal region of FtsZ isimportant for the proteolysis by ClpXP as recently reported(21). However, the rate of FtsZ degradation in the presence of
GTP, the condition favoring polymers, was similar to that in theabsence of GTP, the condition favoring monomers; this isinconsistent with the results of Camberg et al. (21). Although
the reason for this discrepancy is unclear, it might be due to thedifference in the experimental conditions and the methods todetect the degradation of FtsZ. More importantly, the degradation of FtsZ by ClpXP (10% degradation in 2 h) was quite slower
than that of GFP-SsrA (80% degradation in 2 h) under the conditions used (Fig. 8), suggesting that contribution of FtsZ
unfolding by ClpX to the inhibition of FtsZ polymerization by
ClpX is a negligible level (15-min incubation, Fig. 5, A and B).

6654 JOURNAL OF BIOLOGICAL CHEMISTRY

Overexpression of ClpX Induces Cell Division Defect andInhibits Z-ring Formation—Because normal assembly andlocalization of FtsZ are deeply related to correct cell division(32), we examined the effect of ClpX overexpression on the cell
division and Z-ring formation in E. coli (Fig. 9). Cells harboringa vector showed typical rod shape, whereas ClpX-overexpressing cells showed many filaments (Fig. 9A), indicating that overexpression of ClpX leads to the defect in cell division. Next, weinvestigated the effect of overexpression of ClpX variants,
including ClpX⌬N and ClpX-N. ClpX⌬N-overexpressing cells
showed some elongated cells, but rod-type cells were mainly
observed (Fig. 9A). The difference of cell morphology between
ClpX-overexpressing and ClpX⌬N-overexpressing cells was
not due to the different expression levels, because similar
amounts of these proteins were detected by SDS-PAGE. In contrast, ClpX-N-overexpressing cells showed long filaments (Fig.
9A), suggesting that the reason ClpX overexpression leads tocell division defect is not the depletion of endogenous FtsZ
by proteolytic activity of ClpXP. In addition, only a few elongated cells were observed when ClpB and SspB were overexVOLUME 285 • NUMBER 9 • FEBRUARY 26, 2010

Downloaded from www.jbc.org at KUMAMOTO UNIV LIB, on February 24, 2010

FIGURE 8. Degradation of FtsZ by ClpXP. A, FtsZ monomers, preferentiallyformed in the absence of GTP (closed circles), and polymers, preferentiallyformed in the presence of GTP (open circles), were degraded by ClpXP for 120
min. To address the importance of C-terminal region, FtsZ⌬C (closed triangles)
was also analyzed. As positive controls, GFP-SsrA (open triangles) was
degraded by ClpXP in the absence of GTP. At the indicated times, an aliquot of
each reaction was taken and analyzed by SDS-PAGE. B, band intensities of thesubstrate proteins were quantified using ImageQuant.

FIGURE 9. Effects of ClpX overexpression on E. coli cell morphology and
Z-ring formation. A, cell morphologies of vector control (Control), ClpX-overexpressing (ClpX), ClpX⌬N-overexpressing (ClpX⌬N), and ClpX-N-overexpressing (ClpX-N) cells are shown. Protein expression profile of each transformant is analyzed by SDS-PAGE with Coomassie Brilliant Blue staining andattached to the right of each panel. An arrowhead indicates the position corresponding to each overexpressed protein. B, to visualize localization of FtsZ,
the FtsZ-GFPuv expression plasmid was cotransformed with the empty plasmid or ClpX-expression plasmid into E. coli MC4100(DE3). DNA was stainedwith DAPI. Fluorescence of DAPI and GFPuv was captured by fluorescent
microscopy with the respective filters. Merged pictures of DAPI, GFPuv, andphase contrast are also shown. A significant Z-ring is indicated by an arrow. All
scale bars are given as 10 ␮m.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/12/16/M109.080739.DC1.html

ClpX-mediated Inhibition of FtsZ Polymerizationpressed (supplemental Fig. S4). These results of the in vivo
experiments are highly consistent with those of the in vitro
experiments (Fig. 5).
Next, to check the Z-ring formation in vivo, we introduced
two plasmids to express ClpX and FtsZ-GFPuv into E. coli cells.
This enables us to visualize the localization of FtsZ in vivo.
Without ClpX overexpression, typical rod cells with the proper
Z-ring were detected. In contrast, when ClpX was overexpressed, no Z-ring was observed in filamentous cells (Fig. 9B).
Note that almost all FtsZ colocalized with nucleoid because of
unknown reasons. Taken together, these results indicate thatoverexpression of ClpX inhibits the assembly of FtsZ in vivo,
leading to defective cell division.

