Role of the ATP synthase alpha-subunit in conferring sensitivity to tentoxin.

Tentoxin, produced by phytopathogenic fungi, selectively affects the function of the ATP synthase enzymes of certain sensitive plant species. Binding of tentoxin to a high affinity (K(i) approximately 10 nM) site on the chloroplast F(1) (CF(1)) strongly inhibits catalytic function, whereas binding to a second, lower affinity site (K(d) > 10 microM) leads to restoration and even stimulation of catalytic activity. Sensitivity to tentoxin has been shown to be due, in part, to the nature of the amino acid residue at position 83 on the catalytic beta subunit of CF(1). An aspartate in this position is required, but is not sufficient, for tentoxin inhibition. By comparison with the solved structure of mitochondrial F(1) [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], Asp83 is probably located at an interface between alpha and beta subunits on CF(1) where residues on the alpha subunit could also participate in tentoxin binding. A hybrid core F(1) enzyme assembled with beta and gamma subunits of the tentoxin-sensitive spinach CF(1), and an alpha subunit of the tentoxin-insensitive photosynthetic bacterium Rhodospirillum rubrum F(1) (RrF(1)), was stimulated but not inhibited by tentoxin [Tucker, W. C., Du, Z., Gromet-Elhanan, Z. and Richter, M. L. (2001) Eur. J. Biochem. 268, 2179-2186]. In this study, chimeric alpha subunits were prepared by introducing short segments of the spinach CF(1) alpha subunit from a poorly conserved region which is immediately adjacent to beta-Asp83 in the crystal structure, into equivalent positions in the RrF(1) alpha subunit using oligonucleotide-directed mutagenesis. Hybrid enzymes containing these chimeric alpha subunits had both the high affinity inhibitory tentoxin binding site and the lower affinity stimulatory site. Changing beta-Asp83 to leucine resulted in loss of both inhibition and stimulation by tentoxin in the chimeras. The results indicate that tentoxin inhibition requires additional alpha residues that are not present on the RrF(1) alpha subunit. A structural model of a putative inhibitory tentoxin binding pocket is presented.     

Hybrid Rhodospirillum rubrum F(0)F(1) ATP synthases containing spinach chloroplast F(1) beta or alpha and beta subunits reveal the essential role of the alpha subunit in ATP synthesis and tentoxin sensitivity

Trace amounts ( approximately 5%) of the chloroplast alpha subunit were found to be absolutely required for effective restoration of catalytic function to LiCl-treated chromatophores of Rhodospirillum rubrum with the chloroplast beta subunit (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072). To clarify the role of the alpha subunit in the rebinding of beta, restoration of catalytic function, and conferral of sensitivity to the chloroplast-specific inhibitor tentoxin, LiCl-treated chromatophores were analyzed by immunoblotting before and after reconstitution with mixtures of R. rubrum and chloroplast alpha and beta subunits. The treated chromatophores were found to have lost, in addition to most of their beta subunits, approximately a third of the alpha subunits, and restoration of catalytic activity required rebinding of both subunits. The hybrid reconstituted with the R. rubrum alpha and chloroplast beta subunits was active in ATP synthesis as well as hydrolysis, and both activities were completely resistant to tentoxin. In contrast, a hybrid reconstituted with both chloroplast alpha and beta subunits restored only a MgATPase activity, which was fully inhibited by tentoxin. These results indicate that all three copies of the R. rubrum alpha subunit are required for proton-coupled ATP synthesis, whereas for conferral of tentoxin sensitivity at least one copy of the chloroplast alpha subunit is required together with the chloroplast beta subunit. The hybrid system was further used to examine the effects of amino acid substitution at position 83 of the beta subunit on sensitivity to tentoxin.     

