Rotation of a gamma -epsilon Subunit Domain in the Escherichia coli F1F0-ATP Synthase Complex
THE gamma -epsilon SUBUNITS ARE ESSENTIALLY RANDOMLY DISTRIBUTED RELATIVE TO THE alpha 3beta 3delta DOMAIN IN THE INTACT COMPLEX*

(Received for publication, April 21, 1997)

Robert Aggeler , Isla Ogilvie and Roderick A. Capaldi Dagger

From the Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

A triple mutant of Escherichia coli F1F0-ATP synthase, alpha Q2C/alpha S411C/epsilon S108C, has been generated for studying movements of the gamma  and epsilon  subunits during functioning of the enzyme. It includes mutations that allow disulfide bond formation between the Cys at alpha 411 and both Cys-87 of gamma  and Cys-108 of epsilon , two covalent cross-links that block enzyme function (Aggeler, R., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 13888-13891). A cross-link is also generated between the Cys at alpha 2 and Cys-140 of the delta  subunit, which has no effect on functioning (Ogilvie, I., Aggeler, R., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 16652-16656). CuCl2 treatment of the mutant alpha Q2C/alpha S411C/epsilon S108C generated five major cross-linked products. These are alpha -gamma -delta , alpha -gamma , alpha -delta -epsilon , alpha -delta , and alpha -epsilon . The ratio of alpha -gamma -delta to the alpha -gamma product was close to 1:2, i.e. in one-third of the ECF1F0 molecules the gamma  subunit was attached to the alpha  subunit at which the delta  subunit is bound. Also, 20% of the epsilon  subunit was present as a alpha -delta -epsilon product. With regard to the delta  subunit, 30% was in the alpha -gamma -delta , 20% in the alpha -delta -epsilon , and 50% in the alpha -delta products when the cross-linking was done after incubation in ATP + MgCl2. The amounts of these three products were 40, 22, and 38%, respectively, in experiments where Cu2+ was added after preincubation in ATP + Mg2+ + azide. The delta  subunit is fixed to, and therefore identifies, one specific alpha  subunit (alpha delta ). A distribution of the gamma  and epsilon subunits, which is essentially random with respect to the alpha  subunits, can only be explained by rotation of gamma -epsilon relative to the alpha 3beta 3 domain in ECF1F0.


INTRODUCTION

F1F0-type ATPases are found in the plasma membrane of bacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts. These enzymes can both use a proton gradient to synthesize ATP and in the reverse direction hydrolyze ATP to establish a proton gradient for subsequent substrate and ion transport processes (1-3). The F1 part of the enzyme from Escherichia coli, ECF1F0, is composed of alpha , beta , gamma , delta , and epsilon  subunits in the stoichiometry 3:3:1:1:1. This part is linked by a narrow stalk to the F0 part that is composed of a, b, and c subunits in the stoichiometry 1:2:9-12 (3-6). The stalk contains a part of the gamma  and epsilon  subunits (6, 7). Two other subunits, delta  and b, are required for linkage of the F1 and F0 parts. These two subunits may provide a second separate connection (8, 9).

Electron microscopy (10) and, more recently, a high resolution structure of the mitochondrial F1 (11) have shown that the alpha  and beta  subunits alternate in a hexagonal arrangement around a central cavity. These two large subunits have a similar fold, each with three domains, an N-terminal beta  barrel domain on top and away from the F0, a middle nucleotide-binding domain, and a C-terminal alpha -helical domain (11). Three catalytic sites are present and located predominantly on beta  subunits. The other three nucleotide binding sites on the alpha  subunits appear to have mostly a structural role. A part of the gamma  subunit is found within the alpha 3beta 3 barrel and organized as two alpha  helices. A third short alpha  helical segment of this subunit was also resolved in the crystal structure of mitochondrial F1 (11). This lies under the C-terminal domain of one of the beta  subunits where it interacts with the so-called "DELSEED" region (the sequence of residues of this part).

The epsilon  subunit in ECF1 is a two-domain protein (12). The N-terminal 10-stranded beta  barrel region interacts with the gamma  subunit through the length of the stalk (7, 13) and with the c subunit oligomer at one end (14). The helix-loop-helix C-terminal domain of epsilon  lies under the alpha 3beta 3 subunit barrel and interacts with the DELSEED region (15) of a different beta  subunit from the one that binds to the short alpha  helix of gamma  (16).