FEBRUARY 26, 2010 • VOLUME 285 • NUMBER 9

JOURNAL OF BIOLOGICAL CHEMISTRY

6655

Downloaded from www.jbc.org at KUMAMOTO UNIV LIB, on February 24, 2010

DISCUSSION
Here, we examined molecular mechanisms of remodeling of
E. coli cell division protein FtsZ by AAA⫹ chaperone ClpX. We
observed that ClpX shifts the dynamic equilibrium of FtsZ frompolymers toward monomers, working at the initial step of FtsZ
assembly. In this function, the N-terminal domain of ClpX
plays a crucial role in the recognition and remodeling of FtsZ invitro and in vivo. A C-terminal region of FtsZ in part contributes to inhibition of FtsZ polymerization by ClpX as well asdegradation of FtsZ by ClpXP.
High Speed AFM Is a Superior Method for Visualizing FtsZ
Filament Dynamics—In this study, we used a high speed AFM
to visualize FtsZ polymer dynamics on second time scales. Previously, it has been reported that single FtsZ filament dynamics
were visualized with an AFM taking images every 2 min (14).
However, because the time scale of FtsZ filament dynamics ismuch shorter than 2 min (29), an AFM with higher time resolution was desired to visualize FtsZ filament dynamics. Using ahigh speed AFM, we found that FtsZ is in a dynamic equilibrium between monomers and polymers and that those FtsZ
polymers undergo dynamic conformational changes such ascurvature and bundling (Fig. 3 and supplemental Fig. S2). Previous reports suggested that curvature (12) and depolymerization of FtsZ filaments (8, 9) are triggered by GTP hydrolysis
from experimental data with nucleotide analogs. In contrast, a
recent study demonstrated that formation of curved filaments
is independent of GTP hydrolysis (14). Although mechanismsof conformational changes of FtsZ driven by GTP binding andhydrolysis are still unclear (33), our present approach using ahigh speed AFM has the great potential to provide novel
insights into dynamics of GTP-dependent assembly and conformational changes of FtsZ. We are now trying to visualize theinitial process of FtsZ polymerization, ClpX-mediated FtsZ
depolymerization, and ClpXP-mediated FtsZ degradation.
Effect of ATP on ClpX Activity to Regulate FtsZ Polymer
Dynamics—Previously, it has been reported that ATP binding
but not ATP hydrolysis enhances the activity of B. subtilis ClpX
to inhibit FtsZ polymerization, because the addition of ATP,
ADP, and ATP␥S equally exhibited positive effects on it (19).
Recently, by the same group it has been reported that a ClpX
mutant (E182A), which is defective in the ATP hydrolysis,
showed a reduced ability to inhibit FtsZ polymerization in vitro,and thus it has been speculated that ATP hydrolysis is required
for maximum inhibition (20). Therefore, the significance of

