The epsilon subunit as an ATPase inhibitor of the F1-ATPase in Escherichia coli

The isolation of protein ATPase inhibitor was attempted directly from Escherichia coli membrane extracts to examine the possible presence of a Pullman-Monroy-type inhibitor [M. E. Pullman and G. C. Monroy (1963) J. Biol. Chem. 238, 3762-3769] distinct from the epsilon subunit of E. coli ATPase. Purification to homogeneity was achieved in a sequence of steps involving trichloracetic acid precipitation, DEAE-cellulose, Sephadex G75 chromatography, and a terminal isoelectric focusing step. An inhibitory protein was obtained and was identified by its physicochemical and inhibitory properties as the epsilon subunit of E. coli ATPase. The other inhibitory fraction observed in the purification procedure consisted of aggregated epsilon subunits.     

Role of the amino terminal region of the epsilon subunit of Escherichia coli H(+)-ATPase (F0F1).

Escherichia coli strain KF148(SD-) defective in translation of the uncC gene for the epsilon subunit of H(+)-ATPase could not support growth by oxidative phosphorylation due to lack of F1 binding to Fo (M. Kuki, T. Noumi, M. Maeda, A. Amemura, and M. Futai, 1988, J. Biol. Chem. 263, 17, 437-17, 442). Mutant uncC genes for epsilon subunits lacking different lengths from the amino terminus were constructed and introduced into strain KF148(SD-). F1 with an epsilon subunit lacking the 15 amino-terminal residues could bind to F0 in a functionally competent manner, indicating that these amino acid residues are not absolutely necessary for formation of a functional enzyme. However, mutant F1 in which the epsilon subunit lacked 16 amino-terminal residues showed defective coupling between ATP hydrolysis (synthesis) and H(+)-translocation, although the mutant F1 showed partial binding to Fo. These findings suggest that the epsilon subunit is essential for binding of F1 to F0 and for normal H(+)-translocation. Previously, Kuki et al. (cited above) reported that 60 residues were not necessary for a functional enzyme. However, the mutant with an epsilon subunit lacking 15 residues from the amino terminus and 4 residues from the carboxyl terminus was defective in oxidative phosphorylation, suggesting that both terminal regions affect the conformation of the region essential for a functional enzyme.     

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.     

Construction and plasmid-borne complementation of strains lacking the epsilon subunit of the Escherichia coli F1F0 ATP synthase.

Two strains of Escherichia coli that lack the epsilon subunit of the F1F0 ATP synthase have been constructed. They are shown to be viable but with very low growth yields (28%). These strains can be complemented by plasmids carrying wild-type uncC, but not when epsilon is overproduced. These results indicate that epsilon is not essential for growth on minimal glucose medium and that the level of its expression affects the assembly of the ATP synthase.     

Mutations within the uncE gene affecting assembly of the F1F0-ATPase of Escherichia coli.

Activation of protein kinase C (PKC) in human T lymphocytes is an immediate consequence of mitogenic signalling via the antigen-receptor complex and CD2 antigen. In order to investigate further the signal-transduction pathways which result in PKC activation, we have established a novel PKC assay system using streptolysin-O-permeabilized T cells. Known peptide substrates of PKC were introduced into permeabilized cells in the presence of [gamma-32P]ATP, 3 mM-Mg2+ and 150 nM free Ca2+. The peptide found to have the lowest background phosphorylation had the sequence Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys (peptide GS), and the phosphorylation of the peptide was increased up to 6-fold by direct activation of PKC with phorbol 12,13-dibutyrate. Induction of PKC activation with the UCHT1 antibody against the CD3 antigen, or with phytohaemagglutinin (PHA) or guanosine 5'-[gamma-thio]triphosphate (GTP[S]), increased peptide-GS phosphorylation by 2-3 fold. The specificity of PKC action on peptide GS was demonstrated by blocking increases in phosphorylation with a pseudosubstrate peptide PKC inhibitor. PKC activation by this technique could be detected within 1 min of adding external ligand. Dose-response curves revealed that PHA-induced production of inositol phosphates correlated closely with PKC activities, whereas only a partial correlation between these parameters was observed with GTP[S]. Our data are consistent with the presence of more than one G-protein-mediated pathway of PKC regulation in T cells. The quantitative PKC assay system described is both simple and reproducible, and its potential application to a wide range of cell types should prove useful in further investigations of PKC activation mechanisms.     