Reconstitution of the H+-ATPase complex of Rhodospirillum rubrum by the beta subunit of the chloroplast coupling factor 1

A method is described for isolating the beta subunit from spinach chloroplast F1 (CF1). The isolated beta subunit reconstituted an active F1 hybrid with the F1 of Rhodospirillum rubrum chromatophores from which the beta subunit had been removed. The CF1 beta subunit was similar to the isolated beta subunit of Escherichia coli F1 (Gromet-Elhanan, Z., Khananshivili, D., Weiss, S., Kanazawa, H., and Futai, M. (1985) J. Biol. Chem. 260, 12635-12640) in that it restored a substantial rate of ATP hydrolysis and low, but significant light-dependent ATP synthesis to the beta-less chromatophores. The low rate of photophosphorylation observed with the hybrid enzyme probably resulted from a looser coupling of the CF1 beta subunit to proton translocation in the R. rubrum Fo-F1 complex. The hybrid enzyme exhibited a high specificity for Mg2+-ATP as substrate for ATP hydrolysis and both ATP synthesis and hydrolysis were strongly inhibited by the antibiotic tentoxin. In contrast, chromatophores reconstituted with the native R. rubrum beta subunit actively hydrolyzed both Mg2+-ATP and Ca2+-ATP and were insensitive to tentoxin. These results indicate a close functional homology between the beta subunits of the prokaryotic and eukaryotic H+-ATPases and suggest a role for the beta subunit in conferring the different metal ion specificities and inhibitor sensitivities upon the enzymes. They also demonstrate the feasibility of isolating the beta subunit from CF1 in a reconstitutively active form.     

Intertwined translational regulations set uneven stoichiometry of chloroplast ATP synthase subunits

The (C)F1 sector from H(+)-ATP synthases comprises five subunits: alpha, beta, gamma, delta and epsilon, assembled in a 3:3:1:1:1 stoichiometry. Here, we describe the molecular mechanism ensuring this unique stoichiometry, required for the functional assembly of the chloroplast enzyme. It relies on a translational feedback loop operating in two steps along the assembly pathway of CF1. In Chlamydomonas, production of the nucleus-encoded subunit gamma is required for sustained translation of the chloroplast-encoded subunit beta, which in turn stimulates the expression of the chloroplast-encoded subunit alpha. Translational downregulation of subunits beta or alpha, when not assembled, is born by the 5'UTRs of their own mRNAs, pointing to a regulation of translation initiation. We show that subunit gamma, by assembling with alpha(3)beta(3) hexamers, releases a negative feedback exerted by alpha/beta assembly intermediates on translation of subunit beta. Moreover, translation of subunit alpha is transactivated by subunit beta, an observation unprecedented in the biogenesis of organelle proteins.     

Added subunit beta of CF1 as well as gamma/delta/epsilon restore photophosphorylation in partially CF1-depleted thylakoids.

We investigated the ability of subunits beta, gamma, delta, and epsilon of CF1, the F1-ATPase of chloroplasts, to interact with exposed CF0 in EDTA-treated, partially CF1-depleted thylakoid membranes. We measured the ability of subunits beta, gamma, delta, and epsilon to stimulate the rate of photophosphorylation under continuous light and, for subunit beta, also the ability to diminish the proton leakage through exposed CF0 by deceleration of the decay of electrochromic absorption transients under flashing light. The greatest effect was caused by subunit beta, followed by gamma/delta/epsilon. Pairwise combinations of gamma, delta, and epsilon or each of these subunits alone were only marginally effective. Subunit gamma from the thermophilic bacterium PS 3 in combination with chloroplast delta and epsilon was as effective as chloroplast gamma. The finding that the small CF1 subunits in concert and the beta subunit by itself specifically interacted with the exposed proton channel CF0, qualifies the previous concept of subunit delta acting particularly as a plug to the open CF0 channel. The interactions between the channel and the catalytic portion of the enzyme seem to involve most of the small, and at least beta of the large subunits.     

Recent developments on structural and functional aspects of the F1 sector of H+-linked ATPases

This review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of F1 have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An alpha 3 beta 3 gamma delta epsilon stoichiometry is now accepted for MF1 and BF1; the alpha 2 beta 2 gamma 2 delta 2 epsilon 2 stoichiometry for CF1 remains as matter of debate. The major subunits alpha, beta and gamma are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits delta and epsilon. The delta subunit of MF1 corresponds to the epsilon subunit of BF1 and CF1, whereas the mitochondrial subunit equivalent to the delta subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The alpha beta gamma assembly is endowed with ATPase activity, beta being considered as the catalytic subunit and gamma as a proton gate. On the other hand, the delta and epsilon subunits of BF1 and CF1 most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondrial ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the epsilon subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is beta comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The alpha subunit also plays a central role in catalysis, since structural alteration of alpha by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rebuilt 3D structure of the chloroplast f1 ATPase-tentoxin complex

