From the Division of Biological Sciences, Institute
of Scientific and Industrial Research, Osaka University,
Ibaraki, Osaka 567-0047, Japan and the § Department of Life
Sciences, Graduate School of Arts and Sciences, University of Tokyo,
Komaba, Tokyo 153-8902, Japan
Received for publication, January 12, 2001, and in revised form, February 12, 2001
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ABSTRACT |
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ATP synthase
(F0F1) transforms an electrochemical
proton gradient into chemical energy (ATP) through the rotation of a
subunit assembly. It has been suggested that a complex of the ATP is synthesized in chloroplasts, mitochondria, and bacteria by
a ubiquitous ATP synthase (F0F1) coupled with
an electrochemical proton gradient. The F0F1 of
Escherichia coli consists of a catalytic sector,
F1 ( The roles of the In ATP hydrolysis, the mechanical work done by the Studies on the F0 sector involving electron (17) and atomic
force (18, 19) microscopy suggested that the c subunits form
a ring structure and that subunits a and b are
located outside the ring. A ring formed from 12 c subunits
was proposed for the E. coli enzyme from the model of the
solution structure (20, 21) and genetic fusion (22, 23), and the yeast
structure formed from 10 monomers was observed by x-ray diffraction
(24). A ring of 14 monomers was observed more recently for the
chloroplast enzyme (25). The rotation of the c ring with the
As shown previously by Fillingame et al. (34), the membrane
ATPase activity of the c subunit cIle-28
(cI28) to the Thr (cT28) mutant was resistant to
venturicidin, an effective inhibitor of the wild type. In this study,
we observed that the c ring rotation became less sensitive
to venturicidin when the cI28T mutation was introduced into
the c subunit. Furthermore, the
Plasmids, Bacterial Strain, and Growth Conditions--
A plasmid
(pBWU13) carrying the entire gene for F0F1 (35)
was engineered for the rotation of the
Recombinant plasmids were introduced into E. coli strain DK8
( Preparations--
Membrane vesicles were prepared after the
disruption of cells by passage through a French press (35).
Nonengineered F0F1 was purified from membranes
(DK8/pBWU13) as described previously (35). Modified procedures (32)
were used for the engineered enzymes. Membrane vesicles were
centrifuged and then suspended in buffer A (20 mM
MES1-Tricine, pH 7.0, 5 mM MgCl2, and 100 mM KCl). The
suspension (1 ml, 20 mg of protein) was added with 220 µl of 10%
C12E8 (detergent, octaethylene glycol
dodecyl ether). After a 10-min incubation at 4 °C, the suspension
was centrifuged at 220,000 × g for 1 h. 6-{N'-[2-(N-maleimide)ethyl]-N-piperazinylamido}hexyl
D-biotinamide (100 µM) was included in the
suspension for the engineered F0F1 constructed
for the c ring rotation. The supernatant (about 1 ml, 6-12
mg of protein) was applied to a TALON metal affinity column
(1.4 × 1.5 cm; CLONTECH) and eluted with
buffer A containing 50 mM imidazole, 10% glycerol, 0.3%
C12E8, and 0.1% phosphatidylcholine (TypeII-S
from soybean) (Sigma).