There is now considerable evidence that F1F0-type ATPases are highly cooperative enzymes with all three catalytic sites involved (2, 17). This cooperativity of both ATP synthesis and ATP hydrolysis is currently best described by the alternating site model (18). In this model at any time during functioning, one catalytic site is involved in the bond cleavage reaction and is essentially closed; a second is opening to release the tightly bound product, and the third is closing to trap the substrate.

An important tenet of the binding change mechanism is that catalytic sites are sequentially linked to the proton channel for energy coupling by a rotation of the small subunits. Early evidence for such rotation came from cryoelectron microscopy studies on ECF1 that showed the gamma  subunit distributed at all three beta  subunits rather than fixed at one site (19, 20). Consistent with the idea of rotation of gamma  within the alpha 3beta 3 domain, cross-linking of this subunit to alpha  or beta  subunits was found to fully inhibit the functioning of ECF1 (16, 21).

Additional evidence for rotation of the gamma  subunit relative to the alpha 3beta 3 domain has been provided more recently by Duncan et al. (22). These authors isolated a complex containing a (unlabeled) beta  and gamma  subunit, stably linked by a disulfide bond between Cys-87 of gamma  and a Cys introduced at position 380 of beta . They mixed this beta -gamma complex with a 35S-labeled beta  subunit along with alpha , delta , and epsilon  subunits to regenerate a functional F1. This reconstituted enzyme was then shown to undergo subunit switching when the disulfide bond was broken and MgCl2 + ATP was added, i.e. the gamma  moved from unlabeled to labeled beta subunits. Sabbert et al. (23) have also provided evidence of rotation of the gamma  subunit in chloroplast F1. Finally, rotation of the gamma  subunit in the alpha 3beta 3gamma subcomplex of TF1 has been visualized directly in recent elegant single molecule studies (24).

However, all of the above studies have focused on the movements of the gamma  subunit in F1, and in terms of functional relevance it remains important to show that rotation of the gamma  subunit in conjunction with other subunits (e.g. the epsilon  subunit) occurs in the intact F1F0. Also, for an understanding of the coupling mechanisms, it is necessary to establish which subunits are moving with the gamma  subunit and which are fixed with respect to the alpha 3beta 3 subdomain. One attempt to examine rotation in ECF1F0 has been reported recently by Cross and colleagues (25) using the same cross-linking and reconstitution approach they used before for F1. They showed an ATP-driven and dicyclohexylcarbodiimide-sensitive scrambling of gamma  relative to beta  subunits, although the level of this scrambling was lower than would be expected if all enzyme molecules were active.

We have previously observed that disulfide bonds can be formed from an alpha  subunit via a Cys at position 2 to the delta  subunit (in the mutant alpha Q2C (26)) and via a Cys at residue 411 to the gamma  and epsilon  subunits (in the mutant alpha S411C/epsilon S108C (21)). Here, we report cross-linking studies with ECF1F0 from the mutant alpha Q2C/alpha S411C/epsilon S108C where cross-linking from the alpha  subunit to all three small subunits can be obtained at the same time. The combinations of cross-linked products obtained provide information about which subunits have to be moving in the ATP synthase and which do not.


EXPERIMENTAL PROCEDURES

Construction of Mutants and Isolation of Enzymes

The triple mutant pRA170 (alpha Q2C/alpha S411C/epsilon S108C) was obtained by ligating the 5.8-kilobase XhoI/NsiI fragment of pRA140 (21), which contains the mutations alpha S411C and epsilon S108C, to the 6.8-kilobase XhoI/NsiI fragment of pIO1 (26), which contains the mutation alpha Q2C. Mutant pRA141 (alpha S411C) was created by ligation of the 2.9-kilobase SstI/NsiI fragment of pRA100 (27) with the 9.7-kilobase SstI/NsiI fragment of pRA140. We have recently found that both ECF1 and ECF1F0 can be obtained in purer form by using E. coli strains that do not make cytochrome bo (28). For this reason pRA170 was incorporated in RA1, a unc-/cyo- E. coli strain that was created by P1 transduction of Delta (uncB-uncC)ilv::Tn10 from DK8 (29) to GO104 (F-, thi, rpsL, Delta cyo::Kan). GO104 was a kind gift from Dr. Robert Gennis, University of Illinois. Successful transduction was shown by Southern blotting (30). ECF1 and ECF1F0 were isolated as described by Gogol et al. (31) and Aggeler et al. (32), respectively.

