Defining the Domain of Binding of F1 Subunit epsilon  with the Polar Loop of F0 Subunit c in the Escherichia coli ATP Synthase*

Joe Hermolin, Oleg Y. Dmitriev, Ying Zhang, and Robert H. FillingameDagger

From the Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously shown that the E31C-substituted epsilon  subunit of F1 can be cross-linked by disulfide bond formation to the Q42C-substituted c subunit of F0 in the Escherichia coli F1F0-ATP synthase complex (Zhang, Y., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 24609-24614). The interactions of subunits epsilon  and c are thought to be central to the coupling of H+ transport through F0 to ATP synthesis in F1. To further define the domains of interaction, we have introduced additional Cys into subunit epsilon  and subunit c and tested for cross-link formation following sulfhydryl oxidation. The results show that Cys, in a continuous stretch of residues 26-33 in subunit epsilon , can be cross-linked to Cys at positions 40, 42, and 44 in the polar loop region of subunit c. The results are interpreted, and the subunit interaction is modeled using the NMR and x-ray diffraction structures of the monomeric subunits together with information on the packing arrangement of subunit c in a ring of 12 subunits. In the model, residues 26-33 form a turn of antiparallel beta -sheet which packs between the polar loop regions of adjacent subunit c at the cytoplasmic surface of the c12 oligomer.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The H+-transporting, F1F0-ATP synthase of Escherichia coli utilizes an H+ electrochemical gradient to drive ATP synthesis during oxidative phosphorylation (1). Similar enzymes are found in mitochondria, chloroplasts, and other bacteria. The enzymes are composed of two sectors, termed F1 and F0. The F1 sector contains the catalytic sites for ATP synthesis, and when released from membrane, it shows ATPase activity. The F0 sector traverses the membrane and functions as the H+ transporter. When F1 is bound to F0, the complex acts as a reversible, H+-transporting ATP synthase or ATPase. In E. coli, F1 is composed of five types of subunits in an alpha 3beta 3gamma delta epsilon stoichiometry, and F0 is composed of three types of subunits in an a1b2c12 stoichiometry (2-4). The structure of much of the alpha 3beta 3gamma portion of F1 has been solved by x-ray diffraction analysis and shows subunit gamma  extending through the center of a hexamer of the larger, alternating alpha  and beta  subunits (5, 6). During catalysis, the gamma  and epsilon  subunits have been shown to rotate in 120° steps between the three alternating catalytic sites in the beta  subunits (7-13). Subunits gamma  and epsilon  are thought to rotate as a unit because they can be cross-linked to each other with minimal inhibitory effects on ATPase activity (14, 15).

The relation of structure and mechanism in F0 is less thoroughly understood. The largely hydrophobic subunit a folds in the membrane with five transmembrane helices (16, 17), at least two of which likely interact with subunit c during proton transport (18-20). Subunit b is anchored in the membrane via a single transmembrane helix that is connected to a polar, elongated cytoplasmic domain that is thought to play a key role in fixing F1 to F0 (21). Subunit c is a protein of 79-amino acid residues that folds in the membrane in a hairpin-like structure. The two hydrophobic transmembrane alpha -helices are joined by a more polar loop region that is exposed to the F1 binding side of the membrane. Aspartyl 61, lying at the center of transmembrane helix-2, is thought to be the site of H+ binding during transport (22). The polar loop was proposed to play a key role in coupling H+ transport to ATP synthesis or hydrolysis based upon the uncoupled phenotypes of mutants with substitutions in the conserved Arg41-Gln42-Pro43 sequence of the polar loop (22, 23). The "uncoupled" phenotype of the cQ42E mutant proved to be suppressed by second site substitutions in Glu31 of F1 subunit epsilon  (24), and this led to cross-linking studies demonstrating a physical proximity between the polar loop and subunits gamma  and epsilon  of F1 (14, 25, 26). A recently determined NMR structure of monomeric subunit c conforms well with folding predictions made from biochemical and genetic analysis (27).

