From the Research Laboratory of Resources
Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama,
226-8503, Japan, the
Department of Physics, Faculty of Science
and Technology, Keio University, 3-14-1, Hiyoshi, Yokohama 223-8522, Japan, and § CREST (Core Research for Evolutional Science
and Technology) Genetic Programming Team 13, Teikyo University
Biotechnology Research Center 3F, 907 Nogawa, Miyamae,
Kawasaki 216-0001, Japan
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ABSTRACT |
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Rotation of the subunit in
F1-ATPase from thermophilic Bacillus
strain PS3 (TF1) was observed under a fluorescence
microscope by the method used for observation of the
subunit
rotation (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr.
(1997) Nature 386, 299-302). The
3
3
complex of TF1 was
fixed to a solid surface, and fluorescently labeled actin filament was
attached to the
subunit through biotin-streptavidin. In the
presence of ATP, the filament attached to
subunit rotated in a
unidirection. The direction of the rotation was the same as that
observed for the
subunit. The rotational velocity was slightly
slower than the filament attached to the
subunit, probably due to
the experimental setup used. Thus, as suggested from biochemical
studies (Aggeler, R., Ogilvie, I., and Capaldi, R. A. (1997)
J. Biol. Chem. 272, 19621-19624), the
subunit
rotates with the
subunit in F1-ATPase during
catalysis.
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INTRODUCTION |
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F0F1-ATP synthase catalyzes ATP synthesis
coupled with the proton flow across the energy-transducing membranes
such as the plasma membrane of bacteria, mitochondrial inner membrane,
and thylakoid membrane of chloroplast (1-4). F1-ATPase is
the water-soluble portion of F0F1-ATP synthase
and contains a catalytic core for ATP synthesis and hydrolysis. The
F1-ATPase consists of five kinds of subunits with a
stoichiometry of
3
3
1
1
1.
The catalytic sites of ATP synthesis and hydrolysis are located mainly
on the
subunits, and noncatalytic nucleotide binding sites are
located mainly on the
subunits (5). The
3
3
subcomplex of F1-ATPase is regarded as a minimum stable complex which has catalytic features similar to F1-ATPase (6-8). Three catalytic sites of
F1-ATPase exhibit strong negative cooperativity in ATP
binding and positive cooperativity in ATP hydrolysis. To explain these
characteristics, a binding change mechanism was proposed (3, 4) and has
been widely accepted. In the binding change mechanism, all three
subunits in F0F1-ATP synthase are in different
states at a given moment and alternately exchange their states during
ATP synthesis and hydrolysis. The physical rotation of the
subunit
within the
3
3 hexamer was hypothesized as
a mechanism for the binding change to occur (3), and a crystal
structure of bovine mitochondrial F1-ATPase in which a
cylinder of the
3
3 hexamer is penetrated by the coiled-coil structure of the
subunit gave the hypothesis more reality (5). Biochemical (9, 10) and optical (11) analyses
provided support for the rotation of the
subunit, and finally, the
rotation was directly observed as the rotation of a fluorescently
labeled actin filament attached to the
subunit (12). Driven by ATP
hydrolysis, the
subunit rotated for several minutes in the
direction predicted from the crystal structure of bovine mitochondrial
F1. To obtain further insight into the mechanism of this
enzyme, it is necessary to identify each subunit of
F0F1-ATP synthase as either a rotor or stator
subunit.
The subunit, the smallest subunit of bacterial and chloroplast
F1-ATPases, is an endogenous ATPase inhibitor (14-16).
According to recent structural analyses, the
subunit of
Escherichia coli F1-ATPase consists of an
N-terminal
-sandwich and a C-terminal
-helical domain (17, 18).
The
subunit interacts with the
subunit (19) and the analysis of
a chimeric complex from a thermophilic Bacillus PS3
(TF1)1 and
chloroplast F1-ATPase indicated that the
subunit
affects the ATPase activity of F1-ATPase through the
subunit (20). The subunit interface between the
and
subunits
has been explored by the cross-linking and chemical modification (21,
22), and recent work by Aggeler et al. (23) suggested that
the
subunit rotates together with the
subunit. Previously we
reported that the inhibitory effect of the
subunit on ATPase
activity of TF1 was observed only at low concentrations of
ATP. Unlike the case of E. coli F1-ATPase where
the
subunit tends to dissociate from F1-ATPase during
multiple turnovers of ATPase reaction, the
subunit of
TF1 remains associated with the
3
3
portion during catalysis (24).
Taking advantage of this stable association of the
subunit, we
observed directly the rotation of the
subunit in
TF1.