The eluate (500 µl) was applied to a glycerol gradient, 10-30%
glycerol, in 10 mM MES-Tricine, pH 7.0, 5 mM
MgCl2, 0.5 mM dithiothreitol, 0.1%
C12E8, and 0.02% phosphatidylcholine. After centrifugation at 340,000 × g for 5 h, the ATPase
activity (40-150 µg of protein/ml, about 300 µl) was recovered in
the 20-25% glycerol fractions. The F1 sector with the
biotin tag in the Immobilization of F0F1 and Observation of
Rotation--
F0F1 was immobilized on a
nitrocellulose-coated glass surface (cover glass) via the His tag and
Ni2+-nitrilotriacetic acid horseradish peroxidase conjugate
(13). The enzyme was then reacted with streptavidin for 5 min at
20 °C, followed by the attachment of a 10 nM fluorescent
actin filament. The rotation was followed in 10 mM
Hepes-NaOH (pH 7.8) containing 25 mM KCl, 6 mM
MgCl2, 10 mg/ml bovine serum albumin (Sigma), 0.24 mM Triton X-100, pyruvate kinase (50 µg/ml), 1 mM phosphoenolpyruvate, 25 mM glucose, 1 µM biotin, 1% ATPase Assay--
The ATPase activity of the purified
F0F1 or F1 (0.8-1.0 µg of
protein) was assayed in 200 µl of the buffer used for observing rotation (without phosphoenolpyruvate and pyruvate kinase) (32) or in
standard buffer (10 mM Hepes/NaOH, pH 7.2, 6 mM
MgCl2), and the reaction was initiated by adding 5 mM [
The dicyclohexylcarbodiimide (DCCD) sensitivity of the purified
F0F1-ATPase activity was assayed in 10 mM Hepes/NaOH (pH 7.2), 6 mM MgCl2,
and 5 mM Na2Tris ATP (pH 8.0). The
F0F1 was incubated with DCCD (10-40
µM) for 10 min at 20 °C before starting the reaction.
Other Procedures and Materials--
Protein concentrations and
the ATP-dependent formation of an electrochemical proton
gradient were assayed as described previously (35). Fluorescently
labeled biotinylated actin filaments were prepared as described (13).
Venturicidin was kindly supplied by Dr. R. H. Fillingame. Triton
X-100 was obtained from Nacalai Tesque (Kyoto, Japan).
C12E8 was from Calbiochem. DNA-modifying enzymes were obtained from Takara Shuzo C., Ltd. or New England Biolabs
(Beverly, MA). Other chemicals were of the highest grade commercially available.
Properties of the Engineered F0F1 for
Rotation--
Plasmids for two types of engineered
F0F1 were constructed in this study:
(a) the replacement of Glu-2 with Cys in the c
subunit and the introduction of a His tag into the Effects of DCCD and Venturicidin on the Purified Engineered
F0F1--
The engineered
F0F1-ATP synthases could be purified by a
method involving affinity chromatography and glycerol gradient
centrifugation. They comprised eight subunits (Fig.
1) and showed substantial ATPase
activities (30-50% of the nonengineered wild-type enzyme) (Table I),
except that the engineered enzyme with an
Similar to the nonengineered F0F1, the
engineered F0F1 exhibited about 70-80%
inhibition with 40 µM DCCD (assayed after a 10-min
incubation without ATP). The enzyme with the cI28T mutation exhibited reduced sensitivity (Table I), confirming the previous results for membrane ATPase with the same mutation (34).
However, all of the F0F1 was unaffected
by DCCD when assayed in the buffer used for rotation, although it
showed low but significant sensitivity after a 30-min incubation (about
30% inhibition with 40 µM DCCD). This apparent low DCCD
sensitivity may be attributable to the high protein concentration in
the buffer, including serum albumin and the enzymes to regenerate ATP,
or to the presence of a sulfhydryl agent. Therefore, it was not
possible to examine DCCD for the rotation of an actin filament
connected to the c ring because the inhibitor should be
effective right after the addition to the rotating filament.
The inhibitory effect of venturicidin on the ATPase activity was
examined; about 50% of the activity of the engineered
F0F1 (cE2C/
The engineered enzyme with the Rotation of an Actin Filament Connected to the c Ring and Effect of
Venturicidin--
The rotation of actin filaments connected to the
c ring was confirmed, and it was unlikely that the filaments
were connected to the
The rotation was also tested in the presence of venturicidin. On
comparison of the filament rotation before and after the antibiotic
addition, we found that venturicidin increased the pauses in the
wild-type (cI28) engineered F0F1
(Fig. 3a, b, c), confirming the
previous results (32). On the other hand, the filament connected to the
c ring with the cI28T mutation showed apparently
less frequent pauses after venturicidin addition; the ratios of events
(pauses after venturicidin addition and pauses before
venturicidin addition) were 2 for filaments with cI28T and 4 for those without (Fig. 3d).