The F1 part of the chloroplast H+ adenosine triphosphate (ATP)-synthase (CF1) strongly interacts with tentoxin, a natural fungous cyclic tetrapeptide known to inhibit the chloroplast enzyme and not the mammalian mitochondrial enzyme. Whereas the synthesis or the hydrolysis of ATP requires the stepwise rotation of the protein rotor gamma within the (alphabeta)3 crown, only one molecule of tentoxin is needed to fully inhibit the complex. With the help of an original homology modeling technique, based on robust distance geometry protocols, we built a tridimensional model of the alpha3beta3gamma CF1) subcomplex (3200 esidues), in which we introduced three different nucleotide occupancies to check their possible influence on the tentoxin binding site. Simultaneous comparison of three available high-resolution X-ray structures of F1, performed with a local structural alignment search tool, led to characterizing common structural blocks and the distorsions experienced by the complex during the catalytic turnover. The common structural blocks were used as a starting point of the spinach CF1 structure rebuilding. Finally, tentoxin was docked into its putative binding site of the reconstructed structure. The docking method was initially validated in the mitochondrial enzyme by its ability to relocate nucleotides into their original position in the crystal. Tentoxin binding was found possible to the two alpha/beta interfaces associated with the empty and adenosine diphosphate (ADP)-loaded catalytic sites, but not to the one associated with the ATP-loaded site. These results suggest a mechanism of CF1 inhibition by one molecule of tentoxin, by the impossibility of the alpha/beta interface bearing tentoxin to pass through the ATP-loaded state.     

Identification and differential accumulation of two isoforms of the CF1-beta subunit under high light stress in Brassica rapa.

The chloroplast ATP synthase coupling factor CF1 complex contains five nonidentical subunits, alpha, beta, gamma, delta, and epsilon, with a stoichiometry of 3:3:1:1:1. The beta subunit contains the catalytic site for ATP synthesis during photooxidative phosphorylation in the chloroplast. In this study, we have identified two isoforms of the CF1-beta subunit at 56 and 54 kDa in the chloroplast of Brassica rapa, through isolation/purification, proteolytic digestion and internal peptide sequencing. Examining their accumulation pattern demonstrates that both isoforms coexist during chloroplast biogenesis and in mature thylakoid membranes, but the 54 kDa isoform is more apparently upregulated by light or under light stress. LiDS-PAGE shows that the 56 kDa is a major isoform of the CF1-beta subunit under normal light conditions, and its amount was not influenced during high light or other light stress treatments. The 54 kDa isoform is a minor band at normal conditions; however, it significantly increased under excess light stresses, such as high or low light with drought and/or high temperature. Particularly, a ninefold increase was observed after 8-10 h of high light treatment with drought and high temperature. The results suggest that light stress induction of the 54 kDa CF1-beta isoform may present a positive response during chloroplast photoacclimation.     

Trafficking and potential assembly patterns of epsilon-containing GABAA receptors

Incorporation of the epsilon subunit into the GABAA receptor has been suggested to confer unusual, but variable, biophysical and pharmacological characteristics to both recombinant and native receptors. Due to their structural similarity with the gamma subunits, epsilon subunits have been assumed to substitute at the single position of the gamma subunit in assembled receptors. However, prior work suggests that functional variability in epsilon-containing receptors may reflect alternative sites of incorporation and of not just one, but possibly multiple epsilon subunits in the pentameric receptor complex. Here we present data indicating that increased expression of epsilon, in conjunction with alpha2 and beta3 subunits, results in expression of GABAA receptors with correspondingly altered rectification, deactivation and levels of spontaneous openings, but not increased total current density. We also provide data that the epsilon subunit, like the beta3 subunit, can self-export and data from chimeric receptors suggesting that similarities between the assembly domains of the beta3 and the epsilon subunits may allow the epsilon subunit to replace the beta, as well as the gamma, subunit. The substitution of an epsilon for a beta, as well as the gamma subunit and formation of receptors with alternative patterns of assembly with respect to epsilon incorporation may underlie the observed variability in both biophysical and pharmacological properties noted not only in recombinant, but also in native receptors.     