Inhibitory properties of endogenous subunit epsilon in the Escherichia coli F1 ATPase.

Homogeneous ? bound tightly to the purified Escherichia coli ATPase (ECF₁ from which ? had been removed and strongly inhibited its ATPase activity. ECF₁ containing ? had a lower specific activity than ECF₁ missing ?, provided that the ATPase assay was carried out at relatively high concentrations of enzyme. Antiserum specific for the ? subunit stimulated the ATPase, as did diluting the enzyme, apparently by dissociating ?. When the ATPase reaction was started by the addition of enzyme, the rate of ATP hydrolysis increased progressively during the first 3 min until a linear steady-state rate was reached. A prior incubation with ATP abolished the lag period and ADP prevented the ATP effect. ECF₁ missing ? gave a linear rate of ATP hydrolysis without a lag, unless ? was rebound to it before the assay. These results suggest that ECF₁ as purified is in an inhibited state due to the presence of the ? subunit, whose interaction with ECF₁ is governed by an equilibrium binding. ATP appears to convert ECF₁ to a form which more readily binds and releases ?.     

The epsilon subunit and inhibitory monoclonal antibodies interact with the carboxyl-terminal region of the beta subunit of Escherichia coli F1-ATPase

The epitopes of two classes of monoclonal antibody and the binding site for the epsilon subunit have been mapped to the carboxyl-terminal region of the beta subunit of Escherichia coli F1-ATPase using partial CNBr cleavage, weak acid hydrolysis, and Western blots. One class of antibody, B-I, inhibits ATPase activity; the other class, B-II, recognizes an epitope not exposed on the surface of intact F1. Data from two-dimensional gels and blots of beta cleaved with CNBr/weak acid showed that the B-I epitope lies between Asp-381 and the carboxyl-terminal Leu-459, and the B-II epitope lies between Asp-345 and Met-380. Weak acid hydrolysis of the beta-epsilon product obtained by cross-linking F1 with a water-soluble carbodiimide yielded a fragment containing epsilon and a 13-kDa carboxyl-terminal fragment of beta indicating that epsilon interacts with this portion of beta as well. Fab fragments from the B-I antibody beta-6 could be cross-linked to the epsilon subunit in native F1 by various cross-linking agents demonstrating that the antibody and the epsilon subunit occupy adjacent, nonoverlapping sites on the beta subunit. Implications of these results for the roles of the epsilon subunit and of the carboxyl-terminal region of the beta subunit in F1 are discussed.     

The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase.

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.     

Mutants of Escherichia coli H+-ATPase defective in the delta subunit of F1 and the b subunit of F0

Complete nucleotide sequence of the genes for subunits of the H+ ATPase of E.coli has been determined and several hybrid plasmids carrying various portions of these genes have been constructed. Genetic complementation and recombination tests of about forty mutants of E.coli defective in the ATPase were performed using these plasmids for identifying the locations of the mutations. Two mutants defective in the delta subunit and a novel type of mutant defective in the b subunit of F0 were identified. The delta subunit mutants showed no proton conduction, suggesting that this subunit has an important role for the proton conduction. The ATPase of the b subunit mutant has a normal activity of proton channel portion, which phenotype is clearly different from that of mutants of the b subunit reported previously.     

An upstream uncD sequence modulates translation of Escherichia coli uncC.

The effect of upstream uncD sequences on expression of the Escherichia coli uncC gene, encoding the epsilon subunit of F1-ATPase, was studied. uncC expression was reduced severalfold in plasmid constructs bearing, in addition to uncC, a region of uncD located between 85 and 119 bases upstream from the uncC initiation codon. This reduction was independent of in-frame translation of the uncD sequences. An mRNA stem-loop structure in which sequences located within the inhibitory region of uncD base pair with the uncDC intercistronic region is suggested to function in modulating uncC expression.     

Defining the domain of binding of F1 subunit epsilon with the polar loop of F0 subunit c in the Escherichia coli ATP synthase.

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.     

Role of the carboxyl terminal region of H(+)-ATPase (F0F1) a subunit from Escherichia coli.