We examined the effect of the cI28T mutation on the
frictional torque generated by the c ring rotation. Torque
can be determined from the filament rotation rate and viscous drag.
Because the rotational rates of single filaments varied slightly and
paused, as described above, we selected more than 20 linear segments
with no pauses from the rotation time course of each filament and
plotted the average rate (Fig. 3e). The cI28T
mutation showed no significant effect on the filament rotation or
torque generation (Fig. 3e). Furthermore, venturicidin
showed no effect on the torque generation (Fig. 3e,
open and closed triangles).
Rotation of an Actin Filament Connected to the
As predicted by the binding change mechanism for ATP synthesis and
hydrolysis, the continuous rotation of the Fillingame et al. (34) have shown that membrane ATPase
activity (attributable to F0F1) became less
sensitive when the cI28T mutation was introduced. It should
be noted, however, that this antibiotic was not a strong inhibitor even
for the wild-type enzyme (maximum inhibition, about 60%). We observed
lower but significant inhibition of the engineered
F0F1 when ATPase activity was assayed under the
rotation conditions. Consistent with the results for ATPase activity,
this antibiotic did not have a strong effect on the rotation, such as
immediate cessation upon its addition, but rather showed
specific inhibition of F0F1. An actin filament connected to the c ring showed increased pauses upon the
addition of venturicidin, and the cI28T mutation decreased
the inhibitor sensitivity. As described above, this antibiotic had no
effect on the ATPase activity or the We tested a different engineered enzyme for rotation by introducing a
His tag into the c ring and a biotin tag into the
One model for the interaction of the Tsunoda et al. (40) have claimed that they could not connect
an actin filament specifically to the c ring under their
experimental conditions and criticized the experiments by Sambongi
et al. (32) showing c ring rotation. The
protocols used by the two groups are different, including the positions
of Cys residues introduced into the c subunit (Sambongi
et al. (32), Glu-2 In conclusion, we clearly showed that a complex of subunit and c ring (c10-14) of
F0F1 could rotate together during ATP
hydrolysis and synthesis (Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T., Wada, Y., and
Futai, M. (1999) Science 286, 1722-1724). We observed that the rotation of the c ring with the cI28T
mutation (c subunit cIle-28 replaced by Thr)
was less sensitive to venturicidin than that of the wild type,
consistent with the antibiotic effect on the cI28T mutant
and wild-type ATPase activities (Fillingame, R. H., Oldenburg, M.,
and Fraga, D. (1991) J. Biol. Chem. 266, 20934-20939). Furthermore, we engineered F0F1
to see the
3
3 hexamer rotation; a biotin
tag was introduced into the
or
subunit, and a His tag was
introduced into the c subunit. The engineered enzymes could
be purified by metal affinity chromatography and density gradient
centrifugation. They were immobilized on a glass surface through the
c subunit, and an actin filament was connected to the
or
subunit. The filament rotated upon the addition of ATP and
generated essentially the same frictional torque as one connected to
the c ring. These results indicate that the
c10-14 complex is a mechanical unit of
the enzyme and that it can be used as a rotor or a stator
experimentally, depending on the subunit immobilized.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
3
), and a
proton pathway, F0
(ab2c10-14) (for
reviews, see Refs. 1-6). The amino- and carboxyl-terminal
helices
of the
subunit occupy the central space of the
3
3 hexamer, as shown by the high
resolution structure (7). The catalytic sites in the three
subunits
participate alternately in ATP synthesis and also in hydrolysis, as
predicted from the binding change mechanism (6). The mechanism also
proposes that the
subunit rotation plays a major role in the
conformational transmission among the
subunits.
subunit in energy coupling and catalytic
cooperativity have been shown by extensive genetic studies (2). Mutation and suppression studies have suggested that the
subunit carboxyl-terminal domain and amino-terminal Met-23 interact
through long range conformational transmissions involving the
movement of the two helices (8, 9).
subunit rotation has been
suggested by
-
cross-linking followed by dissociation and
reconstitution (10) and the movement of a probe attached to the
carboxyl terminus of the
subunit (11). Continuous rotation has been
observed directly as the movement of an actin filament connected to the
subunit in the F1 sector (12-14). The rotation of the
subunit with the
subunit was also shown subsequently (15).
