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J. Biol. Chem., Vol. 275, Issue 37, 28353-28355, September 15, 2000
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§,,
From The Medical Research Council Dunn Human
Nutrition Unit, Cambridge CB2 2XY and ¶ The Medical Research
Council Laboratory of Molecular Biology,
Cambridge CB2 2QH, United Kingdom
Received for publication, June 30, 2000, and in revised form, July 25, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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In mitochondria, the hydrolytic activity of ATP
synthase is regulated by a natural inhibitor protein,
IF1. The binding of IF1 to ATP synthase
depends on pH values, and below neutrality, IF1 forms a
stable complex with the enzyme. Bovine IF1 has two oligomeric states, dimer and tetramer, depending on pH values. At pH
6.5, where it is active, IF1 dimerizes by formation of an antiparallel The F1F0-ATP synthase complex (also called
F1F0-ATPase) plays a central role in energy
transformation in most living organisms. It is composed of two major
domains, a globular F1 catalytic domain and a
membrane-bound F0 proton-translocating domain linked
together by a central stalk. The synthesis of ATP requires an
electrochemical proton gradient across the inner mitochondrial
membrane, which is driven by the transport of protons back into the
matrix through the F0 domain. When a cell is deprived of
oxygen, the electrochemical gradient across the inner membrane
collapses, and the enzyme switches its catalytic activity from ATP
synthesis to ATP hydrolysis. Under these conditions,
F1-ATPase, the catalytic domain of ATP synthase, catalyzes
the hydrolysis of ATP to ADP and phosphate. In mitochondria, this
hydrolytic activity is regulated by a natural inhibitor protein, IF1. In bovine mitochondria, IF1 is a basic
protein of 84 amino acids long (1). The binding of IF1 to
ATP synthase depends on pH values and, below neutrality, its inhibitory
capacity increases (2). Recently, we have shown that bovine
IF1 has two oligomeric states, tetramer and dimer, favored
by pH values above and below 6.5, respectively (3). Activation is
accompanied by a decrease in IF1 helicity relative to the
inactive form between residues 35 and 47, which are involved in the
formation of the inactive tetramers. At a pH value of about 6.5, IF1 forms an active antiparallel dimer, and this
arrangement places the inhibitory N-terminal regions in opposition. At
higher pH values, two dimers associate into the inactive tetramer. An
important implication of this model is that dimeric IF1 is
capable of binding to two F1 domains simultaneously.
In this paper, we describe experiments using gel filtration
chromatography, analytical ultracentrifugation, and electron microscopy that validate this prediction.
Purification of F1-ATPase and the IF1-F1
Complex--
The purification of bovine F1-ATPase (4) and
recombinant bovine IF1 (3) were carried out as described
previously. The IF1-F1 complex was prepared as
follows: purified F1-ATPase (25 mg), stored as an ammonium
sulfate precipitate, was collected by centrifugation (31,000 × g) at 4 °C, re-dissolved in minimal buffer (20 mM MOPS1-NaOH, pH
6.6, 1 mM EDTA, 10% glycerol, 0.001% (w/v)
phenylmethylsulfonyl fluoride), and desalted on a PD-10 column
(Amersham Pharmacia Biotech). The enzyme was mixed with a 5-fold molar
excess of IF1 over F1 and kept for 20 min at
37 °C. A portion of MgATP was added at 5, 10, and 15 min to give a
final concentration of 0.5 mM. Then, the mixture was loaded
onto a HiLoad 26/60 Superdex 200 column to separate the
IF1-F1 complex from F1-ATPase and
free IF1.