Significance of the epsilon subunit in the thiol modulation of chloroplast ATP synthase

To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity.     

Chemical cross-linking studies of chloroplast coupling factor 1.

Cross-linking reagents have been used to link covalently adjacent subunits of solubilized spinach chloroplast coupling factor 1, which is a latent ATPase. 1,5-Difluoro-2,4-dinitrobenzene, dimethyl-3,3'-dithiobispropionimidate, and dimethylsuberimidate are able to form bridges of 3 to 11 A between amino groups, and hydrogen peroxide and the o-phenanthroline-cupric ion complex catalyze the oxidation of intrinsic sulfhydryl groups. The five individual subunit bands (alpha, beta, gamma, delta, and epsilon) and several new aggregate bands can be separated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same four fastest moving aggregate bands, as characterized by their mobilities, migrate more slowly than the heaviest subunit band and appear with all of the cross-linkers employed. The subunit composition of the aggregate bands has been determined through the use of the reversible cross-linkers, dimethyldithiobispropionimidate, (o-phenanthroline)2Cu(II), and H2O2, and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which aggregates are separated in the first dimension, the disulfide cross-links are cleaved, and the individual subunits present in the aggregates are separated in the second dimension. The subunits are detected by Coomassie brilliant blue staining and by labeling some of the sulfhydryl groups of the gamma and epsilon subunits with radioactive N-ethylmaleimide. The results obtained indicate that the alpha and beta subunits can cross-link directly with each of the other subunits, that two beta subunits are adjacent, and that gamma epsilon, gamma epsilon 2, alpha delta, and beta delta aggregates are present. A minimal subunit stoichiometry consistent with these results is alpha 2 beta 2 gamma delta epsilon 2. A possible structural model of the coupling factor is derived from the data. Similar, but less extensive, experiments have been carried out with the heat-activated coupling factor (which is an ATPase); no differences in the spatial arrangement of subunits are detected from the two-dimensional gel electrophoresis analysis of the cross-linked aggregates.     

Assembled F1-(alpha beta ) and Hybrid F1-alpha 3beta 3gamma -ATPases from Rhodospirillum rubrum alpha, wild type or mutant beta, and chloroplast gamma subunits. Demonstration of Mg2+versus Ca2+-induced differences in catalytic site structure and function

Refolding together the expressed alpha and beta subunits of the Rhodospirillum rubrum F(1)(RF(1))-ATPase led to assembly of only alpha(1)beta(1) dimers, showing a stable low MgATPase activity. When incubated in the presence of AlCl(3), NaF and either MgAD(T)P or CaAD(T)P, all dimers associated into closed alpha(3)beta(3) hexamers, which also gained a low CaATPase activity. Both hexamer ATPase activities exhibited identical rates and properties to the open dimer MgATPase. These results indicate that: a) the hexamer, as the dimer, has no catalytic cooperativity; b) aluminium fluoride does not inhibit their MgATPase activity; and c) it does enable the assembly of RrF(1)-alpha(3)beta(3) hexamers by stabilizing their noncatalytic alpha/beta interfaces. Refolding of the RrF(1)-alpha and beta subunits together with the spinach chloroplast F(1) (CF(1))-gamma enabled a simple one-step assembly of two different hybrid RrF(1)-alpha(3)beta(3)/CF(1)gamma complexes, containing either wild type RrF(1)-beta or the catalytic site mutant RrF(1)beta-T159S. They exhibited over 100-fold higher CaATPase and MgATPase activities than the stabilized hexamers and showed very different catalytic properties. The hybrid wild type MgATPase activity was, as that of RrF(1) and CF(1) and unlike its higher CaATPase activity, regulated by excess free Mg(2+) ions, stimulated by sulfite, and inhibited by azide. The hybrid mutant had on the other hand a low CaATPase but an exceptionally high MgATPase activity, which was much less sensitive to the specific MgATPase effectors. All these very different ATPase activities were regulated by thiol modulation of the hybrid unique CF(1)-gamma disulfide bond. These hybrid complexes can provide information on the as yet unknown factors that couple ATP binding and hydrolysis to both thiol modulation and rotational motion of their CF(1)-gamma subunit.     