ATP hydrolysis remains to be elucidated. Based on the previous
finding that at least ATP binding has a role for modulation of
the ClpX activity (19), they expected that ATP binding triggers
the hexamer formation of ClpX, leading to a maximum activityof ClpX. However, they did not show the oligomeric state of B.
subtilis ClpX under the conditions tested. More importantly, if
ATP affects the oligomer formation of ClpX, light scattering
assay is not available to evaluate FtsZ polymerization in thepresence of ClpX, because ClpX oligomerization and the formation of complexes between FtsZ and ClpX severely affectlight scattering signal of FtsZ polymers. Therefore, we alternatively employed a sedimentation assay in this study.
We found that the N-terminal domain of ClpX has a majorrole for regulating FtsZ polymer dynamics in vitro and in vivo
(Figs. 5 and 9). This is highly correlated with the finding that, forinhibition of FtsZ polymerization, ClpX does not require ATPand energy-consuming steps such as protein unfolding andtranslocation (Fig. 5) (19). Surprisingly, ATP and ATP␥S
reduced the ability of ClpX to inhibit FtsZ polymerization (Fig.
5); this is inconsistent with the previous report by Weart et al.
(19). Based on our data with E. coli proteins, we propose a possible explanation for the effect of ATP on ClpX activity toinhibit FtsZ assembly; ATP binding induces conformationalchanges of ClpX leading to unsuitable structures of the N-terminal domain of ClpX for binding to FtsZ. Actually, binding
and/or hydrolysis of ATP in the AAA⫹ domain of ClpX induces
a large movement of the N-terminal domain (34). Because the
N-terminal domain of ClpX forms dimers (4) and has the ability
to inhibit the FtsZ assembly, large oligomer formation in thepresence ATP or stable hexamer formation in the presence ofATP␥S (Fig. 4) would lead to the reduction of the freely accessible N-terminal domain of ClpX. A recent finding that a mutation into the Walker B motif (E182A) reduced the ClpX activity
to inhibit FtsZ assembly in vitro (20) can also be accounted forby our proposed molecular mechanism, because the ATP hydrolysis mutant, ClpX (E182A), might be kept in a state of ATPbound hexamer. Taking into consideration that the negative
effect of ADP was smaller than that of ATP and ATP␥S, theADP-bound but not ATP-bound ClpX is in a preferred conformation for FtsZ recognition.
ClpX Recognizes the C Terminus and Other Parts of FtsZ—To
date, several FtsZ modulators have been identified in E. coli.
MinC, one of the negative regulators for FtsZ assembly, recognizes a C-terminal unstructured region of FtsZ (supplemental
Fig. S5) (17). FtsA and ZipA, which collaborate in anchoring
FtsZ to membrane and stabilize the Z-ring, also interact with
the C-terminal region of FtsZ (supplemental Fig. S5) (15, 16).
SulA, a cell division inhibitor of the SOS response that prevents
FtsZ assembly and facilitates FtsZ depolymerization, interacts
with the C-terminal domain of FtsZ (supplemental Fig. S5) (18).
In the case of FtsZ degradation by ClpXP, the C-terminal
unstructured region of FtsZ is important for the recognition by
ClpX (Fig. 10) (21). Similarly, the C-terminal region of FtsZ acts
as a recognition signal for the inhibition of FtsZ polymerization
by ClpX (Fig. 7), but other parts of FtsZ are also recognized by
the N-terminal domain of ClpX. Previously, it has beenreported that the N-terminal domain of ClpX preferentiallyrecognizes several signal sequences containing hydrophobicSupplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/12/16/M109.080739.DC1.html

ClpX-mediated Inhibition of FtsZ Polymerization

6656 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 285 • NUMBER 9 • FEBRUARY 26, 2010