Gel Filtration Chromatography and SDS Polyacrylamide Gel
Electrophoresis--
Protein samples (5 mg/ml; 100 µl) prepared as
described above were chromatographed at room temperature at a flow rate
of 0.2 ml/min on a Superose 6 HR 10/30 column (Amersham Pharmacia
Biotech) pre-equilibrated in buffer containing 50 mM
MOPS-NaOH, pH 6.5, 10 mM magnesium sulfate, 1 mM EDTA, 0.02% sodium azide,10% glycerol, and 0.001%
phenylmethylsulfonyl fluoride. The absorbance of the eluant was
monitored at 280 nm. Thyroglobulin, ferritin, catalase, and aldolase
(molecular masses of 669, 440, 232, and 158 kDa, respectively) were
used to calibrate the column. Its void volume (V0) was determined using blue dextran 2000. The
Kav for the individual proteins was calculated
from the following expression: Kav = (Ve
Denaturing SDS-PAGE gels containing a 12-22% (w/v) acrylamide
gradient separating gel and a 4% (w/v) stacking gel
(acrylamide:N,N'-methylenebisacrylamide), 30:0.8
(w/w) were cast in 10-cm × 10-cm × 0.6-mm format and run in
the buffer system of Laemmli (5).
Sedimentation Velocity--
Sedimentation velocity runs were
performed at 20.0 °C and at various rotor speeds (finally using
40,000 rpm for F1-ATPase and 30,000 rpm for the
IF1-F1 complex) in a Beckman AN-60Ti rotor and
a Beckman XL-A ultracentrifuge. The samples were dissolved in buffer
consisting of 50 mM MOPS-NaOH, pH 6.5, 10 mM
magnesium sulfate, 1 mM EDTA, 0.02% sodium azide, and
0.001% phenylmethylsulfonyl fluoride. Scans were taken as frequently
as possible (i.e. with a zero-interval setting, which gives
~4.5 min with 3 cells in the rotor). Data were analyzed
initially by plotting g(s*) against s*, where
g(s*) is the fraction of material sedimenting between s* and (s* + Electron Microscopy--
Grids were prepared by evaporating
carbon onto 400-mesh copper/rubidium grids (Maxtaform, Graticules,
Tombridge, U.K.). They were covered by a thin film of a 0.5% Formvar
solution and then washed with chloroform. Grids were glow-discharged
for 30 s to make the carbon film hydrophilic. A solution of the
purified IF1-F1 complex (5 µl, 0.01 mg/ml)
was applied to the grid and left for 2 min. Then the grid was washed
with three drops of gel filtration buffer and stained with a solution
of 4% (w/v) methylamine tungstate (Agar, Stansted, U.K.) in water (pH
6.5).
Images were recorded at a magnification of 67,000 on a Philips Tecnai
12 electron microscope operating at 100 kV using low dose conditions
(approximately 10 e Apparent Molecular Masses of F1-ATPase and the
IF1-F1 Complex in Solution--
The apparent molecular
masses of F1-ATPase and IF1-F1
complex in solution were determined by gel filtration chromatography (Fig. 1) and sedimentation velocity
analysis (see Fig. 3). F1-ATPase eluted from a Superose 6 column as a single peak of 367 kDa, whereas under the same conditions,
the IF1-F1 complex eluted at 696 kDa. The small
shoulder contains monomeric F1-ATPase (Fig. 1A).
Fractions from both peaks were analyzed by SDS-PAGE (Fig.
2). The gels confirmed the presence of
IF1 (9.6 kDa) in the IF1-F1
complex. The separation of the F1 and
IF1-F1 complexes by gel filtration has been
observed before (11).
Sedimentation velocity data for F1-ATPase and the
IF1-F1 complex are summarized in Fig.
3. Direct dc/dt
analysis showed that F1 alone had
s20,w = 12.1 (± 0.7) S and
F1-IF1 complex had
s20,w = 17.0 (± 0.1) S. To test
whether there might be more than one component in the boundary, plots
of g(s*) against s*20,w
were also made. As a further test, model fitting of the
dc/dt data with models with either one or two
components was carried out. The best fits were given by the
single-component model for both data sets, and plots of the fit
residuals against radius are shown (Fig. 3). This increase in
sedimentation coefficient is compatible with IF1 binding
two F1-ATPase units in the complex, and this is the only
possibility, as any shape change that produced such an increase in
sedimentation would have led to a lower apparent molecular mass during
elution from the column. Thus all of the hydrodynamic data are only
explained by a dimerization of F1-ATPase on forming a
complex with IF1.