Thus, this enzyme can be defined as a biological nanomotor carrying
out rotational catalysis (16).
subunit rotation
should be transmitted to the F0 sector for
ATP-dependent proton transport. During ATP synthesis,
proton transport should drive the
subunit rotation, which causes
the
subunit conformational change to release the product ATP.
Therefore, it is essential to determine how the
subunit rotation is
coupled to the proton transport through F0.
subunit has been proposed (26-29) and has been suggested by
chemical cross-linking (30, 31). We recently provided the first direct
evidence that the c ring rotates continuously together with
the
subunit in F0F1 (32);
F0F1 was immobilized on a glass surface through
His tags connected to the
subunits, and an actin filament was
attached to the c ring. Pänke et al. (33)
have also demonstrated the c ring rotation more recently
using a different experimental system. The x-ray structure of the yeast
enzyme showed the tight association between the
subunit and the
c ring, also suggesting that they can rotate together as an
assembly (24).
3
3 hexamer could rotate when
F0F1 was immobilized through the c
ring. These results indicate that a complex of the
subunit and
the c ring is a mechanical unit of the nanomotor and can be
a rotor or stator in an experimental system, depending on the subunit immobilized.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
3
hexamer. Codons for the (His)6-Leu-His (His tag) were
introduced between Met-1 and Asn-3 of the c subunit, and
codons for the 123 (Val-18-Tyr-140) and 105 (Lys-20-Leu-124) amino
acid residues of the transcarboxylase biotin binding domain (biotin
tag) (including multicloning sites from the PinPoint Xa-1 vector
(Promega)) were inserted, respectively, into the amino-terminal regions
of the
and
subunits (both between the 1st and 2nd codons).
Plasmid pBWU13 was also engineered for the c ring rotation,
as described previously (32): a His tag, downstream of the initiation
codon of the
subunit; cGlu-2
Cys in the c
subunit; and
Cys-87
Ala and
Cys-112
Ala in the
subunit (32). The cI28T (cIle-28 replaced by Thr)
mutation was further introduced into the engineered
F0F1 for the c ring rotation by a
polymerase chain reaction-based method.
unc), which lacks the
F0F1 gene (36). Cells were grown at 37 °C in
a rich medium (L-broth) supplemented with ampicillin (50 µg/ml), a
synthetic medium containing 0.5% glycerol for enzyme purification, or
the same medium containing 0.5% succinate for testing growth as to
oxidative phosphorylation (35). Biotin (2 µM) was
included in the synthetic medium for growth of the strain expressing
the enzyme with the biotin tag.
subunit was purified as described previously
(13).
-mercaptoethanol, glucose oxidase (216 µg/ml), and catalase (36 µg/ml). Immediately after the addition of
5 mM ATP, the rotating filament in a 1-mm2 area
was observed at 20 °C with a Zeiss Axiovert 135 equipped with an
ICCD camera (Atto Instruments). The filament images were video recorded
and analyzed (13).
-32P]ATP. When indicated, various
concentrations of venturicidin were included in the mixtures followed
by incubation for 5 min before the addition of ATP. After 20 min of
incubation at 20 °C, the reaction was terminated by adding 0.3 N trichloroacetic acid. Each mixture was centrifuged at
3000 × g for 5 min, and 100 µl of the supernatant
was mixed with 250 µl of 1.25% ammonium molybdate containing 3.8%
HCl. The [32P]phosphomolybdate was extracted in 500 µl
of isobutyl alcohol-benzene-acetone (3:3:1, v/v), and then the
radioactivity was determined with a liquid scintillation counter
(37).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit
(
His tag) with or without the c subunit Ile-28 to Thr
(cI28T)
mutation2 and
(b) the introduction of a His tag into the c
subunit and a biotin tag into the
(or
) subunit. The recombinant
plasmids encoding these enzymes were introduced into E. coli
strain DK8 lacking the entire F0F1 gene (36).