Recent experiments now indicate that the c12 oligomer of F0 is organized in a ring with transmembrane helix-1 on the inside and transmembrane helix-2 on the outside (2, 4, 28) and with the a and b subunits associating at the periphery of the ring (2, 20). Such an arrangement is consistent with low resolution electron and atomic force microscopic images (29-31). The structural data fit well with rotary models where H+ transport at the a-c interface is proposed to drive rotation of the oligomeric c ring as the Asp61 carboxylate is protonated and deprotonated from alternate access channels on each side of the membrane (10, 32-34). The rotation of subunit c is proposed to drive the rotation of the gamma epsilon unit in F1 via a fixed linkage between subunit c and the gamma  and epsilon  subunits (14) although other explanations have been proposed (22, 25). The elongated cytoplasmic domain of subunit b is thought to extend from the membrane surface to the top of F1 as a "second stalk", or stator, to hold F1 fixed as subunit gamma  rotates at the center of the molecule (21, 35-37).

In this study, the interacting regions of the polar loop of subunits c and subunit epsilon  are more thoroughly defined by disulfide cross-linking of Cys introduced into the two subunits. The experimental design and interpretation was aided by a structural model for subunit epsilon  derived by NMR (38) and x-ray crystallography (39). The Cys residues of subunit epsilon  that form cross-links with subunit c localize to a span of residues 26-33 which fold as two strands of antiparallel beta -sheet connected by a loop. A structural model for the subunit-subunit interaction is developed from the structures of the monomeric subunits and information on the organization of the c-oligomer, using distance constraints from the cross-linking data reported here. The model indicates that the segment of antiparallel beta -sheet encompassing residues 26-33 of subunit epsilon  packs in a space between neighboring polar loops of the subunit c oligomer.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutant Construction and Expression-- The plasmids constructed in this study are derivatives of plasmid pYZ201 (25), which carries the eight structural genes of unc operon coding F1F0 (bases 870-10172; Ref. 40).1 The Cys substitutions in subunit c and subunit epsilon  were introduced by oligonucleotide-directed mutagenesis using the strategy described previously (25). The plasmids were expressed in strain OM204 (41), a strain in which the unc operon is deleted from the chromosome.

Membrane Preparations and Cross-link Analysis-- Cells were grown and membranes prepared by the methods described (25). To catalyze disulfide bond formation, aliquots of 200-µl membrane vesicles at 10 mg/ml in TMG buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 10% (v/v) glycerol) were treated with 20 µl of a mixture of 15 mM CuSO4 and 45 mM 1,10-phenanthroline in 50% ethanol. After a 1-h incubation at room temperature, the reaction was stopped by addition of 20 µl of 0.5 M Na2EDTA and 20 µl of 0.5 M N-ethylmaleimide (NEM)2 in dimethyl sulfoxide, and the sample was incubated for a further 60 min. The sample was then diluted with 230 µl of 2× SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 0.02% bromphenol blue) and incubated at 30 °C for 1 h. The solubilized membrane proteins were separated by SDS-polyacrylamide gel electrophoresis using a 7.5-15% acrylamide gradient and the Tris-Tricine buffer described by Schägger and von Jagow (42). After electrophoretic transfer to nitrocellulose paper (43), immunostaining was carried out using the Enhanced Chemiluminescence System (Amersham Pharmacia Biotech). The rabbit antiserum to subunit c used was that described by Girvin et al. (44). Antibodies that nonspecifically cross-reacted with E. coli membrane proteins were removed by preabsorption with membranes prepared from a mutant strain with a deleted unc operon (44). The mouse monoclonal antibody to subunit epsilon  (13-A7, epsilon II; Ref. 45) was a gift from Dr. R. Capaldi (University of Oregon, Eugene, OR).