Downloaded from www.jbc.org at KUMAMOTO UNIV LIB, on February 24, 2010

dimers, in vitro (35, 36). However,taking into consideration that tubulins are also in a dynamic equilibrium between monomers and polymers (37) like FtsZ, Spastin andKatanin may have the potential toshift the equilibrium toward monomers from polymers by inhibiting
assembly and reassembly of ␣/␤-tubulin dimers in a manner similar to
ClpX-mediated inhibition of FtsZ
polymerization. Recently, we havereported that the N terminus followed by the AAA domain of
SPAS-1, the Spastin homolog in
Caenorhabditis elegans, interacts
with ␣/␤-tubulin dimers (38). It isinteresting to mention that theeffect of AAA⫹ protein on the protein polymerization has been demonstrated by using yeast prion polymers. Both in vivo and in vitro datasuggest that Hsp104 disassembles
amyloid-like aggregates of Sup35,
Ure2, and Rnq1 (39 – 41). Importantly, similar to our findings, theseeffects of Hsp104 are also modulated by its N-terminal domain (42).
Molecular Mechanisms for the
Remodeling of FtsZ Polymer
FIGURE 10. Proposed molecular mechanisms for the remodeling of FtsZ polymer dynamics by ClpX. FtsZ Dynamics by ClpX—Based on ouris in GTP-dependent polymer dynamics. GTP binding triggers FtsZ polymerization, whereas hydrolysis or data, we propose molecular mecharelease of GTP induces depolymerization. Reassembly of the disassembled FtsZ filaments and/or monomers is
induced by GDP/GTP exchange or GTP rebinding. ClpX binding to FtsZ monomers or short polymers results in nisms for the remodeling of FtsZ
prevention of assembly and reassembly of FtsZ. In the absence of ATP, ClpX predominantly forms dimers or polymer dynamics by ClpX (Fig. 10).
small oligomers, and many N-terminal domains of ClpX seem to be accessible for FtsZ. Therefore, strong FtsZ is in a dynamic equilibrium
inhibition of FtsZ polymerization is archived. In the presence of ATP, ClpX forms hexamers or more large
oligomers. Some of them can slightly unfold FtsZ monomers or polymers by using the energy from ATP between monomers and polymers
hydrolysis, but its contribution to prevention of FtsZ assembly would be a negligible level. Alternatively, (Fig. 3 and supplemental Fig. S2),
FtsZ-accessible N-terminal domains in ClpX hexamers and large oligomers are limited, and thus inhibition ofwhich is triggered by GTP binding/
FtsZ polymerization is weakened.
hydrolysis and GDP/GTP exchange.
In the absence of ATP, ClpX forms
residues (34). Based on their results, we searched for ClpX predominantly dimers (Fig. 4) and binds to FtsZ monomers or
recognition signals in FtsZ and found several candidates small oligomers. The binding is achieved by the interaction(supplemental Fig. S5). When the sequences buried inside between the N-terminal domain of ClpX and the C-terminal
the folded structure are eliminated, we identified only three domain and other parts of FtsZ. Importantly, an effective inhisequences (supplemental Fig. S5). Interestingly, all of them bition of FtsZ polymerization is provided only by binding ofare located in the structures forming binding interfaces ClpX with FtsZ. Similarly, ClpX interacts with spontaneously
between FtsZ protomers. It is therefore reasonable to spec- depolymerized FtsZ, including a variety of oligomeric struculate that ClpX recognizes one or more regions facing tures ranging from monomers to small oligomers, and subsequently it prevents reassembly of them. If FtsZ assembly is unitoward neighbor protomers.
Action of ClpX toward Preformed FtsZ Polymers—Interest- directional, ClpX binding to either end of FtsZ filament isingly, we found that ClpX can act on preformed FtsZ polymers enough to inhibit the short filament reassembly. In contrast, if it(Fig. 7). Based on the current finding that FtsZ is in a dynamic is bidirectional, binding to both ends is required for strong inhiequilibrium between monomers and polymers (Fig. 3 and sup- bition. In the presence of ATP, ClpX unfolds FtsZ monomers,
plemental Fig. S2), ClpX seems to inhibit reassembly of FtsZ but the efficiency of FtsZ unfolding by ClpX is very low. Altermonomers and short filaments rather than to disassemble or natively, ClpX forms hexamers and large oligomers (Fig. 4), andsever FtsZ polymers. In eukaryotes, it has been shown that AAA hence, freely accessible N-terminal domains of ClpX are limfamily proteins Spastin and Katanin disassemble and sever ited, resulting in a decrease in the ClpX activity to suppress FtsZ
taxol-stabilized microtubules, the polymer of ␣/␤-tubulin polymerization.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/12/16/M109.080739.DC1.html

ClpX-mediated Inhibition of FtsZ Polymerization
Acknowledgments—We acknowledge Dr. T. Okuno for FtsZ preparations; Dr. B. Bukau and Dr. S. Hishida for providing plasmids; Dr. K.
Sonomoto and S. Matsumoto for sending bacterial strains; Dr. A.
Nagafuchi and Dr. T. Seki for SEM operation; Dr. D. Yamamoto fortechnical advice regarding high speed AFM; Dr. M. Wachi for stimulating discussions, and C. Ichinose for secretarial assistance.
REFERENCESFEBRUARY 26, 2010 • VOLUME 285 • NUMBER 9