The effects of amino acid substitutions in the carboxyl terminal region of the H(+)-ATPase a subunit (271 amino acid residues) of Escherichia coli were studied using a defined expression system for uncB genes coded by recombinant plasmids. The a subunits with the mutations, Tyr-263----end, Trp-231----end, Glu-219----Gln, and Arg-210----Lys (or Gln) were fully defective in ATP-dependent proton translocation, and those with Gln-252----Glu (or Leu), His-245----Glu, Pro-230----Leu, and Glu-219----His were partially defective. On the other hand, the phenotypes of the Glu-269----end, Ser-265----Ala (or end), and Tyr-263----Phe mutants were essentially similar to that of the wild-type. These results suggested that seven amino acid residues between Ser-265 and the carboxyl terminus were not required for the functional proton pathway but that all the other residues except Arg-210, Glu-219, and His-245 were required for maintaining the correct conformation of the proton pathway. The results were consistent with a report that Arg-210 is directly involved in proton translocation.     

Isolated epsilon subunit of thermophilic F1-ATPase binds ATP

F1-ATPase, a soluble part of the F0F1-ATP synthase, has subunit structure alpha3beta3gammadeltaepsilon in which nucleotide-binding sites are located in the alpha and beta subunits and, as believed, in none of the other subunits. However, we report here that the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus strain PS3 can bind ATP. The binding was directly demonstrated by isolating the epsilon subunit-ATP complex with gel filtration chromatography. The binding was not dependent on Mg2+ but was highly specific for ATP; however, ADP, GTP, UTP, and CTP failed to bind. The epsilon subunit lacking the C-terminal helical hairpin was unable to bind ATP. Although ATP binding to the isolated epsilon subunits from other organisms has not been detected under the same conditions, a possibility emerges that the epsilon subunit acts as a built in cellular ATP level sensor of F0F1-ATP synthase.     

Interaction between Glu-219 and His-245 within the a subunit of F1F0-ATPase in Escherichia coli

Oligonucleotide-directed mutagenesis was used to generate mutations in the a subunit gene (uncB) altering the glutamic acid 219 and the histidine 245 codons. Substitutions of aspartic acid, glutamine, histidine, and leucine for glutamic acid at position 219 neither alter the hydrolytic activity of membrane-bound F1 nor the association of F1 with F0. However, the efficiency of F0-mediated proton translocation was reduced to varying degrees. Replacement of glutamic acid 219 with leucine reduced the ATP-driven proton pumping activity of intact F1F0 to undetectable levels. Roughly 5% of normal activity was observed when glutamine occupied position 219. Surprisingly higher activity, approaching 20% of wild type levels, is seen when histidine replaced glutamic acid 219. The aspartic acid substitution resulted in little loss of enzyme function. Substitution of glutamic acid for histidine 245 results in a reduction to about 45% of normal proton translocation. Construction of the double mutant with substitution of histidine at position 219 and glutamic acid at position 245 yields a complex with better proton translocation than with either mutant separately. The possibility that a functionally important interaction between histidine 245 and glutamic acid 219 of the a subunit may be directly involved in the proton translocation mechanism of F1F0-ATP synthase is discussed.     

F1F0-ATPase from Escherichia coli with mutant F0 subunits. Partial purification and immunoprecipitation of F1F0 complexes

Previously identified mutations in subunits a and b of the F0 sector of the F1F0-ATPase from Escherichia coli are further characterized by isolating detergent-solubilized, partially purified F1F0 complexes from cells bearing these mutations. The composition of the various F1F0 complexes was judged by quantitating the amount of each subunit present in the detergent-solubilized preparations. The composition of the F0 sectors containing altered polypeptides was determined by quantitating the F0 subunits that were immunoprecipitated by antibodies directed against the F1 portion. In this way, the relative amounts of F0 subunits (a, b, c) which survived the isolation procedure bound to F1 were determined for each mutation. This analysis indicates that both missense mutations in subunit a (aser206----leu and ahis245----tyr) resulted in the isolation of F1F0 complexes with normal subunit composition. The nonsense mutation in subunit a (atyr235----end) resulted in isolation of a complex containing the b and c subunits. The bgly131----asp mutation in the b subunit results in an F0 complex which does not assemble or survive the isolation. The isolated F1F0 complex containing the mutation bgly9----asp in the b subunit was defective in two regards: first, a reduction in F1 content relative to F0 and second, the absence of the a subunit. Immunoprecipitations of this preparation demonstrated that F1 interacts with both c and mutant b subunits. A strain carrying the mutation, bgly9----asp, and the compensating suppressor mutation apro240----leu (previously shown to be partially unc+) yielded an F1F0 ++ complex that remained partially defective in F1 binding to F0 but normal in the subunit composition of the F0 sector. The assembly, structure, and function of the F1F0-ATPase is discussed.     