Structural Modeling of Subunit-Subunit Interaction-- A model for subunit c interaction in the c12 oligomer has been derived from the NMR model (27) using distance constraints derived from the cross-linking data of Jones et al. (28).3 A subunit c dimer, taken from the oligomer model, was manually docked to the N-terminal domain of subunit epsilon  (residues 1-87) so that the distances between the alpha -carbons of cross-linked residues were <12 Å. The range for alpha -carbon distances in naturally occurring disulfide bonds in proteins is 4-7.5 Å (46). A somewhat wider distance constraint range of 4-11 Å was used in the molecular mechanics calculations done here to allow for possible thermal motions and distortions of structure on cross-link formation. The shortest of the two distances between the Cys alpha -carbon in each of the two subunits c and the Cys alpha -carbon in epsilon  in the manually docked structure was used to impose a distance constraint. The positions of all the atoms of the c subunits were fixed except for residues 39-45, which were left unrestrained. The backbone angles in subunit epsilon  were restrained to their value in the x-ray structure (39) using quadratic restraints. Energy minimization was performed with CVFF (constant valence force field) using the steepest descents and then conjugated gradient methods as implemented in DISCOVER 3.0 (Molecular Simulations Inc.) until the maximum derivative was below 0.1 kcal mol-1 Å-1.4

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Properties of Cys-substituted Double Mutants-- All of the mutants constructed grew on succinate minimal medium, which indicates formation of a functional ATP synthase (Table I). Two of the epsilon -substituted, cQ42C mutants showed very little membrane ATPase activity and nondetectable amounts of epsilon  subunit on immunoblotting (Table I). In these two cases, epsilon V25C/cQ42C and epsilon G27C/cQ42C, we conclude that the F1-F0 interaction is probably stable under in vivo conditions but that the F1-ATPase and epsilon  subunit disassociate from the membrane during membrane preparation.

                              
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Table I
Cross-linking of cysteine-substituted epsilon  subunits to Q42C subunit c

Cross-linking of Cysteine in Various epsilon -Substituted/cQ42C Mutants-- We had previously shown that Cys at position 31 in subunit epsilon  cross-links with Cys in positions 40, 42, or 43 in the polar loop of subunit c (25). In this study, Cys was substituted in a series of positions proximal to position 31 in subunit epsilon , and cross-link formation was tested with Cys at position 42 in subunit c. An experiment comparing cross-linking in cQ42C/epsilon E29C and cQ42C/epsilon E31C mutant membranes illustrates a number of typical features (Fig. 1). An epsilon -c cross-linked product was observed in membranes prepared from both mutants. The cross-linked product identified as epsilon -c was found at an identical position on blots stained with either anti-epsilon or anti-c antibody.5 Comigration of the product on the two types of blots was confirmed using several types of gels of varying acrylamide concentration. The epsilon -c product seen in untreated membranes is presumed to form by autoxidation as the membranes are isolated from the cell. The extent of cross-link formation was enhanced by Cu(II)(phenanthroline)2 (CuP) catalyzed oxidation. Cross-linking was largely reversed by subsequent treatment with 0.5 mM dithiothreitol.


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Fig. 1.   Immunoblots showing cross-link formation between subunits c and epsilon  in membrane vesicles of Cys-substituted mutants. Mutations are indicated by referring to the positions of the Cys residues of subunit c and subunit epsilon . Membranes, prepared in the absence of dithiothreitol, were either treated with CuP (+Cu) or not treated (0Cu). Following quenching of the reaction with EDTA and NEM, the treated or untreated membrane vesicles were centrifuged and resuspended in TMG buffer containing (+DTT) or lacking (0DTT) 0.5 mM dithiothreitol and incubated for 30 min at 22 C. Following solubilization with SDS and electrophoresis of 50-µg samples, acrylamide gels were blotted to nitrocellulose paper and probed with anti-subunit epsilon  or anti-subunit c antibodies. The positions of subunit epsilon , the epsilon -c dimer, and subunit c monomer, dimer (c2), trimer (c3), and tetramer (c4) are indicated. Some of the dimers, trimers, and tetramers of subunit c result from incomplete disaggregation of the c12 oligomer in SDS sample buffer. Most of the staining seen at the position of c4 is because of an immunoartifact in the membrane. The positions of molecular mass markers, with the molecular mass given in kDa, are shown at the side of the blots. Note that subunit c electrophoreses anomalously relative to these markers.

