Existence of stoichiometric amounts of an intrinsic ATPase inhibitor and two stabilizing factors with mitochondrial ATP synthase in yeast

Two proteinaceous factors, 15K and 9K proteins, which acted together to stabilize the inactivated yeast F1F0-ATPase-inhibitor complex [Hashimoto, T., et al. (1984) J. Biochem. 95, 131-136] were hardly distinguishable from the sigma and epsilon subunits, respectively, of yeast F1-ATPase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. However, they were clearly distinguishable from these subunits by analyses of the sequences at their amino terminals and by immunoblotting combined with SDS polyacrylamide gel electrophoresis. The two stabilizing factors and an ATPase inhibitor existed in mitochondria in equimolar ratios to F1-ATPase. These three protein factors were not present in purified F1-ATPase or in F1F0-ATPase preparations, but remained in the mitochondrial membranes after extraction of F1F0-ATPase with Triton X-100. These observations strongly suggest that the two stabilizing factors and the ATPase inhibitor form a regulatory substructure of mitochondrial ATP synthase, in addition to the F1 and F0 subunits.     

The inhibitor peptide of the mitochondrial F1.F0-ATPase interacts with calmodulin and stimulates the calmodulin-dependent Ca2+-ATPase of erythrocytes

The binding of calmodulin to the mitochondrial F1.F0-ATPase has been studied. [125I]Iodoazidocalmodulin binds to the epsilon-subunit and to the endogeneous ATPase inhibitor peptide in a Ca2+-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca2+-ATPase of erythrocytes has also been analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca2+-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the two mechanisms of activation are different. These in vitro effects of the two regulatory proteins may reflect a common origin of the two ATPases considered and/or of the regulatory proteins.     

Partial amino terminal sequence of the precursor of mitochondrial ATPase inhibitor protein synthesized with mRNA partially purified by gel permeation chromatography

Messenger RNA coding mitochondrial ATPase inhibitor protein, a small peptide comprised of 63 amino acid residues, was separated from a large quantity of mRNAs of larger molecules by high speed gel permeation chromatography. Messenger RNA coding a small stabilizing factor of inactivated F1F0-ATPase complex, which is also comprised of 63 amino acids, was recovered in the same fraction as the ATPase inhibitor, whereas mRNA for a large stabilizing factor with an apparent molecular weight of 15,000 was recovered in a fraction of slightly larger molecules. ATPase inhibitor precursor labeled with various kinds of radioactive amino acids was prepared separately by cell-free translation with the purified mRNA, and the amino terminal sequence of the precursor was examined. It was demonstrated that an extra peptide of 21 amino acid residues, including 5 leucine, 4 serine, 1 glycine, and 1 methionine residues, is located at the amino terminus of the ATPase inhibitor precursor.     

Binding properties of an intrinsic ATPase inhibitor and occurrence in yeast mitochondria of a protein factor which stabilizes and facilitates the binding of the inhibitor to F1F0-ATPase

The content of an intrinsic ATPase inhibitor in mitochondria was determined by a radioimmunoassay procedure which showed the molar ratio of the inhibitor to ATPase to be 1:1. The ratio in submitochondrial particles, where half of the enzyme was activated, was the same as that of mitochondria, indicating that the inhibitor protein has affinity for the mitochondrial membrane as well as for F1-ATPase. The inhibitor protein could be removed from the mitochondrial membrane by incubation with 0.5 M Na2SO4 and concomitantly the enzyme was fully activated. The enzyme fully activated by the salt treatment was inactivated again by the externally added ATPase inhibitor in the presence of ATP and Mg2+. The enzyme-inhibitor complex (inactive) on the mitochondrial membrane was more stable than the solubilized enzyme-inhibitor complex but gradually dissociated in the absence of ATP and Mg2+. However, in mitochondria, the enzyme activity was inhibited even in the absence of the cofactors. A protein factor stabilizing the enzyme-inhibitor complex on the mitochondrial membrane was isolated from yeast mitochondria. This factor stabilized the inhibitor complex of membrane-bound ATPase while having no effect on that of purified F1-ATPase. It also efficiently facilitated the binding of the inhibitor to membrane-bound ATPase to form the complex, which reversibly dissociated at slightly alkaline pH.     