Other Methods

ECF1F0 was reconstituted in egg lecithin on a Sephadex G-50 column (medium, 1.5 × 60 cm) in 50 mM Tris, pH 7.5, 2 mM MgCl2, 2 mM DTT1 and 10% glycerol, and cross-linking of the enzyme was carried out with CuCl2 in 50 mM MOPS, pH 7.0, 2 mM MgCl2, 2 mM ATP, 10% glycerol as described by Aggeler et al. (16). Cross-linked products were separated by electrophoresis on SDS-containing polyacrylamide gels according to Laemmli (33). Two-dimensional SDS-polyacrylamide gel electrophoresis was carried out by resolving cross-linked products in a first dimension without prior treatment with reducing agents on an 8% polyacrylamide gel. A portion of a lane was cut out and exposed to 50 mM DTT in dissociation buffer for 2 h at room temperature. The gel piece was rotated 90° and positioned with agarose on a stacking gel of a 10-18% polyacrylamide gel for the second dimension. Protein concentration was determined with the BCA protein assay from Pierce. Gels were stained with Coomassie Brilliant Blue R (34). Cross-linked products were identified with Western blotting, using monoclonal antibodies against F1 subunits (35).


RESULTS

The experiments described here utilize the mutant alpha Q2C/alpha S411C/epsilon S108C. The ATP hydrolysis rates of this mutant were in the range of 25-30 µmol of ATP hydrolyzed per min per mg, which is the same as for wild-type enzyme. Also, the ECF1F0 displayed efficient proton pumping activity as determined by ATP-dependent 9-amino-6-chloro-2-methoxyacridine fluorescence quenching in inner membranes (result not shown). Therefore, the introduction of three different mutations did not disrupt the functioning of the enzyme. Previous work with ECF1 and ECF1F0 from the mutant alpha S411C/epsilon S108C has shown that CuCl2 treatment induces cross-linking between the Cys at 411 of one alpha  subunit and the intrinsic Cys-87 of the gamma  subunit and between the Cys-411 of a second alpha  subunit and Cys-108 of epsilon  (21). Cross-linking of alpha  to gamma , as obtained in an alpha S411C mutant, or of alpha  to epsilon  obtained at low CuCl2 concentrations in the alpha S411C/epsilon S108C mutant fully inhibited ATPase activity. Studies using the ECF1 and ECF1F0 from the mutant alpha Q2C have shown that CuCl2 causes full yield cross-linking between an alpha  and the delta  subunit involving the Cys at position 2 of alpha  and the two intrinsic Cys residues of delta , i.e. Cys-64 and Cys-140. The cross-linking of alpha  to delta  is without significant effect on either ATPase activity or ATP-coupled proton translocation as measured by the 9-amino-6-chloro-2-methoxyacridine fluorescence quenching method (26).

The effect of CuCl2 treatment on ECF1F0 isolated from the triple mutant alpha Q2C/alpha S411C/epsilon S108C is shown in Fig. 1. These experiments were conducted using 50 or 200 µM CuCl2 with incubation for 1 h at room temperature. All of the subunits of the complex are resolved in Fig. 1A. The delta  and epsilon  subunits are each essentially missing, and the gamma  subunit is much reduced in the gel, having been cross-linked with the alpha  subunit. The band of the alpha  subunit is also greatly reduced (by around 85%). In Fig. 1B, the cross-linked products are optimally resolved. Each was identified by immunoblotting with monoclonal antibodies against each of the subunits. Five main cross-linked products were generated; in order of decreasing apparent molecular weight, these are alpha -delta -gamma , alpha -gamma , alpha -delta -epsilon , alpha -delta , and alpha -epsilon , respectively. Other cross-linked products were obtained but only in small amounts. These include an alpha -alpha product that has also been resolved after cross-linking of the alpha Q2C mutant and probably involves a disulfide bridge from Cys-2 of one alpha  and Cys-90 of a second alpha  subunit.