The intensity of the epsilon -c-immunostained product that is detected with anti-epsilon antibody does prove to be misleading. Beginning with the anti-epsilon blot shown in Fig. 1, note the much greater intensity of staining of the epsilon -c product in cQ42C/epsilon E29C versus cQ42C/epsilon E31C membranes despite the loading of equal amounts of membrane protein in all lanes. Note also for the epsilon E29C membranes that the changes in intensity of epsilon -c are considerably greater than the changes in intensity of monomeric epsilon , i.e. the changes are not in the inverse proportions expected in a precursor-product relationship. We interpret this to mean that the epsilon -c heterodimer of the epsilon E29C mutant protein binds antibody better than the epsilon E29C monomer and also better than the epsilon -c heterodimer of the epsilon E31C mutant. This interpretation is qualitatively confirmed by the relative intensities of the bands on the anti-c blot. In the untreated membrane samples, the intensity of the anti-c immunostained epsilon -c product is much greater for epsilon E31C than for epsilon E29C, i.e. just the reverse of the pattern seen with anti-epsilon antibody. We have concluded that the best way to approximate the extent of epsilon -c formation may be to compare the amounts of monomeric epsilon  remaining after various treatments. Using this criteria, the epsilon E31C mutant would appear to form at least as much and possibly more epsilon -c product by either autoxidation or CuP-catalyzed oxidation.

Other mutants also show epsilon -c products whose staining intensities differ considerably with the two antibodies. In the experiment shown in Fig. 2, membranes were washed with 1 mM Tris-HCl, pH 8.0, 0.5 mM Na2EDTA, a procedure which removes F1 and uncross-linked subunit epsilon  and other nonspecific immunoreactive proteins. The epsilon S28C, epsilon E29C and epsilon G30C epsilon -c products stain much more intensely with anti-epsilon antibody than with anti-c antibody. Conversely, the epsilon -c product in the epsilon G33C mutant stains much less intensely with anti-epsilon body than with anti-c antibody. Other epsilon -c products stain with nearly equivalent intensities in the two blots. In summary, the blots do not provide a good quantitative means of distinguishing the extent of cross-link formation in different mutant membranes.


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Fig. 2.   CuP-catalyzed cross-linking of Cys42 in subunit c with Cys at various positions in subunit epsilon  analyzed by immunoblots of "stripped" membranes. The cross-linking reaction was carried out with whole membranes, and the reaction was quenched with EDTA and NEM. Membranes were then stripped of F1 by incubation in 1 mM Tris-HCl, pH 8, 0.5 mM EDTA, 10% (v/v) glycerol, and the stripped membranes were collected by centrifugation and solubilized in SDS sample buffer. Immunoblots prepared with anti-epsilon and anti-c antibodies are marked as described for Fig. 1. Numbers at the top indicate the position of the Cys in subunit epsilon .

A survey of epsilon -c cross-linking in a series of double-Cys-substituted pairs is shown in Fig. 3. In the presence of CuP, most or all of the Cys-substituted subunit epsilon  was cross-linked with Cys42-substituted subunit c when Cys was substituted as positions 26, 28, 29, 30, 32, and 33 in subunit epsilon . Detectable cross-linking also occurred in the absence of CuP in each of these mutants. No cross-linking was observed with Cys substituted at positions 24, 25, 27, 34 and 38 of subunit epsilon . In the case of the epsilon V25C mutant, subunit epsilon  was not incorporated into the membrane. This was also the case for the epsilon G27C mutant (not shown). In other experiments, the cQ42C/epsilon S10C pair was also shown to not form a cross-link (Table I). Most of the epsilon -c-cross-linked product formed in the various membranes shown in Fig. 3 was reduced by treatment with dithiothreitol. The results from several similar experiments are summarized in Table I.


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Fig. 3.   CuP-catalyzed cross-linking of Cys42 in subunit c with Cys at various positions in subunit epsilon . Immunoblots of whole membranes are shown after probing with anti epsilon  antibody. Whole membranes, prepared in the absence of dithiothreitol, were resuspended in TMG buffer and treated with CuP (+) or not treated (0), and the reaction was quenched with EDTA and NEM. Cross-linked products in CuP-treated membranes were reduced with 25 mM dithiothreitol (+DTT) in SDS gel sample buffer containing 4 M urea. The positions of the Cys substitutions in subunit epsilon  are indicated.

