From the Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, D-49069 Osnabrück, Germany
Received for publication, February 26, 2003 , and in revised form, April 29, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During ATP synthesis, the rotation of subunits and
is driven
by proton translocation through F0. Both subunits are known to
contact the subunit c oligomer at the cytoplasmic surface of
F0 (2), and during
coupled catalysis, a
c10 subcomplex rotates
relative to the remainder of the F0F1 complex driven by
successive protonation/deprotonation of amino acid residue cAsp-61
located in the second transmembrane helix of subunit c
(3,
5). Because of the rotational
movement of the central stalk, a second stalk is necessary for the
stabilization of the F0F1 complex, which is built up at
least of the two copies of subunit b
(6,
7). During rotational
catalysis, elastic torque is generated, which is thought to be stored in the
two stalks, and as a consequence drives ATP synthesis in F1 or
proton pumping in F0. Good candidates for elastic deformation are
the intertwined helices of subunit
as a torsional spring and the
parallel helices of the subunit b dimer topped by subunit
and
bottomed by subunit a serving as a parallelogram-like spring
(3).
Dividing the subunits of the ATP synthase into structural elements of rotor
and stator, there is general agreement that the
3
3 complex and subunit
of
F1 as well as subunits a and b of F0
belong to the stator (1,
3). Thereby, the subunit
b dimer was shown to be a prerequisite for the binding of
F1 to F0, and close proximity between subunit b
and subunits
,
, and
of F1 was very well
documented arguing in favor of direct interactions
(8). Within the F0
complex, subunits a and b are located outside the ring-like
subunit c oligomer (9)
and cross-linking between both subunits has been observed but mostly without
defining the contact sites (2,
1013).
In this study, the purification of a stable ab2 subcomplex isolated by Ni-NTA1 affinity chromatography after addition of a His6 tag to the N terminus of subunit a is described, clearly demonstrating strong interactions between both F0 subunits without further manipulation. The ab2 subcomplex was reconstituted together with purified subunit c into phospholipid vesicles and exhibited passive proton translocation rates and DCCD-sensitive coupled ATPase activities after rebinding of F1 comparable with those of wild type F0.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparative ProceduresF1, F0, and subunit c were purified as described previously (1820). Proteoliposomes were prepared according to Okamoto et al. (21) with the following modifications. E. coli lipids (Avanti Pro Lipid) present in chloroform were dried under a stream of argon and redissolved in buffer by sonication at a concentration of 30 mg/ml. The weight ratio of phospholipid to protein was 1:266. Dialysis was carried out at 4 °C for 40 h changing the buffer once. For reconstitution of subunit c present in chloroform/methanol/H2O 5:5:1, the protein was added to E. coli lipids prior to the removal of the organic solvent. K+-loading of proteoliposomes was carried out as described previously (22) with the following modifications. Proteoliposomes (usually 180 µl) and 0.45 M K2SO4 in 66 mM sodium phosphate buffer, pH 7.0, were mixed in a ratio of 2:1, and the sonication/freezing (liquid nitrogen)/thawing cycle was repeated twice.
Purification of ab2
SubcomplexFor purification of the ab2
subcomplex, everted membrane vesicles were prepared at 4 °C as described
by Schneider and Altendorf
(19) using 0.1 mM
TES/NaOH, pH 7.0, 40 mM -amino-n-caproic acid, 250
mM sucrose, 0.25 mM EGTA, 20 mM magnesium
acetate, 5 mM p-aminobenzamidine, and 1 mM
dithiothreitol as buffer system
(23). For the removal of
F1, membranes were washed once with each of the buffers described
previously (23). Subsequently,
membranes were incubated overnight in 1 mM Tris/HCl, pH 7.5, 0.5
mM EDTA, and 10% (v/v) glycerol, collected by centrifugation, and
washed twice with 0.1% (w/v) sodium deoxycholate
(19) before resuspension in 50
mM Tris/HCl, pH 8.0, and 10% (v/v) glycerol.