The transformed cells could grow on succinate through oxidative
phosphorylation similar to those harboring the control plasmid
encoding the nonengineered F0F1 (Table
I). These results indicate that the
genetic modification for observing rotation did not affect the
catalysis by or energy coupling of the enzyme.
Properties of F0F1 engineered for rotation
subunit biotin tag was
difficult to purify for an unknown reason and was obviously less pure
than the other enzymes (Fig. 1, lane 3). The positions of
the
and
subunits with a biotin tag and biotinylation of
c subunit cE2C were confirmed by
immunoblotting with streptavidin (data not shown). The c
subunit with a His tag showed significantly stronger staining than that
without the tag.
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Fig. 1.
Polyacrylamide gel electrophoresis of
engineered F0F1. The engineered
F0F1 (1.0 µg) was subjected to polyacrylamide
gel electrophoresis (5-20% linear gradient) in the presence of 0.1%
sodium dodecyl sulfate. The gel was stained with Coomassie Brilliant
Blue: lane 1, nonengineered F0F1;
lane 2, F0F1 with a His tag in the
c ring and a biotin tag in the ; lane 3,
F0F1 with a His tag in the c ring
and a biotin tag in the
; lane 4,
F0F1 with cE2C and a His tag
in the
; and lane 5, F0F1 with
cE2C, a His tag in
, and the cI28T
mutation.
His tag) as to
the c ring rotation was inhibited by 8 µM venturicidin, whereas ~30% of the activity of cI28T was
inhibited (Fig. 2a),
confirming the previous results for membrane enzymes (34). These
enzymes became less sensitive when assayed in the buffer for rotation
(Fig. 2b). However, this antibiotic can be considered as a
specific inhibitor for the engineered F0F1
under the rotation conditions because it was less inhibitory for the enzyme with the cI28T mutation and not inhibitory for the
F1 at all. It should be noted that specific inhibition was
observed after a 5-min incubation, suggesting that venturicidin can be tested upon addition to the rotating enzyme.
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Fig. 2.
Effects of venturicidin on the ATPase
activity of engineered F0F1. The ATPase
activities of engineered F0F1 as to
c ring and 3
3 hexamer rotation
were assayed in the presence of various concentrations of venturicidin:
engineered F0F1 for c ring rotation,
open circles; engineered F0F1 with
the cI28T mutation for c ring rotation,
closed circles; engineered F1 for
subunit
rotation, triangles; and F0F1
engineered for
subunit rotation, squares. Various
concentrations of venturicidin were added 5 min before the addition of
ATP. ATPase activity was examined under two sets of assay conditions: 5 mM ATP and 6 mM MgCl2 in Hepes
buffer (a) and the same substrate concentration in the
buffer used for rotation (b). The specific activities
of all enzymes without venturicidin addition are shown in Table
I.
biotin tag/cHis tag for
3
3 hexamer rotation was significantly
less sensitive to venturicidin; only about 15% inhibition was observed
with excess venturicidin (Fig. 2a). This result may be
attributable to the introduction of the His tag into the c
subunit. Therefore, the antibiotic could not be tested for the rotation
of this enzyme.
subunit of the contaminating F1,
as discussed previously (32). As a control, F1 (with no
cysteine in the
subunit) was isolated from the engineered
F0F1 to test whether the actin filament can
bind to the Cys-less
and rotate with the addition of ATP. We could
find no rotating filament, indicating that the contaminating Cys-less
F1, if any, is not responsible for the rotation
observed.
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Fig. 3.