Correspondence of minor subunits of plant mitochondrial F1ATPase to F1F0ATPase subunits of other organisms

In addition to two major alpha- and beta-subunits, the soluble oligomycin-insensitive F1ATPase purified from sweet potato root mitochondria contains four different minor subunits of gamma (Mr = 35,500), delta (Mr = 27,000), delta' (Mr = 23,000), and epsilon (Mr = 12,000) (Iwasaki, Y., and Asashi, T. (1983) Arch. Biochem. Biophys. 227, 164-173). Among these minor subunits, the delta-subunit specifically cross-reacted with an antibody against the delta-subunit of maize mitochondrial F1 which contains only three minor gamma-, delta- and epsilon-subunits like F1ATPases from other organisms, indicating that the delta'-subunit is an extra subunit of sweet potato F1 which is absent in the maize F1. All of the four minor subunits of sweet potato F1 were purified and their N-terminal amino acid sequences of 30-36 residues were determined. The N-terminal sequence of gamma-subunit was homologous to those of the gamma-subunits of bacterial F1 and mammalian mitochondrial F1. The N-terminal sequence of the delta-subunit was homologous to those of the delta-subunits of bacterial F1, chloroplast CF1, and oligomycin sensitivity conferring protein of bovine mitochondrial F1F0. A sequence homology was also observed between the sweet potato epsilon-subunit and the epsilon-subunit of bovine mitochondrial F1. The N-terminal sequence of the delta'-subunit did not show any significant sequence homology to known protein sequences. These subunit correspondences place plant mitochondrial F1 at an unique position in the evolution of F1ATPase.     

Simultaneous bindings of ATPase inhibitor and 9K protein to F1F0-ATPase in the presence of 15K protein in yeast mitochondria

Yeast mitochondrial ATP synthase has three regulatory proteins, ATPase inhibitor, 9K protein, and 15K protein. The 9K protein binds directly to purified F1-ATPase, as does the ATPase inhibitor, but the 15K protein does not [Hashimoto, T. et al. (1987) J. Biochem. 102, 685-692]. In the present study, we found that 15K protein bound to purified F1F0-ATPase, forming an equimolar complex with the enzyme. The apparent dissociation constant was calculated to be 1.4 x 10(-5) M. The ATPase inhibitor and 9K protein also bound to F1F0-ATPase in the presence of ATP and Mg2+, and the dissociation constants of their bindings were about 3 X 10(-6) M. They bound to the enzyme competitively in the absence of 15K protein, but in its presence, they bound in equimolar amounts to the enzyme. The ATP-hydrolyzing activity of the enzyme-ligand complex was greatly influenced by the order of bindings of ATPase inhibitor and 9K protein: when the ATPase inhibitor was bound first, the activity of the enzyme was inhibited completely and was not restored by 9K protein, but when 9K protein was added first, the activity was inhibited only partially even after equimolar binding of the ATPase inhibitor to the enzyme. These observations strongly suggest that the 15K protein binds to the F0 part and functions to hold the ATPase inhibitor or 9K protein on the F1 subunit.     

Diethylstilbestrol. A novel F0-directed probe of the mitochondrial proton ATPase

At low concentrations, diethylstilbestrol (DES) is shown to be a potent F0-directed inhibitor of the F0F1-ATPase of rat liver mitochondria. In analogy to other F0-directed inhibitors, DES inhibits both the ATPase and ATP-dependent proton-translocation activities of the purified and membrane bound enzyme. When added at low concentrations with dicyclohexylcarbodiimide (DCCD), a covalent inhibitor, DES acts synergistically to inhibit ATPase activity of the complex. At higher concentrations, DES restores DCCD-inhibited ATPase activity. However, there is no restoration of ATP-dependent proton translocation. Under these conditions DCCD remains covalently bound to the F0F1-ATPase complex and F1 remains bound to Fo. Significantly, when the F0F1-ATPase is inhibited by the Fo-directed inhibitor venturicidin rather than DCCD, DES is also able to restore ATPase activity. In contrast, DES is unable to restore ATPase activity to F0F1 preparations inhibited by the Fo-directed inhibitors oligomycin or tricyclohexyltin. However, combinations of [DES + DCCD] or [DES + venturicidin] can restore ATPase activity to F0F1 preparations inhibited by either oligomycin or tricyclohexyltin. Results presented here indicate that the F0 moiety of the rat liver mitochondrial proton ATPase contains a distinct binding site for DES. In addition, they suggest that at saturating concentrations simultaneous occupancy of the DES binding site and sites for either DCCD or venturicidin promote "uncoupled" ATP hydrolysis.     