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EXPERIMENTAL PROCEDURES |
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Preparation of the Mutant
3
3
Complex and
Subunit of
TF1--
Wild-type
subunit of TF1 does not
contain cysteine. To ensure specific modification, a mutant
subunit
(H38C) of TF1 was generated by the method of Kunkel
et al. (25). A primer oligonucleotide (5'-AAGCGGAATGCATCCCGGCAAAATG-3') which contained substitution corresponding to H38C mutation and a new EcoT22I site, was
used to introduce mutation to the expression plasmid of
TF1
, pTE2 (24). The mutant
subunit was expressed in
E. coli BL21(DE3) and purified as described previously (20)
except that all buffers contained 1 mM DTT. The expression
plasmid for the cysteine-less, His-tagged mutant (
C193S,
His10-tags in
N termini)
3
3
complex was generated from the
expression plasmid used for the observation of the rotation of the
subunit (12). Its BglII-NheI fragment was
exchanged by that of wild-type plasmid (pKABG1) (7), and the mutation
S107C was reverted to serine. The
3
3
complex was purified as described
previously (7).
Preparation of Streptavidin-attached
3
3
Complex of
TF1--
Purified (H38C)
subunit was incubated at
23 °C with 2 mM DTT for 15 h and passed through a
Sephadex G-25M column equilibrated with 50 mM Tris-HCl (pH
8.0) and 100 mM KCl. Then, 50 mM
N'-[2-(N-maleimido)-ethyl]-N-piperazinyl D-biotinamide (Dojindo) dissolved in dimethyl sulfoxide was
added to the
subunit solution (100 µM) to give a
final concentration of 1 mM and incubated for 2 h at
23° C. The reaction was quenched by the addition of 7 mM
DTT. The biotinylated
subunit was allowed to bind to the
3
3
complex, and the
3
3
complex formed was purified as
described previously (24). The
3
3
complex obtained was then mixed with 10 molar excess of streptavidin
(SA) and incubated for 20 min at 23° C. Excess streptavidin was
removed by G4000SWXL (Tosoh) gel filtration high performance liquid
chromatography, and the fraction containing the
3
3
SA (the superscript
SA designates the subunit labeled with biotin-streptavidin) complex was
concentrated by Microcon-100 (Amicon).
Observation of Rotation--
The rotation of the and
subunits was observed by the same experimental setup as that used for
the rotation of the
subunit in the previous report (12, 13, Fig.
1). The ATP concentration was fixed at 2 mM in an ATP
regenerating system, containing 0.2 mg/ml creatine kinase and 2.5 mM creatine phosphate. Rotation was observed at 23° C on
an inverted fluorescence microscope (IX70, Olympus), and images were
recorded with an SIT camera (C2741-08, Hamamatsu Photonics) on an 8-mm
video tape. The rotation angle of the filament was estimated from the
circular movement of the centroid of the filament image calculated
using a digital image processor (DIPS-C2000, Hamamatsu Photonics) (12,
13).
Other Materials and Procedures--
For a control,
3
3
SA in which streptavidin
was attached to the biotinylated
-Cys-107 was prepared from the
mutant (
C193S,
S107C, His10-tags in
N termini)
3
3
complex as described (12, 13). A
3
3
SA
complex was
reconstituted from
3
3
SA
and the wild-type
subunit (24). The purity of the complexes was
checked by 6% polyacrylamide gel electrophoresis without a denaturing
reagent (24). ATPase activity was measured at 23° C in the presence
of an ATP regenerating system in 10 mM MOPS-KOH (pH 7.0)
buffer containing 50 mM KCl, 4 mM
MgCl2, 50 µg/ml pyruvate kinase, 50 µg/ml lactate
dehydrogenase, 2.5 mM phosphoenolpyruvate, 0.2 mM NADH, and 2 mM ATP. Steady-state ATPase
activities of the (
C193S, His-tag)
3
3
complex and the (
C193S, His-tag)
3
3
complex were almost the same; 57 s
1 and 58 s
1 (expressed as a turnover
rate), respectively. Rabbit skeletal actin filaments were biotinylated
and stained with phalloidin-tetramethylrhodamine B isothiocyanate
conjugate (as in Ref. 12 but without cross-linking). Streptavidin was
purchased from Sigma as lyophilized powder. Experimental procedures of
recombinant DNA were performed as described in a manual (26). E. coli strain JM109 (27) was used for the preparation of plasmids,
and the strain CJ236 (25) was used for generating uracil-containing
single-stranded plasmids for site-directed mutagenesis. Protein
concentrations were determined by the method of Bradford (28) using
bovine serum albumin as a standard or from the UV absorbance using an
absorbance 0.45 at 280 nm for 1 mg/ml of the subunit complexes of
TF1 (29). Polyacrylamide gel electrophoresis in the
presence of 0.1% sodium dodecyl sulfate was performed as described by
Laemmli (30).