Rotation of an actin
filament connected to the c ring with or without the
cI28T mutation. a, time courses of the
rotations of actin filaments (1.2 µm) connected to the c
ring. The rotation of the actin filament connected to the
cI28 (wild type) or cT28 (mutant) c
ring of engineered F0F1 was followed before and
after the addition of venturicidin (Vent). We slowly
replaced the buffer for rotation with the same solution containing 70 µM venturicidin, waited about 30 s until the
movement of the liquid had completely stopped, and then started
recording the rotation. b, increase of pauses after
venturicidin addition. The boxed areas (20 rounds) in
panel a are expanded, and the beginnings of pauses are
indicated by arrows. cI28, left;
cT28, right; Vent ( ),
before venturicidin addition; and Vent (+), after
the addition. c, examples of pauses in expanded scale.
Boxed areas in panel b are further expanded,
showing how pauses were defined. We defined pauses as described
previously (32); the raw data plots (black dots) were median
filtered (with rank 5) (gray line), and the resulting
linear segments that had R2 values
greater than 0.96 with a rotational rate between
0.2 and 0.2 (sec
1 and dwell durations longer than 67 ms were
defined as pauses. Arrowheads pointing down and
up indicate the beginning and end of pauses, respectively.
(cI28, left; cT28, right;
Vent (
), before venturicidin addition; and
Vent (+), after the addition.) d,
increase in pauses after venturicidin addition. The rotation of 10 actin filaments (<2 µm) connected to F0F1
(cI28, cT28) was recorded, and the numbers of
pauses (per round) were recorded after and before venturicidin
addition. The ratios of event numbers (pauses after
venturicidin addition and pauses before venturicidin addition) are
shown. e, rotations versus filament length. The
rotational rates of actin filaments were obtained and plotted against
filament lengths. Open circles, cI28; open
triangles, cI28 after venturicidin addition;
closed circles, cT28; and closed
triangles, cT28 after venturicidin addition. The
dotted lines represent the calculated rotational rate of
filaments with a constant torque value of 40 piconewton·nm.
3
3 Hexamer in
F0F1--
The results obtained for the
cT28 mutant strengthen the notion that a complex of
the c ring and the
subunit rotates during ATP
hydrolysis, and this prompted us to examine a further possibility. If
the
subunit and c ring form a mechanical unit, the
3
3 hexamer rotates when the c
ring is immobilized on a glass surface (Fig. 4a). We introduced a His tag
into the c subunit and a biotin tag into the
or
subunit to examine this possibility. As expected, an actin filament
connected to the
3
3 hexamer rotated upon
ATP addition (Fig. 4b). The rotation direction of the
hexamer (counterclockwise viewed from the F1 side)
was consistent with that of the c ring (clockwise from the
F1) in F0F1 immobilized on a glass
surface through the
or
subunit. The rotation of the
3
3 hexamer generated essentially the same
torque (~40 piconewton·nm) as that of the c ring.
An effect of venturicidin on the
3
3
hexamer rotation was not apparent, which is consistent with its slight
effect on the ATPase activity of the F0F1
engineered for
3
3 rotation (Fig. 2). As a
control, we purified F1 with a biotin tag in the
subunit (but no His tag in the
subunit) and connected an actin
filament to it. We found no rotating filament among 10,000 attached
nonspecifically to the glass surface, supporting the results described
above.
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Fig. 4.
Rotation of an actin filament connected to
the or
subunit. a, experimental system for observing the
rotation of filaments connected to
subunits. b, time
courses of rotating filaments (1.3 and 1.9 µm) connected to c,
, and
subunits.
c, effect of the actin filament length on the rotational
rate. The rotational rates of actin filaments connected to
(open circles) or
(closed circles) subunits
were plotted against filament length. The dotted line
represents the calculated rotational rate of the filaments with a
constant torque value of 40 piconewton·nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit in the
F1 sector has been observed during ATP hydrolysis (12-14). The
subunit rotation should be transmitted to the F0
sector for coupling with proton transport. We provided the first
evidence that the c subunit ring rotates with the
subunit in F0F1 (32). Pänke et
al. (33) have also demonstrated the c ring rotation more recently. The present study further confirmed the co-rotation of
the
subunit with the c ring.
rotation of the engineered
F1. These results suggest that venturicidin inhibits
F0F1 rotation by binding to the c
ring and that a complex of the
subunit and c ring was rotating.