Cross-linking from the Opposite Side of Subunit c-- In the NMR structure of subunit c, Gln42 lies on a flattened face of the loop region opposite Ala40 and Asp44. We introduced Cys into positions 40 and 44 of the loop to see if the pattern of cross-linking to residues surrounding epsilon Glu31 differed from that with Cys at cQ42C. As shown in Fig. 4, Cys substituted at either position 40 or 42 was readily cross-linked to Cys at positions 28, 31, and 32 of subunit epsilon . We conclude that subunit epsilon  must be able to interact with either face of the loop region when it binds to the oligomer of subunit c.


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Fig. 4.   Cu-phenanthroline-catalyzed cross-linking from Cys40 and Cys44 in subunit c to Cys at various positions in subunit epsilon . Immunoblots of whole membranes are shown after probing with anti-epsilon antibody. Whole membranes, prepared in the absence of dithiothreitol, were treated with CuP (+) or not treated (0), and the reaction was quenched with EDTA and NEM. Cross-linked products in CuP-treated membranes were reduced with dithiothreitol (+DTT) as described in Fig. 3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The surface of subunit epsilon  lying proximal to subunit c has been mapped by cross-linking experiments to a region encompassing residues 26-33, which in the NMR and x-ray diffraction structures of subunit epsilon  (38, 39) reside in a loop of antiparallel beta -sheet (Fig. 5A). The NMR and x-ray diffraction structures agree closely and show subunit epsilon  to be a protein of two distinct domains. The N-terminal domain of 86 residues folds in a 10-stranded beta -sandwich and the C-terminal domain of 45 residues is formed from two alpha -helices arranged in an antiparallel coiled coil. Much of the C-terminal domain appears to be nonessential because it can be deleted without effect on ATP synthase function (47). Cross-linking and chemical modification experiments indicate that the surface of the beta -sandwich, including residues His38 and Ser10, neighbor the gamma  subunit and that His38 lies close to the surface of F0 (14, 38, 39, 48). Residue 31 of epsilon  must also lie proximal to the surface of F0 because the epsilon E31C-substituted protein can be cross-linked to Cys at positions 40, 42, and 43 of subunit c (25). The loop including residues 26-33 protrudes from the "bottom" of subunit epsilon  as a well defined lobe (Fig. 5A), and most Cys replacements in this loop were cross-linked to Q42C subunit c in the experiments described here. In addition, Cys at positions 28, 31, and 32 in subunit epsilon  were shown to cross-link to Cys lying on opposing flattened faces of the polar loop of subunit c in the NMR structure (Ref. 27; Fig. 5B), i.e. at either position 42 or at positions 40 and 44 in the loop.


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Fig. 5.   Monomeric structures of subunit epsilon  and subunit c showing positions of cross-linkable residues. A, ribbon depiction of the beta -barrel domain of subunit epsilon  (residues 1-87) showing positions of cysteine residues studied here (Protein Data Bank code 1aqt). Cysteine within the yellow loop (positions 26-33) cross-link with subunit c. Cysteine bordering the yellow loop at positions 24, 25, and 34 (indicated by magenta) do not cross-link with subunit c. Cys at positions 10 and 38 (green) can be cross-linked to subunit gamma  but not to subunit c. B, structure of loop region of monomeric subunit c showing side chain orientation of Ala40, Gln42, and Asp44 on opposite faces of the polar loop (Protein Data Bank code 1a91, model 1).

The interacting surfaces of subunit epsilon  and subunit c have been modeled beginning with a model for the c12 oligomer described elsewhere.3 In the modeling, equivalent distance constraints were imposed for each cross-link formed because of difficulties in quantitatively distinguishing the extent of cross-link formation. In the model, the loop of antiparallel beta -sheet that is centered around epsilon Glu29 packs between the polar loops of two c subunits (Fig. 6A). The model also depicts the epsilon Glu31 residue lying close enough to the conserved and essential cArg41 residue to interact electrostatically and also close to cGln42 (Fig. 6B). The positioning of these side chains in the model should be interpreted with caution because the model is derived without use of side chain distance constraints. However, with these precautions, the general proximity of residues in the model does provide a reasonable explanation for the uncoupling effects of the cQ42E mutation (23) in that charge-charge repulsion would be expected between the cGlu42 and epsilon Glu31 carboxylates. The charge-charge repulsion explanation is supported by the differences in pH dependence of neutral versus positively charged epsilon -31 suppressor substitutions in restoring function to the cQ42E mutant (24). The smaller uncoupling effects of some substitutions in epsilon Glu31 (49), versus the cQ42E mutation, are not as easily rationalized by the model.