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RESULTS AND DISCUSSION |
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Experimental Setup--
Three kinds of subunit complexes were used
for experiments; the (C193S,
H38C, His-tag)
3
3
complex for observing the rotation of the
subunit, and the (
C193S,
S107C, His-tag)
3
3
and the (
C193S,
S107C,
His-tag)
3
3
complexes for observing the rotation of the
subunit. Ten histidine tags at the N termini of
the
subunits were used for the immobilization of the complex on a
solid surface, and
-C193S was for elimination of unwanted cysteine
residues to ensure the specificity of the biotinylation (among five
kinds of subunits of the wild-type TF1, only the
subunit contains a cysteine residue,
-Cys-193). In addition to these
two mutations, we introduced a cysteine at the position of
-His38
which is supposed to face the F0 side (opposite to the N
termini of
and
subunits) in the structure of
F1-ATPase (31). The biotinylation at
-Cys-38 did not
impair the ability of the
subunit to associate with the
3
3
complex to form the
3
3
complex. Polyacrylamide gel
electrophoresis without a denaturing reagent, in which two
(
3
3
and
3
3
) complexes were electrophoresed as separate bands (24), gave only a single band at a corresponding position, ensuring the homogeneity of each complex prepared. After streptavidin was bound to the introduced biotin, we fixed
3
3
SA to the beads which
adhered on the glass plate, and a fluorescently labeled, biotinylated
actin filament was attached to the
subunit through
biotin-streptavidin-biotin (Fig. 1).
Fixation and actin filament attachment to
3
3
SA and
3
3
SA
were carried out
in the same way.
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Characteristics of the Rotation--
When 2 mM ATP was
supplied, continuous rotation of the actin filament attached to
3
3
SA was observed (Fig.
2). Rotation was absolutely dependent on
ATP hydrolysis. In the absence of ATP, or in the presence of 2 mM ATP + 10 mM NaN3 which inhibits
ATPase activity of TF1, no continuous rotation was observed
(data not shown). The number of the rotating actin filaments per total
actin filaments was less than 1%, 4- to 5-fold lower than the number
observed for actin filaments attached to
3
3
SA. If the connection
between the
and
subunits is weak, a rotating actin filament
would be detached in a short time from the complex by the hydrodynamic
frictional load. However, because the duration of the rotation which
sometimes continued more than 5 min was apparently similar to the case
when an actin filament was attached to the
subunit, the
-
intersubunit connection might be strong enough to bear the hydrodynamic
friction on the actin filament. The rotation was anti-clockwise when
viewed from the membrane side. This direction is the same as that
observed for the rotation of an actin filament attached to the
subunit (12). According to the crystal structure of the bovine
mitochondrial F1 (5), when the
(and
) subunit(s)
rotates in this direction, one
subunit experiences the transition
in the order expected in the ATP hydrolysis reaction, AMPPNP-bound
form, ADP-bound form, and empty form.
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FOOTNOTES |
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* This work was supported in part by Grant 07458158 from the Ministry of Education, Science, and Culture of Japan and grants from CREST (Core Research for Evolutional Science and Technology).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.
¶ Supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.
** To whom correspondence should be addressed: Research Laboratory of Resources Utilization, R-1, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama, 226-8503, Japan. Tel.: 81-45-924-5233; Fax: 81-45-924-5277; E-mail: myoshida{at}res.titech.ac.jp.
1
The abbreviations used are: TF1,
F1-ATPase from the thermophilic Bacillus PS3;
MOPS, 3-(N-morpholino)propanesulfonic acid; 3
3
SA, a mutant (
C193S,
S107C, His10-tag in N terminus of the
subunit)
3
3
complex of TF1 with
streptavidin bound to biotinylated
-Cys-107;
3
3
SA
, a complex of
3
3
SA and the wild-type
subunit;
3
3
SA, a mutant
(
C193S,
H38C, His10-tag in N terminus of the
subunit)
3
3
complex of
TF1 with streptavidin bound to biotinylated
-Cys-38; SA,
streptavidin; DTT, dithiothreitol; AMPPNP, adenosine 5'-(
,
-imino)triphosphate; Ni-NTA, nickel-nitrilotriacetic
acid.
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REFERENCES |
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