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

F1-ATPase from different submitochondrial particles.

1. F1-ATPase has been extracted by the diphosphatidylglycerol procedure from mitochondrial ATPase complexes that differ in ATPase activity, cold stability, ATPase inhibitor and magnesium content. 2. The ATPase activity of the isolated enzymes was dependent upon the activity of the original particles. In this respect, F1-ATPase extracted from submitochondrial particles prepared in ammonia (pH 9.2) and filtered through Sephadex G-50 was comparable to the enzyme purified by conventional procedures (Horstman, L.L. and Racker, E. (1970) J. Biol. Chem. 245, 1336--1344), whereas F1-ATPase extracted from submitochondrial particles prepared in the presence of magnesium and ATP at neutral pH was similar to factor A (Andreoli, T.E., Lam, K.W. and Sanadi, D.R. (1965) J. Biol. Chem. 240, 2644--2653). 3. No systematic relationship has been found in these F1-ATPase preparations between their ATPase inhibitor content and ATPase activity. Rather, a relationship has been observed between this activity and the efficiency of the ATPase inhibitor-F1-ATPase association within the membrane. 4. It is concluded that the ATPase activity of isolated F1-ATPase reflects the properties of original ATPase complex provided a rapid and not denaturing procedure of isolation is employed.     

F0F1-ATPase of plant mitochondria: isolation and polypeptide composition

A simple and high yield purification procedure for the isolation of F0F1-ATPase from spinach leaf mitochondria has been developed. This is the first report concerning purification and composition of the plant mitochondrial F0F1-ATPase. The enzyme is selectively extracted from inner membrane vesicles with the zwitterionic detergent, 3-[(3-cholamidopropyl) dimethyl ammonio]-1- propane sulfonate (CHAPS). The purified enzyme exhibits a high oligomycin-sensitive ATPase activity (3,6 mumol.min-1.mg-1). SDS-PAGE of the purified F0F1-ATPase complex reveals protein bands of molecular masses of 54 kDa (F1 alpha,beta), 33 kDa (F1 gamma), 28 kDa, 23 kDa, 21 kDa (F1 delta), 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa (F1 epsilon) and 8.5 kDa. All polypeptides migrate as one complex in a polyacrylamide gradient gel under non-denaturing conditions in the presence of 0.1% Triton X-100. Five polypeptides could be identified as subunits of F1. Polypeptides of molecular masses 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa constitute the F0 part of the complex. Our results show that polypeptide composition of the plant mitochondrial F0 differs from other eukaryotic F0 of yeast, mammals and chloroplasts.     

Cold lability of membrane-bound F1-ATPase.

1. Preincubation of MgATP submitochondrial particles with EDTA or Tris.HCl liberated a measurable amount of ATPase inhibitor that could be rapidly purified using only trichloroacetic acid precipitation and heat treatment. 2. In spite of the emergence of high ATPase activity, a considerable amount of ATPase inhibitor was left in the particles. Comparative analysis of other submitochondrial preparations indicated that only AS-particles were effectively depleted. 3. The high ATPase activity of inhibitor-deficient particles, was labile at low temperature provided that the exposure to cold was done in the presence of MgATP. Other nucleotides could not substitute for ATP. Glycerol inhibited and salts enhanced the cold inactivation of membrane-bound F1-ATPase. Isolation of F1-ATPase from cold-inactivated particles yielded a soluble preparation of correspondingly lower activity. 4. It is concluded that together with the increase of ATPase activity, the ATP-dependent cold lability of membrane-bound F1-ATPase and the dislocation of ATPase inhibitor at non operative sites reveal the extent of ATPase complex disorganization.     

The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.     

Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase

The F(0)F(1)-ATPase complex of yeast mitochondria contains three mitochondrial and at least 17 nuclear gene products. The coordinate assembly of mitochondrial and cytosolic translation products relies on chaperones and specific factors that stabilize the pools of some unassembled subunits. Atp10p was identified as a mitochondrial inner membrane component necessary for the biogenesis of the hydrophobic F(0) sector of the ATPase. Here we show that, following its synthesis on mitochondrial ribosomes, subunit 6 of the ATPase (Atp6p) can be cross-linked to Atp10p. This interaction is required for the integration of Atp6p into a partially assembled subcomplex of the ATPase. Pulse labeling and chase of mitochondrial translation products in vivo indicate that Atp6p is less stable and more rapidly degraded in an atp10 null mutant than in wild type. Based on these observations, we propose Atp10p to be an Atp6p-specific chaperone that facilitates the incorporation of Atp6p into an intermediate subcomplex of ATPase subunits.     