For solubilization, membranes (10 mg/ml) were stirred with 0.8% (w/v)
n-dodecyl--D-maltoside (DM) (Anatrace) for 12
h on ice. After centrifugation, 150 mM NaCl and 10 mM
imidazole were added to the supernatant prior to incubation with
Ni-NTA-agarose (Qiagen) equilibrated in 50 mM Tris/HCl, pH 8.0, 150
mM NaCl, 10% (v/v) glycerol, 0.1 mM phenylmethylsulfonyl
fluoride, 0.05% (w/v) DM, and 10 mM imidazole. Supernatant and
Ni-NTA-agarose were mixed in amounts corresponding to 915 mg of
membrane protein/ml gel matrix and incubated by end-over-end shaking for
12 h at 4 °C. Subsequently, the loaded gel matrix was packed into a
column and washed with 10 volumes of buffer and with 10 volumes of the same
buffer containing 20 mM imidazole. To allow subsequent ammonium
sulfate precipitation of the protein and reconstitution into liposomes by
dialysis, the detergent was exchanged by washing the column with 10 volumes of
50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 10% (v/v) glycerol,
0.1 mM phenylmethylsulfonyl fluoride, 1% (w/v) sodium cholate, and
20 mM imidazole. Finally, the ab2 subcomplex
was eluted with the same buffer containing 100 mM imidazole,
precipitated with 35% (w/v) ammonium sulfate
(19), and resuspended in the
cholate-containing buffer without imidazole.
AssaysProtein concentrations were determined with the BCA assay (Pierce) used as recommended by the supplier. Proteins were separated by SDS-PAGE and detected by silver staining (17). Densitometric analyses of band intensities of silver-stained SDS-gels were performed with ImageQuant (Amersham Biosciences). Immunoblotting was performed according to Birkenhäger et al. (24). Passive proton translocation rates were measured (25) using 2 µM valinomycin for induction of the diffusion potential, and the initial rates were calculated according to Dmitriev et al. (26). DCCD-sensitive ATPase activities of F1-reconstituted proteoliposomes were determined as described previously (27).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Reconstitution of His Tag-modified F0
ComplexesIn both cases, the a N-His6 and
the b N-His6 construct, solubilization of membranes as
described by Schneider and Altendorf
(19) allowed the isolation of
F0 complexes comparable with wild type (data not shown). The
F0 complexes were incorporated into liposomes prepared from E.
coli lipids and assayed for passive proton translocation by imposing a
K+/valinomycin diffusion potential. The resulting initial rates of
H+ uptake were comparable for F0 complexes prepared from
both the a N-His6 construct and the wild type strain,
whereas the net initial rate of F0 isolated from membranes
containing b N-His6 amounts only to 15% of that value
(Table I, compare
values in parentheses). In addition, after the binding of
F1 to F0-containing proteoliposomes, DCCD-sensitive
coupled ATPase activity was again comparable for a N-His6
and wild type while b N-His6 exhibited only 6070%
ATPase activity (Table I). The
results obtained indicate that the additional histidine residues at the N
terminus of subunit a have no influence on the function of
F0 in proton translocation and F1 binding. However, an
addition of histidine residues to the N terminus of subunit b affects
at least the reconstitution of the protein complex into phospholipid vesicles,
since growth of cells and ATPase activities in native membranes remained
unchanged.
|
Purification of ab2 SubcomplexIncubation of F1-depleted everted membrane vesicles of the a N-His6 construct with 0.8% DM resulted in solubilization of >90% of the F0 subunits present. Surprisingly, the use of Ni-NTA affinity chromatography allowed the purification of an ab2 subcomplex instead of single subunit a carrying the His tag, whereas subunit c was completely removed during the washing steps prior to elution of the protein with 100 mM imidazole (Fig. 2). The protein complex obtained is homogeneous with the exception of a protein with an apparent molecular mass of 15,000 present in low variable amounts (Fig. 2), which could be identified by immunoblot analysis as a degradation product of subunit b (Fig. 1B, compare lane 6). The quantification of band intensities of subunits a and b in silver-stained SDS gels at different protein concentrations revealed almost identical densitometric proportions in both wild type F0 and the purified subcomplex (data not shown). Therefore, the stoichiometry of both proteins remains unchanged during the purification process, resulting in the isolation of an ab2 subcomplex.