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Fig. 6.   Model for structural interaction of subunit epsilon  between loop regions of subunit c. A, surface representation of beta -barrel domain of subunit epsilon  (residues 1-87) packed between two subunits c. The view shown is from the outside of the c-oligomeric ring. The two subunits c are distinguished by different shades of blue. The approximate positions of epsilon Glu29 and polar loop residues in subunit c are indicated. B, side chain interactions at the epsilon -c interface. The projection shown is rotated by approximately 180° about the y axis relative to the view shown in panel A. Backbone positions in the loop of antiparallel beta -sheet of subunit epsilon  are numbered. Backbone traces of subunit c are shown in ribbon format. The side chains of cArg41 (cR41), cGln42 (cQ42), and epsilon Glu31 (epsilon E31) are depicted using Corey-Pauling-Koltun colors to emphasize the guanidino, carboxyamide, and carboxylate groups, respectively.

In the model shown in Fig. 6A, it is notable that the space between loops of subunit c is essentially filled by the packing with subunit epsilon . Because residue 205 of subunit gamma  is also known to cross-link with residues 42, 43, and 44 of subunit c (14, 26), it seems likely that the region around gamma -205 packs between a different set of c subunits than those interacting with epsilon . The shielding of two separate pairs of subunit c by the binding of subunits gamma  and epsilon , respectively, may explain the observations of Watts and Capaldi (50) on the functional effects of NEM modification of Cys42 in cQ42C mutant membranes. Function was retained during the initial phase of modification of approximately 60% of the subunits and then lost during modification of the last 40%. The inhibitory phase of NEM modification may correspond to reaction with Cys42 at the c-gamma -c or c-epsilon -c interfaces. In the currently envisioned rotary models, subunits gamma  and epsilon  remain fixed to a set of c subunits and turn as the c-oligomer rotates (14, 33, 34). The binding interaction must be of sufficient strength to withstand a considerable torque, estimated at exceeding 40 pN nm under load, or approximately 12 kcal mol-1 for each 120° step in which ATP is synthesized (8, 34, 51-53). From the structural model deduced here, it seems likely that the binding energy is derived from the combined interaction of subunit epsilon  with the loop region of one set of subunit c and of subunit gamma  with an adjacent set of subunit c.

    ACKNOWLEDGEMENT

We thank Dr. Rod Capaldi (University of Oregon) for the gift of anti-epsilon monoclonal antibody.

    FOOTNOTES

* This work was supported by United States Public Health Service Grant GM23105 from the National Institutes of Health.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: Dept. of Biomolecular Chemistry, 587 Medical Sciences Bldg., University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-262-1439; Fax: 608-262-5253.

1 The unc DNA numbering system corresponds to that used by Walker et al. (40).

3 O. Y. Dmitriev, M. E. Girvin, P. C. Jones, and R. H. Fillingame, submitted for publication.

4 A coordinate file of the final model is available by E-mail at dmitriev{at}iris.bmolchem.wisc.edu.

5 A number of minor bands were also detected with the anti-epsilon antibody, the intensity and number of which vary from experiment to experiment. They appear to be artifactual because similar bands are detected in mutant membranes lacking subunit epsilon . Most of the proteins reacting nonspecifically are removed by the stripping procedure used to remove F1, i.e. washing membranes with 1 mM Tris-HCl and 0.5 mM EDTA, but this treatment also removes monomeric epsilon  (see Fig. 2).

    ABBREVIATIONS

The abbreviations used are: NEM, N-ethylmaleimide; CuP, Cu(II) (phenanthroline)2; Tricine, N-tris(hydroxymethyl)methylglycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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