Electron Microscopic Examination of the IF1-F1
Complex--
Samples of the IF1-F1 were
diluted in the gel filtration buffer to a protein concentration of 0.01 mg/ml and stained with methylamine tungstate (4%, w/v). Methylamine
tungstate, which has a pH value of 6.5 in water solution, has been
reported to preserve delicate complexes such as viruses, and pH
6.0-6.5 is optimal for formation of the IF1-F1
complex (12).
Electron microscopic analysis of the samples (Fig.
4) revealed the presence of dimeric
F1 complexes. They represented about 70% of the particles
present in the fields that were examined, the other particles being
mostly monomeric F1-ATPase. As the starting material was
the main peak obtained by gel filtration of the
F1-IF1 complex, it was devoid of monomeric
F1-ATPase. Therefore, the monomers must have arisen by
disruption of the dimeric complex during the dilution and staining
procedures. A few particles appear to be trimeric. As there was only
evidence for dimer formation by gel filtration and sedimentation
analysis, these apparent trimers in the electron microscopy pictures
are unlikely to represent biologically significant assemblies. In a
control experiment, particles of F1 alone were examined in
the same way. About 94% of them were clearly monomeric.
Implications for Regulation and Quaternary Structure of ATP
Synthase--
Bovine IF1 exists in two oligomeric states
depending on the pH value. At low pH values, IF1 forms an
active dimer held together by an antiparallel
At present, it is not known whether IF1 can bring about the
dimerization of the F1F0-ATPase complex and
whether it is involved in formation of dimeric complexes of bovine and
yeast F1F0-ATPases that have been detected by
native gel electrophoresis in mild detergents (13, 15). The dimeric
yeast enzyme contains membrane bound subunits (e and g) that are not
present in the monomer, and they appear to be necessary for
dimerization to take place. However, the possible involvement of the
inhibitor protein (and of two other proteins known as 9 and 15K
proteins, which are both required for the action of the yeast
IF1) have not been investigated. It may be that dimeric
F1F0-ATP synthase forms specific supramolecular complexes with the respiratory complexes in the inner mitochondrial membrane (13).
-helical coiled-coil in its C-terminal region. This arrangement places the inhibitory N-terminal regions in opposition, implying that active dimeric IF1 can bind two
F1 domains simultaneously. Evidence of dimerization of
F1-ATPase by binding to IF1 is provided by gel
filtration chromatography, analytical ultracentrifugation, and electron
microscopy. At present, it is not known whether IF1 can
bring about the dimerization of the F1F0-ATPase complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
V0)/(Vt V0), where Vt and
Ve are the elution volume for the protein and
the total bed volume, respectively. A plot of
Kav versus log(molecular weight) with
standards gave a straight line with a correlation coefficient of 0.99. Linear regression gave the equation log(molecular weight) = 2.239 Kav + 3.427, which was used to calculate
the apparent molecular weight.
s*) (6, 7), using the
DCDT+ software package (Version 1.05) (8). This software was also used
for direct fitting of simple Gaussian functions to
dc/dt versus s curves (9)
to test for the number of components giving a "best fit" to the
data. Sedimentation coefficients were converted from s* to
s20,w, taking values of 0.759 ml/g
for the partial specific volume, 1.00213 g/ml for the solvent density,
and 1.0103 (entipoise) for the viscosity, calculated with
SEDNTERP (Version 1.03) (10). All plots were produced from the data
using the program ProFit (QuantumSoft). For plots of the original
scans, scans at equal time intervals were selected (630 s for
F1 alone and 666 s for
F1-IF1complex), to give visual separation
between the traces drawn.
/Å2). The quality of the
images was checked on an optical difractometer. The defocus was about
1000 nm. The images were collected with a Zeiss-SCAI scanner using a
step size of 7 µm (pixel size, 1.04 Å). Images were demagnified by
linear interpolation on the computer to a pixel size of 5.2 Å.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (7K):
[in a new window]
Fig. 1.
Gel filtration chromatography of
F1-ATPase and of the IF1-F1
complex. A and B, elution profiles of the
IF1-F1 complex and F1-ATPase,
respectively. C, separation of a mixture of the
IF1-F1 complex and F1-ATPase.
View larger version (100K):
[in a new window]
Fig. 2.