The polar domain of the b subunit of Escherichia coli F1F0-ATPase forms an elongated dimer that interacts with the F1 sector.

A soluble form of the b subunit of the F0 sector of the F1F0-ATPase of Escherichia coli has been produced, purified, and characterized. In this form of the protein, designated bsol, residues 25-146 (the carboxyl terminus) of b have been fused to an amino-terminal octapeptide extension derived from the vector pUC8. The inferred subunit molecular weight of bsol is 15,459. bsol protein was expressed in E. coli as a soluble cytoplasmic protein and was readily purified to homogeneity by conventional methods. The molecular weight of bsol, determined by sedimentation equilibrium, was 31,200, indicating that the protein is dimeric. Chemical cross-linking studies supported this conclusion. However, bsol sedimented with a coefficient of just 1.8 S and behaved on size exclusion chromatography with an apparent molecular weight of 80,000-85,000. These results indicate that the protein exists in solution as a highly elongated dimer. The circular dichroism spectrum indicated that bsol is highly alpha-helical. Binding of bsol to F1-ATPase was directly demonstrated by size exclusion chromatography. bsol also inhibited the binding of F1-ATPase to F1-depleted membrane vesicles, as measured by reconstitution of energy-dependent quinacrine fluorescence quenching. This result implies that bsol and F0 compete for binding to the same site on F1. The apparently normal interaction of bsol with F1-ATPase strongly suggests that the recombinant protein assumes the correct structure. No substantial effects of bsol on the ATPase activity of purified F1 were observed.     

Suppression mutations in the defective beta subunit of F1-ATPase from Escherichia coli

The Escherichia coli mutant of the proton-translocating ATPase KF11 (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., and Futai, M. (1980) J. Biochem. (Tokyo) 88, 695-703) has a defective beta subunit with serine being replaced by phenylalanine at codon 174. Four suppression mutants (RE10, RE17, RE18, and RE20) from this strain capable of growth on minimal plate agar supplemented by succinate were isolated. The original point mutation at codon 174 was intact in these strains. Additional point mutations, Ala-295 to Thr, Gly-149 to Ser, Leu-400 to Gln, Ala-295 to Pro, for RE10, RE17, RE18, and RE20, respectively, were identified by the polymerase chain reaction and sequencing. These mutations, except for RE10, were confirmed as a single mutation conferring a suppressive phenotype by genetic suppression assay using KF11 as the host cells. The results indicated that Ser-174 has functional interaction with Gly-149, Ala-295, and Leu-400. The residues are located within the previously estimated catalytic domain of the beta subunit, indicating that this domain is indeed folded for the active site of catalytic function. Growth rates of the revertants in the minimal medium with succinate increased compared with that of KF11, showing that ATP synthesis recovered to some extent. The ATP hydrolytic activity in the revertant membranes increased in RE17 and RE20 but did not in RE10 and RE18, suggesting that synthesis and hydrolysis are not necessarily reversible in the proton-translocating ATPase (F1F0).     

Isolated epsilon subunit of Bacillus subtilis F1-ATPase binds ATP

Previously, we demonstrated ATP binding to the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus PS3 [Kato-Yamada Y., Yoshida M. (2003) J. Biol. Chem. 278, 36013]. However, whether it is a general feature of the epsilon subunit from other sources is yet unclear. Here, using a sensitive method to detect weak interactions between fluorescently labeled epsilon subunit and nucleotide, it was shown that the epsilon subunit of F1-ATPase from Bacillus subtilis also bound ATP. The dissociation constant for ATP binding at room temperature was calculated to be 2 mM, which may be suitable for sensing cellular ATP concentration in vivo.     

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.