Reconstitution of F0 SubcomplexesFor the reconstitution of an F0 complex, functional in both passive proton translocation and coupled ATPase activity after binding of F1, all of the three F0 subunits are necessary (17, 19, 28). Therefore, also for the ab2 subcomplex, passive proton translocation (Fig. 3) and functional binding of F1 (Table II) could only be observed after co-reconstitution with subunit c, whereas the rates measured either for reconstituted ab2 subcomplex or subunit c alone (even in the presence of a 6-fold excess) were comparable with those of plain liposomes.
|
|
To obtain maximum rates of proton translocation and ATPase activity, the amounts of subunit c necessary for co-reconstitution are higher as calculated (Table II and Fig. 3). Therefore, co-reconstitution was performed with varying amounts of subunit c. A possible explanation might be that subunit c is present in different conformations after chloroform/methanol extraction and that it cannot completely refold after incorporation into the lipid environment or during assembly with the other F0 subunits. A second explanation might be that subunit c is integrated into the lipid bilayer in different orientations so that only half of the protein is suitable for reconstitution of F0. Furthermore, the relatively low amount of ab2 subcomplex present in the proteoliposomes can only be saturated with subunit c in the presence of an excess of proteolipid. In addition, because of the extreme hydrophobicity of subunit c, a correct determination of the protein concentration is generally difficult.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A first hint for a possible interaction between both subunits has been indicated years ago by the mutation of aPro-240 to alanine or leucine, which suppresses the effects observed by mutation bG9D (29). However, the evaluation of second site suppressors can be delusive without an additional approach demonstrating direct interaction, because mutations can cause long distance conformational changes (30). Early data on chemical cross-linking of F0F1 only indicated the proximity of subunits a and b without defining possible contact sites (11, 12), whereas recently, cross-linking within region bPro-28 to bGly-43 of cysteine-substituted subunit b with subunit a was demonstrated (10, 13).2 However, the counterparts in subunit a remained unidentified. In addition, in a review, a cross-link between the N terminus of subunit b (bN2C) and residues aG227C or aL228C of subunit a has been reported previously (2). The formation of the ab2 subcomplex isolated in this study is not triggered by any cross-linking reagent and therefore reflects subunit interactions occurring within the F0 complex in vivo. Nevertheless, a determination of distinct contact sites between subunits a and b is still of great interest.
Studies on solubilization and purification of F0 allowed the isolation of several F0 subcomplexes in addition to single subunits (17, 19, 28). In each case, after the addition of the missing subunit(s), the subcomplexes can be co-reconstituted to form an F0 complex functional in proton translocation and F1 binding. The strong interaction between subunits a and b as part of the stator has been demonstrated in this study. The formation of a stable ab2 subcomplex seems reasonable, because both subunits belong to the stator part of the F0 complex and are supposed to withstand mechanical torque built up during rotational catalysis. In addition, subunits a and c cooperatively catalyze proton translocation during ATP synthesis/hydrolysis (3, 5, 31). The interaction of both subunits was shown by purification of a stable ac10 subcomplex (19) as well as by cross-linking between subunit a and at least one copy of subunit c, revealing that the C-terminal helix of subunit c and the penultimate helix of subunit a pack close enough to interact during functional catalysis (5, 32). However, the isolation of a subcomplex consisting of subunits b and c has not yet been achieved, although disulfide bond formation between the N terminus of subunit b (bN2C) and the C terminus of subunit c (cV78C) indicate a close proximity of both subunits (33) arguing for only a reasonably weak interaction between stator and rotor at the subunit bc interface (see Ref. 10).
![]() |
FOOTNOTES |
---|
To whom correspondence should be addressed: Universität Osnabrück,
Abteilung Mikrobiologie, D-49069 Osnabrück, Germany. Tel.:
49-541-969-2809; Fax: 49-541-969-2870; E-mail:
deckers-hebestreit{at}biologie.uni-osnabrueck.de.
1 The abbreviations used are: Ni-NTA, nickel-nitrilotriacetic acid;
a N-His6, His6 tag introduced at the N terminus
of subunit a; a C-His5, His5 tag at
the C terminus of subunit a; b N-His6,
His6 tag at the N terminus of subunit b; DCCD,
N,N'-dicyclohexylcarbodiimide; DM,
n-dodecyl--D-maltoside; TES,
N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid.
2 S. Konrad, J.-C. Greie, K. Altendorf, and G. Deckers-Hebestreit,
unpublished results.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|