Effect of proteolytic digestion on the Ca2+-ATPase activity and subunits of latent and thiol-activated chloroplast coupling factor 1

The activation by proteases of the Ca2+-dependent ATPase of chloroplast coupling factor 1 (CF1) has been investigated. Using low concentrations of papain and trypsin, the increase in ATPase activity and the degradation of the five subunits of CF1 were compared. Sodium dodecyl sulfate-gel electrophoresis of protease-treated CF1 revealed that the delta subunit was very rapidly degraded and that the alpha and beta subunits were clipped. The gamma and epsilon subunits were more resistant to digestion. The modification of the alpha subunit of latent CF1 most closely correlated with the activation of Ca2+-ATPase activity. Trypsin treatment of dithiothreitol-activated CF1 resulted in a very rapid increase in Ca2+-ATPase activity and a corresponding rapid cleavage of the gamma subunit to a 25,000-dalton species. With more prolonged treatment, the 25,000-dalton species was cleaved to fragments of 14,000 and 11,000-daltons. Dithiothreitol treatment did not alter the rate of attack on the other subunits. The gamma subunit of heat-activated CF1 was also more susceptible to protease digestion. The increased protease sensitivity of the gamma subunit of soluble CF1 after treatment with dithiothreitol or heat mimics the increased protease sensitivity of the gamma subunit of bound CF1 when thylakoids are treated with trypsin during illumination (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5915-5920). These results suggest that the conformational changes that occur when purified CF1 is exposed to dithiothreitol are similar to those that CF1 bound to thylakoid membranes undergoes under illumination.     

Important subunit interactions in the chloroplast ATP synthase

General structural features of the chloroplast ATP synthase are summarized highlighting differences between the chloroplast enzyme and other ATP synthases. Much of the review is focused on the important interactions between the epsilon and gamma subunits of the chloroplast coupling factor 1 (CF(1)) which are involved in regulating the ATP hydrolytic activity of the enzyme and also in transferring energy from the membrane segment, chloroplast coupling factor 0 (CF(0)), to the catalytic sites on CF(1). A simple model is presented which summarizes properties of three known states of activation of the membrane-bound form of CF(1). The three states can be explained in terms of three different bound conformational states of the epsilon subunit. One of the three states, the fully active state, is only found in the membrane-bound form of CF(1). The lack of this state in the isolated form of CF(1), together with the confirmed presence of permanent asymmetry among the alpha, beta and gamma subunits of isolated CF(1), indicate that ATP hydrolysis by isolated CF(1) may involve only two of the three potential catalytic sites on the enzyme. Thus isolated CF(1) may be different from other F(1) enzymes in that it only operates on 'two cylinders' whereby the gamma subunit does not rotate through a full 360 degrees during the catalytic cycle. On the membrane in the presence of a light-induced proton gradient the enzyme assumes a conformation which may involve all three catalytic sites and a full 360 degrees rotation of gamma during catalysis.     

Substitution of a single amino acid switches the tentoxin-resistant thermophilic F1-ATPase into a tentoxin-sensitive enzyme

In contrast to the homologous bacterial and mitochondrial enzymes the chloroplast F(1)-ATPase (CF(1)) is strongly affected by the phytopathogenic inhibitor tentoxin. Based on structural information obtained from crystals of a CF(1)-tentoxin co-complex (Groth, G. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3464-3468) we have replaced residues betaSer(66) and alphaArg(132) in the alpha(3)beta(3)gamma subcomplex of the thermophilic F(1)-ATPase from Bacillus PS3 by the corresponding residues of the chloroplast ATPase to confer tentoxin sensitivity to the thermophilic enzyme. The mutation alphaArg(132) --> Pro, proposed to relieve steric constraints on tentoxin binding, did not have any significant effect. However, mutation betaSer(66) --> Ala, predicted to provide a crucial hydrogen bond with the inhibitor, resulted in tentoxin inhibition of ATP hydrolysis comparable with the situation found with the chloroplast enzyme.     