Regulatory role of the ATPase inhibitor protein on proton conduction by mitochondrial H+-ATPase complex

This study shows that the natural inhibitor protein of mitochondrial H+-ATPase complex (IF1) inhibits, in addition to the catalytic activity, the proton conductivity of the complex. The inhibition of ATPase activity by IF1 is less effective in the purified F1 than in submitochondrial particles where F1 is bound to F0. No inhibition of H+ conductivity by F0 is observed in F1-depleted particles.     

Similarity of lysosomal H+-ATPase to mitochondrial F0F1-ATPase in sensitivity to anions and drugs as revealed by solubilization and reconstitution

Lysosomal H+-translocating ATPase (H+-ATPase) was solubilized with lysophosphatidylcholine and reconstituted into liposomes (Moriyama, Y., Takano, T. and Ohkuma, S. (1984) J. Biochem. (Tokyo) 96, 927-930). In this study, the sensitivities of membrane-bound, solubilized and liposome-incorporated ATPase to various anions and drugs were measured in comparison with those of similar forms of mitochondrial H+-ATPase (mitochondrial F0F1-ATPase) with the following results. (1) Bicarbonate and sulfite activated solubilized lysosomal H+-ATPase, but not the membrane-bound ATPase or ATPase incorporated into liposomes. All three forms of mitochondrial F0F1-ATPase were activated by these anions. (2) All three forms of both lysosomal H+-ATPase and mitochondrial F0F1-ATPase were strongly inhibited by SCN-, NO3- and F-, but scarcely affected by Cl-, Br- and SO2-4. (3) The solubilized lysosomal H+-ATPase was strongly inhibited by azide, quercetin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and oligomycin. Its sensitivity was almost the same as that of mitochondrial F0F1-ATPase. Neither membrane-bound ATPase nor ATPase incorporated into liposomes was affected appreciably by these drugs. These results indicate that the sensitivity to anions and drugs of lysosomal H+-ATPase depends on the form of the enzyme and that the sensitivity of the solubilized lysosomal H+-ATPase is very similar to that of mitochondrial F0F1-ATPase. On the other hand, the two ATPases differ in their sensitivity to N-ethylmaleimide and pyridoxal phosphate: only the mitochondrial ATPase is inhibited by pyridoxal phosphate whereas only the lysosomal ATPase is inhibited by N-ethylmaleimide.     

The hydrogen ion-pumping adenosine triphosphatase of platelet dense granule membrane. Differences from F1F0- and phosphoenzyme-type ATPases

Using a coupled transport assay which detects only those ATPase molecules functionally inserted into the platelet dense granule membrane, we have characterized the inhibitor sensitivity, substrate specificity, and divalent cation requirements of the granule H+ pump. Under identical assay conditions, the granule ATPase was insensitive to concentrations of NaN3, oligomycin, and efrapeptin which almost completely inhibit ATP hydrolysis by mitochondrial membranes. The granule ATPase was inhibited by dicyclohexylcarbodiimide but only at concentrations much higher than those needed to maximally inhibit mitochondrial ATPase. Vanadate (VO3-) ion and ouabain also failed to inhibit granule ATPase activity at concentrations which maximally inhibited purified Na+,K+-ATPase. Two alkylating agents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and N-ethylmaleimide both completely inhibited H+ pumping by the granule ATPase under conditions where ATP hydrolysis by mitochondrial membranes or Na+,K+-ATPase was hardly affected. These results suggest that the H+-pumping ATPase of platelet granule membrane may belong to a class of ion-translocating ATPases distinct from both the phosphoenzyme-type ATPases present in plasma membrane and the F1F0-ATPases of energy-transducing membranes.     

Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase

Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 +/- 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum raised against the beta-subunit of mitochondrial ATPase indicates that the ATPase activity in the peroxisomal fractions can not be ascribed to contamination with mitochondria or other subcellular organelles. From the sensitivity of the ATPase present in the peroxisomal fraction towards a variety of ATPase inhibitors, we conclude that it displays both V-type and F-type features and is distinguishable from both the mitochondrial F1F0-ATPase and the lysosomal V-type ATPase.     