3
3 hexamer. Thus,
F0F1 was immobilized on a glass surface and
connected with an actin filament without using chemistry such as
cysteine residue modification with biotin-maleimide (12-15, 32). Upon the addition of ATP, the actin filament connected to the
3
3 hexamer rotated counterclockwise when
viewed from the F1 side and generated similar torque to the
rotation. These results strongly suggest that the
c10-14 complex is a mechanical unit and
rotates during ATP hydrolysis and that the rotor and stator of
the entire complex are interchangeable experimentally, depending on the
subunit immobilized.
subunit with the c
ring during ATP synthesis and hydrolysis predicts that the
subunit interacts with the hydrophilic loop between the transmembrane helices
of each c subunit one by one during rotation (discussed in
Refs. 38 and 39). The rotation of the actin filament connected to the
c ring indicates that this model will not work.
However, it may be possible to argue that the c ring rotated
with the
subunit as a part of the fluorescent probe (actin
filament). The rotation of the filament connected to the
3
3 hexamer finally excluded this
possibility because the model predicts that the filament should not
rotate if the c ring is immobilized and the
subunit
moves on the ring.
Cys; Tsunoda et al. (40),
Cys inserted between Glu-2 and Asn-3), the conditions for maleimide
modification (Sambongi et al. (32), 4 °C at pH 7.0;
Tsunoda et al. (40), 25 °C at pH 7.5), and the detergent used for F0F1 preparation. Furthermore, in most
experiments, Tsunoda et al. (40) reacted
F0F1 with fluorescein-5-maleimide and then biotinylated anti-fluorescein IgG and the actin filament through streptavidin. On the other hand, Sambongi et al. (32)
reacted F0F1 with biotin-maleimide and then
connected an actin filament through streptavidin. Thus, the arguments
raised by Tsunoda et al. (40), which are based on mostly
negative observations, may reflect the differences in the experimental
systems. As described in this study, the conclusion of Sambongi
et al. (32) is supported by Pänke et al.
(33) and the effect of venturicidin on the cI28T mutant, as
described above. Furthermore, the actin filament connected to the
3
3 hexamer in the immobilized
F0F1 rotated, as discussed above.
and
the c ring is a mechanical unit and that it rotates as an
assembly. The preparations we used have all subunits of
F0F1, and previous studies indicated that it is
not easy to dissociate F0F1 even under
conditions that release F1 from membranes (41). Genetic (42) and reconstitution (43) studies have suggested that the three
F0 subunits (a, b, and c) are
required for the formation of the F0F1 complex.
However, we could not prove definitely that the rotating enzyme during
video recording has the original integrity of
F0F1. In this regard, Sambongi et
al. (32) did not conclude that the rotating enzyme had all the
subunits. Pänke et al. (33) stated more clearly that
they might have observed the rotation of the c ring in
incomplete F0F1. Although we accept this
reservation as to the integrity of the rotating complex, we strongly
suggest that the rotation of a complex of the
subunit and
c ring is related to the fully functional
F0F1. The obvious next step is to examine
subunit rotation during ATP synthesis and to detect ATP synthesis when
the c ring or
subunit is artificially rotated.
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ACKNOWLEDGEMENTS |
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We thank Le Phi Nga for technical assistance during the early stage of this work.
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FOOTNOTES |
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* This work was supported in part by the Japanese Ministry of Education, Science, and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Div. of Biological Sciences, ISIR, Osaka University, Ibaraki, Osaka 567-0047, Japan. Tel.: 81-6-6879-8480; Fax: 81-6-6875-5724; E-mail: m-futai@sanken.osaka-u.ac.jp.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M100289200
2
The subunit cysteine residues were removed
from this enzyme.
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ABBREVIATIONS |
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The abbreviations used are: MES, 4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DCCD, dicyclohexylcarbodiimide.
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