JOURNAL OF BIOLOGICAL CHEMISTRY

6657

Downloaded from www.jbc.org at KUMAMOTO UNIV LIB, on February 24, 2010

1. Tucker, P. A., and Sallai, L. (2007) Curr. Opin. Struct. Biol. 17, 641– 652
2. Flynn, J. M., Neher, S. B., Kim, Y. I., Sauer, R. T., and Baker, T. A. (2003)
Mol. Cell 11, 671– 683
3. Martin, A., Baker, T. A., and Sauer, R. T. (2008) Nat. Struct. Mol. Biol. 15,
1147–1151
4. Wojtyra, U. A., Thibault, G., Tuite, A., and Houry, W. A. (2003) J. Biol.
Chem. 278, 48981– 48990
5. Goehring, N. W., and Beckwith, J. (2005) Curr. Biol. 15, R514 –R526
6. Margolin, W. (2005) Nat. Rev. Mol. Cell Biol. 6, 862– 871
7. Adams, D. W., and Errington, J. (2009) Nat. Rev. Microbiol. 7, 642– 653
8. Mukherjee, A., and Lutkenhaus, J. (1994) J. Bacteriol. 176, 2754 –2758
9. Bramhill, D., and Thompson, C. M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91,
5813–5817
10. Romberg, L., Simon, M., and Erickson, H. P. (2001) J. Biol. Chem. 276,
11743–11753
11. Yu, X. C., and Margolin, W. (1997) EMBO J. 16, 5455–5463
12. Lu, C., Reedy, M., and Erickson, H. P. (2000) J. Bacteriol. 182, 164 –170
13. Beuria, T. K., Krishnakumar, S. S., Sahar, S., Singh, N., Gupta, K., Meshram, M., and Panda, D. (2003) J. Biol. Chem. 278, 3735–3741
14. Mingorance, J., Tadros, M., Vicente, M., González, J. M., Rivas, G., andVélez, M. (2005) J. Biol. Chem. 280, 20909 –20914
15. Ma, X., and Margolin, W. (1999) J. Bacteriol. 181, 7531–7544
16. Hale, C. A., Rhee, A. C., and de Boer, P. A. (2000) J. Bacteriol. 182,
5153–5166
17. Shen, B., and Lutkenhaus, J. (2009) Mol. Microbiol. 72, 410 – 424
18. Cordell, S. C., Robinson, E. J., and Lowe, J. (2003) Proc. Natl. Acad. Sci.
U.S.A. 100, 7889 –7894
19. Weart, R. B., Nakano, S., Lane, B. E., Zuber, P., and Levin, P. A. (2005) Mol.
Microbiol. 57, 238 –249

20. Haeusser, D. P., Lee, A. H., Weart, R. B., and Levin, P. A. (2009) J. Bacteriol.
191, 1986 –1991
21. Camberg, J. L., Hoskins, J. R., and Wickner, S. (2009) Proc. Natl. Acad. Sci.
U.S.A. 106, 10614 –10619
22. Kanemori, M., Nishihara, K., Yanagi, H., and Yura, T. (1997) J. Bacteriol.
179, 7219 –7225
23. Woo, K. M., Kim, K. I., Goldberg, A. L., Ha, D. B., and Chung, C. H. (1992)
J. Biol. Chem. 267, 20429 –20434
24. Mukherjee, A., and Lutkenhaus, J. (1998) EMBO J. 17, 462– 469
25. Ando, T., Kodera, N., Takai, E., Maruyama, D., Saito, K., and Toda, A.
(2001) Proc. Natl. Acad. Sci. U.S.A. 98, 12468 –12472
26. Ando, T., Uchihashi, T., Kodera, N., Yamamoto, D., Miyagi, A., Taniguchi,M., and Yamashita, H. (2008) Pflugers Arch. 456, 211–225
27. Sugimoto, S., Higashi, C., Saruwatari, K., Nakayama, J., and Sonomoto, K.
(2007) FEBS Lett. 581, 2993–2999
28. Hiraga, S., Ichinose, C., Niki, H., and Yamazoe, M. (1998) Mol. Cell 1,
381–387
29. Chen, Y., and Erickson, H. P. (2005) J. Biol. Chem. 280, 22549 –22554
30. Grimaud, R., Kessel, M., Beuron, F., Steven, A. C., and Maurizi, M. R.
(1998) J. Biol. Chem. 273, 12476 –12481
31. Levchenko, I., Seidel, M., Sauer, R. T., and Baker, T. A. (2000) Science 289,
2354 –2356
32. Sugimoto, S., Saruwatari, K., Higashi, C., and Sonomoto, K. (2008) Microbiology 154, 1876 –1885
33. Oliva, M. A., Cordell, S. C., and Löwe, J. (2004) Nat. Struct. Mol. Biol. 11,
1243–1250
34. Thibault, G., Yudin, J., Wong, P., Tsitrin, V., Sprangers, R., Zhao, R., andHoury, W. A. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 17724 –17729
35. McNally, F. J., and Vale, R. D. (1993) Cell 75, 419 – 429
36. Roll-Mecak, A., and Vale, R. D. (2008) Nature 451, 363–367
37. Kueh, H. Y., and Mitchison, T. J. (2009) Science 325, 960 –963
38. Matsushita-Ishiodori, Y., Yamanaka, K., Hashimoto, H., Esaki, M., andOgura, T. (2009) Genes Cells 14, 925–940
39. Chernoff, Y. O., Lindquist, S. L., Ono, B., Inge-Vechtomov, S. G., andLiebman, S. W. (1995) Science 268, 880 – 884
40. Shorter, J., and Lindquist, S. (2004) Science 304, 1793–1797
41. Tipton, K. A., Verges, K. J., and Weissman, J. S. (2008) Mol. Cell 32,
584 –591
42. Hung, G. C., and Masison, D. C. (2006) Genetics 173, 611– 620