Fig. 1. CuCl2-induced cross-linking of ECF1F0 from alpha Q2C/alpha S411C/epsilon S108C. ECF1F0, reconstituted into egg lecithin vesicles, was centrifuged twice in 50 mM MOPS, pH 7.0, 2 mM MgCl2, and 10% glycerol to remove DTT before 2 mM ATP was added, and the enzyme (at 0.45 mg/ml) was incubated for 10 min at room temperature. A, 240-µl aliquots were supplemented with 0 (lane 1), 50 (lanes 2 and 3), and 200 µM CuCl2 (lanes 4 and 5) and incubated at room temperature for 1 h. After addition of 7 mM EDTA, 120-µl aliquots were either treated with 25 mM DTT for 2 h (lanes 3 and 5) or not (lanes 2 and 4). 60 mM N-ethylmaleimide was added and followed by dissociation buffer without reducing agent. 53-µg samples were loaded on 10-18% polyacrylamide gels. B, 120-µl aliquots were treated as above, and 26 µg of protein was loaded on a 8% polyacrylamide gel. The gels in panels A and B are stained with Coomassie Brilliant Blue. C, 53 µg of cross-linked protein as in lane 2 was applied on a 5-cm wide lane on a 8% polyacrylamide gel. After transfer onto a nitrocellulose membrane monoclonal antibodies against the F1 subunits were used (delta  is shown).
[View Larger Version of this Image (40K GIF file)]

In several different experiments the cross-linked products involving the delta  subunit, i.e. alpha -delta -gamma , alpha -delta -epsilon , and alpha -delta , appeared to be in almost equal amounts based on immunostaining of the Western blots with the anti-delta monoclonal antibody (e.g. Fig. 1C). To better quantitate the yields of these cross-linked products, CuCl2-treated enzyme was subjected to two-dimensional SDS-polyacrylamide gel electrophoresis with separation in the first dimension in the absence of DTT and in the second dimension in the presence of DTT. A typical result is shown in Fig. 2A. It is clear from these data that the amounts of gamma , delta , and epsilon  not in the major products identified in the figure are very small, i.e. less than 5% for each subunit. A scan of the three peaks of delta  subunit released from alpha -delta -gamma , alpha -delta -epsilon , and alpha -delta products by DTT dissociation is shown in Fig. 2B. The relative percentage of these three peaks was 30, 20, and 50% (averages of two experiments), respectively. Similar quantitation of percentages of the gamma  subunit in the alpha -delta -gamma and alpha -gamma bands gave 31 and 69%, respectively, and for the epsilon  subunit in the alpha -delta -epsilon and alpha -epsilon gave 19 and 81%, respectively. The experiment in Fig. 2 involved cross-linking of ECF1F0 after preincubation in MgCl2 + ATP. When cross-linking was performed after preincubation in MgCl2 + NaN3 + ATP the distribution of alpha -delta -gamma , alpha -delta -epsilon , and alpha -delta was 40.3 ± 1.0, 21.7 ± 0.9, and 38.0 ± 0.2% (averages of three experiments), respectively.


Fig. 2. Resolution and quantitation of subunits involved in CuCl2 induced cross-link of ECF1F0 from alpha Q2C/alpha S411C/epsilon S108C by two-dimensional SDS-polyacrylamide gel electrophoresis. ECF1F0 in egg lecithin vesicles in 50 mM MOPS, 2 mM MgCl2, 2 mM ATP, and 10% glycerol was cross-linked at a concentration of 0.46 mg/ml with 100 µM CuCl2 for 1 h at room temperature. The reaction was stopped with 7 mM EDTA, and 20 mM N-ethylmaleimide was added before the dissociation buffer without reducing agent. For the first dimension, 70 µg of protein was applied on a 8% polyacrylamide gel. After electrophoresis the top 12.5 cm of the 18-cm long resolving gel of a lane was cut out, soaked in 12 ml of dissociation buffer with 50 mM DTT for 2 h, and placed on a 10-18% polyacrylamide gel for the second dimension. A, the bands were visualized with Coomassie Brilliant Blue. B, relative intensities were determined by scanning the gel with Adobe Photoshop and NIH Image. The cross-linked products giving rise to the peaks are indicated.
[View Larger Version of this Image (38K GIF file)]