Analysis of column fractions by
SDS-PAGE. Panels A and B, fractions from
peaks A and B, respectively, in Fig. 1. The
apparent molecular masses of the standard proteins are: 83, 62, 47.5, 32.5, 25, and 16.5 kDa.
View larger version (28K):
[in a new window]
Fig. 3.
Sedimentation velocity analysis of
F1-ATPase and of the purified
IF1-F1 complex. The data are shown (at
equal time intervals for each sample) as A280
against radius for each sample. Analysis of the data is shown as plots
of g(s*) against s* (showing the distribution of
the sample with sedimentation coefficient) and also as the residuals
(in A280 against
s*20,w) for fitting of a model with a
single component, distributed as a Gaussian function, to the
dc/dt curves. Panels A and
B, F1-ATPase and IF1-F1
complex, respectively.
View larger version (123K):
[in a new window]
Fig. 4.
Electron microscopy of
IF1-F1 complexes in negative stain.
Samples were diluted at 0.01 mg/ml in gel filtration buffer and stained
with a solution of 4% (w/v) methylamine tungstate in water (pH
6.5).
-helical coiled-coil
from residues around 46-84
(3).2 In this arrangement,
the N-terminal inhibitory domains are exposed, and each is capable of
interacting with an F1-ATPase complex, as the results
presented here now demonstrate. It is thought that the interaction
between IF1 and F1 involves the C-terminal
region of one or more of the 
-subunits (14).
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ACKNOWLEDGEMENTS |
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We thank M. Montgomery for excellent technical assistance.
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FOOTNOTES |
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* 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 during part of this work by an European Molecular Biology Organization fellowship and by a Training and Mobility of Researchers Marie Curie research training grant from the European Community.
To whom correspondence should be addressed: The Medical
Research Council Dunn Human Nutrition Unit, Hills Road,
Cambridge CB2 2XY, U.K. Tel.: 0044-1223-252701; Fax:
0044-1223-252705; E-mail: walker@mrc-dunn.cam.ac.uk.
Published, JBC Papers in Press, July 28, 2000, DOI 10.1074/jbc.C000427200
2 D. J. Gordon-Smith, R. J. Carbajo, M. J. Runswick, J. E. Walker, and D. Neuhaus, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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| 1. | Walker, J. E. (1994) Curr. Opin. Struct. Biol. 4, 912-918 |
| 2. | Panchenko, M. V., and Vinogradov, A. D. (1985) FEBS Lett. 184, 226-230 |
| 3. | Cabezon, E., Butler, P. J. G., Runswick, M. J., and Walker, J. E. (2000) J. Biol. Chem. 275, 25460-25464 |
| 4. | Orris, G. L., Leslie, A. G. W., Braig, K., and Walker, J. E. (1998) Structure 6, 831-837 |
| 5. | Laemmli, U. K. (1970) Nature 227, 680-685 |
| 6. | Stafford, W. F., III (1992) Anal. Biochem. 203, 295-301 |
| 7. | Stafford, W. F., III (1994) Methods Enzymol. 240, 478-501 |
| 8. | Philo, J. S. (2000) Anal. Biochem. 279, 151-163 |
| 9. | Stafford, W. F., III (1997) Curr. Opin. Biotechnol. 8, 14-24 |
| 10. | Laue, T. M., Shah, B. D., Ridgeway, T. M., and Pelletier, S. L. (1992) in Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S. E. , Rowe, A. J. , and Horton, J. C., eds) , pp. 90-125, Royal Society of Chemistry, Cambridge, U.K. |
| 11. | Walker, J. E., Fearnley, I. M., Gay, N. J., Gibson, B. W., Northrop, F. D., Powell, S. J., Runswick, M. J., Saraste, M., and Tybulewicz, V. L. (1985) J. Mol. Biol. 184, 677-701 |
| 12. | Faberge, A. G., and Oliver, R. M. (1974) J. Microscopie (Paris) 20, 241-246 |
| 13. | Schagger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783 |
| 14. | Jackson, P. J., and Harris, D. A. (1988) FEBS Lett. 229, 224-228 |
| 15. | Arnold, I., Pfeiffer, K., Neupert, W., Stuart, R. A., and Schagger, H. (1998) EMBO J. 17, 7170-7178 |
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