Binding properties of 9K protein to F1-ATPase: a counterpart ligand to the ATPase inhibitor

A regulatory subunit of yeast mitochondrial ATP synthase, 9K protein, formed an equimolar complex with F1-ATPase in the presence of ATP and Mg2+, indicating that the binding of the protein to the enzyme took place in a similar manner to that of ATPase inhibitor. The ATP-hydrolyzing activity of F1-ATPase decreased 40% on binding of the 9K protein, and the remaining activity was resistant to external ATPase inhibitor. The apparent dissociation constant of the F1-ATPase-9K complex was determined by gel permeation chromatography to be 3.7 X 10(-6) M, which was in the same order of magnitude as that of enzyme-ATPase inhibitor complex (4.2 x 10(-6) M). When added simultaneously the binding of the inhibitor and 9K protein to F1-ATPase were competitive and the sum of their bindings did not exceed 1 mol per mol of enzyme. However, the binding of each protein ligand to F1-ATPase took more than 1 min for completion, and when one of these two proteins was added 10 min after the other, it did not replace the other. These observations strongly suggest that membrane-bound F1-ATPase always binds to either the 9K protein or ATPase inhibitor in intact mitochondria and that the complexes with the two ligands are active and inactive counterparts, respectively.     

Activation and inhibition of the Escherichia coli F1-ATPase by monoclonal antibodies which recognize the epsilon subunit

The properties of two monoclonal antibodies which recognize the epsilon subunit of Escherichia coli F1-ATPase were studied in detail. The epsilon subunit is a tightly bound but dissociable inhibitor of the ATPase activity of soluble F1-ATPase. Antibody epsilon-1 binds free epsilon with a dissociation constant of 2.4 nM but cannot bind epsilon when it is associated with F1-ATPase. Likewise epsilon cannot associate with F1-ATPase in the presence of high concentrations of epsilon-1. Thus epsilon-1 activates F1-ATPase which contains the epsilon subunit, and prevents added epsilon from inhibiting the enzyme. Epsilon-1 cannot bind to membrane-bound F1-ATPase. The epsilon-4 antibody binds free epsilon with a dissociation constant of 26 nM. Epsilon-4 can bind to the F1-ATPase complex, but, like epsilon-1, it reverses the inhibition of F1-ATPase by the epsilon subunit. The epsilon subunit remains crosslinkable to both the beta and gamma subunits in the presence of epsilon-4, indicating that it is not grossly displaced from its normal position by the antibody. Presumably the activation arises from more subtle conformational effects. Antibodies epsilon-4 and delta-2, which recognizes the delta subunit, both bind to F1F0 in E. coli membrane vesicles, indicating that these subunits are substantially exposed in the membrane-bound complex. Epsilon-4 inhibits the ATPase activity of the membrane-bound enzyme by about 50%, and Fab prepared from epsilon-4 inhibits by about 40%. This inhibition is not associated with any substantial change in the major apparent Km for ATP. These results suggest that inhibition of membrane-bound F1-ATPase arises from steric effects of the antibody.     

Amino acid substitutions in the epsilon-subunit of the F1F0-ATPase of Escherichia coli

A mutant strain of Escherichia coli was isolated in which Gly-48 of the mature epsilon-subunit of the energy-transducing adenosine triphosphatase was replaced by Asp. This amino acid substitution caused inhibition of ATPase activity (about 70%), loss of ATP-dependent proton translocation and lowered oxidative phosphorylation, but did not affect proton translocation through the F0. Purified F1-ATPase from the mutant strain bound to stripped membranes with the same affinity as the normal F1-ATPase. Partial revertant strains were isolated in which Pro-47 of the epsilon-subunit was replaced by Ser or Thr. Pro-47 and Gly-48 are predicted to be residues 2 and 3 in a Type II beta-turn and the Gly-48 to Asp substitution is predicted to cause a change from a Type II to a Type I or III beta-turn. Space-filling models of the beta-turn (residues 46-49) in the normal, mutant and partial revertant epsilon-subunits indicate that the peptide oxygen between Pro-47 and Gly-48 is in a different position to the peptide oxygen between Pro-47 and Asp-48 and that the substitution of Pro-47 by either Ser or Thr restores an oxygen close to the original position. It is suggested that the peptide oxygen between Pro-47 and Gly-48 of the epsilon-subunit is involved either structurally in inter-subunit H-bonding or directly in proton movements through the F1-ATPase.