DISCUSSION

The question of whether there is rotation of the gamma  subunit between the three alpha -beta pairs during the functioning of F1 has been convincingly answered as reviewed in the Introduction. ATP hydrolysis clearly drives a movement of the gamma  subunit such that it visits all three alpha -beta subunit pairs during cooperative catalysis (23, 24). A priori, this rotational movement could be an artifact of a freedom of the gamma  subunit that is allowed only when the F1 is dissociated from the F0. However, the results of Zhou et al. (25) and the data presented in this study are evidence that this is not the case. The key observations here are that in ECF1F0 from the mutant alpha Q2C/alpha S411C/epsilon S108C there is cross-linking of gamma and epsilon  subunits each separately to the same alpha  subunit that binds the delta  subunit (Fig. 3). However, the gamma  and epsilon  subunits are never bound to the same alpha  subunit. The significance of these results is clear when the activity effects of cross-linking of the gamma , delta , or epsilon  subunits to alpha  subunits are considered. Covalent cross-linking of the delta  to an alpha  subunit has been found to have little or no effect on either cooperative ATP hydrolysis or on the proton pumping function of ECF1F0 (26). This is in contrast to the cross-linking of gamma  or epsilon  to alpha  subunits, which completely blocks functioning (21). The conclusion from these activity data is that movements of gamma and epsilon  but not delta  are an essential part of the functioning of the enzyme. It follows that the delta  subunit must be fixed with its interaction thereby identifying one of the three alpha  subunits (alpha delta ) that can be visited by both gamma  and epsilon  since both alpha -delta -gamma and alpha -delta -epsilon cross-linked products were obtained.


Fig. 3. Rotation of gamma  and epsilon  in ECF1F0. The gamma  and epsilon  subunits (shaded) rotate relative to delta  subunit. Cysteines involved in cross-links were either introduced by site-directed mutagenesis at positions 2 and 411 of the alpha  subunit and 108 of the epsilon  subunit or were endogenous in positions 87 of gamma  and 140 of delta .
[View Larger Version of this Image (14K GIF file)]

The distribution of cross-linked products observed is understandable when it is considered that the experiments reported here involve a population of ECF1F0 molecules that are not synchronized. Thus, at any time during enzyme turnover after ATP hydrolysis has stopped, around one-third of any rotating subunits should be at each of the three alpha -beta pairs. This is approximately the observed distribution of both gamma  and epsilon  subunits in our experiments whether enzyme activity was ended by substrate depletion or by addition of azide. In previous studies it was shown that cross-linking of epsilon  to the gamma  subunit did not block activity (7, 27), indicating that these two subunits move together as a mobile domain. In electron microscopy studies (19), we have seen movements of the gamma  subunits relative to epsilon , but this is because the antibody fragments to alpha and epsilon  used to tag specific subunits release epsilon  from gamma  so that it is fixed at one alpha -beta subunit pair.

In summary, the scrambling of gamma  and epsilon  subunits with respect to the three alpha  subunits, one of which is clearly distinguished by interaction of the delta  subunit, is evidence for rotational movements of the main stalk forming subunits in ECF1F0. The only other explanation of the data, that ECF1F0 assembles with a fixed random distribution of the small subunits, does not seem tenable. The gamma  and epsilon  subunits appear to move as one domain, although there may be small movements of the two relative to one another as part of the coupling between catalytic sites and proton channel functioning (6).

For rotational movements of the gamma  and epsilon  subunits to occur within ECF1F0, the alpha 3beta 3 domain must be fixed relative to the F0 part by a stator. Recent evidence suggests that this stator is contributed by the delta  with the b subunits (9, 26, 36). For coupling, ATP hydrolysis-driven movements of the gamma -epsilon domain must be linked to proton translocation. It has been established that both the gamma  and epsilon  (Refs. 7 and 14, respectively) interact directly with the c subunit oligomer of the F0 subunit. The covalent cross-linking of gamma  or epsilon  to the c subunit ring does not block ATP hydrolysis (7, 14), which implies that the rotatory element in ECF1F0 is a gamma -epsilon -c oligomer domain moving relative to the alpha 3-beta 3-delta -a-b2 complex.


FOOTNOTES

*   This research was supported by National Institutes of Health Grant HL 24526.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.
Dagger    To whom correspondence should be addressed. Tel.: 541-346-5881; Fax: 541-346-4854.
1   The abbreviations used are: DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid.

ACKNOWLEDGEMENT

The excellent technical assistance of Kathy Chicas-Cruz is gratefully acknowledged.


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