ATPase activity of a highly stable alpha(3)beta(3)gamma subcomplex of thermophilic F(1) can be regulated by the introduced regulatory region of gamma subunit of chloroplast F(1)

A mutant F(1)-ATPase alpha(3)beta(3)gamma subcomplex from the thermophilic Bacillus PS3 was constructed, in which 111 amino acid residues (Val(92) to Phe(202)) from the central region of the gamma subunit were replaced by the 148 amino acid residues of the homologous region from spinach chloroplast F(1)-ATPase gamma subunit, including the regulatory stretch, and were designated as alpha(3)beta(3)gamma((TCT)) (Thermophilic-Chloroplast-Thermophilic). By the insertion of this regulatory region into the gamma subunit of thermophilic F(1), we could confer the thiol modulation property to the thermophilic alpha(3)beta(3)gamma subcomplex. The overexpressed alpha(3)beta(3)gamma((TCT)) was easily purified in large scale, and the ATP hydrolyzing activity of the obtained complex was shown to increase up to 3-fold upon treatment with chloroplast thioredoxin-f and dithiothreitol. No loss of thermostability compared with the wild type subcomplex was found, and activation by dithiothreitol was functional at temperatures up to 80 degrees C. alpha(3)beta(3)gamma((TCT)) was inhibited by the epsilon subunit from chloroplast F(1)-ATPase but not by the one from the thermophilic F(1)-ATPase, indicating that the introduced amino acid residues from chloroplast F(1)-gamma subunit are important for functional interaction with the epsilon subunit.     

The epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases. Structure, arrangement, and role of the epsilon subunit in energy coupling within the complex

Recent studies show that the epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases is a component of the central stalk that links the F(1) and F(0) parts. This subunit interacts with alpha, beta and gamma subunits of F(1) and the c subunit ring of F(0). Along with the gamma subunit, epsilon is a part of the rotor that couples events at the three catalytic sites sequentially with proton translocation through the F(0) part. Structural data on the epsilon subunit when separated from the complex and in situ are reviewed, and the functioning of this polypeptide in coupling within the ATP synthase is considered.     

Evidence for the existence of protomers in the assembly of encephalomyocarditis virus.

Two capsid precursor subunits, which sediment on glycerol gradients at 13S and 14S, respectively, have been identified in cytoplasmic extracts of encephalomyocarditis virus-infected HeLa cells. The 13S subunit, which was detected after a 10-min pulse label with -3H-labeled amino acids, contained only capsid precursor chain A (mol wt 100,000). When the 10-min pulse label in such cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic extracts was replaced by a 14S subunit containing equimolar proportions of three chains: epsilon, gamma, and alpha. This (epsilon, gamma, alpha)-containing 14S subunit could be dissociated into 6S subunits with the same polypeptide composition. The sedimentation properties and the polypeptide stoichiometry of these three precursor subunits, when compared with those of the 13S, (beta, gamma, alpha)(5), and 5S, (beta, gamma, alpha), subunits derived by acid dissociation of purified virions, suggest the following structural assignments: 13S, (A)(5); 14S, (epsilon, gamma, alpha)(5), 6S, (epsilon, gamma, alpha). The molecular weights of the individually isolated capsid chains were determined by gel filtration in 6 M guanidine hydrochloride to be: epsilon, 36,000; alpha, 32,000; beta, 29,500; gamma, 26,500; and delta, 7,800. With the exception of the delta-chain, these values are in reasonable agreement with the values previously determined by electrophoresis on sodium dodecyl sulfatepolyacrylamide gels. These data support the hypothesis that picornavirus capsids are assembled from identical protomers according to the following scheme: (A) leads to (A)(5) leads to (epsilon, gamma, alpha)(5) leads to (delta, beta, gamma, alpha)60-n(epsilon, gamma, alpha)n where n is the number of immature protomers per virion.     

Extraction and purification of the beta subunit and an active alpha beta-core complex from the spinach chloroplast CFoF1-ATP synthase.

Incubation of Rhodospirillum rubrum chromatophores with 2 M LiCl in the presence of MgATP has been shown to remove their F1 beta subunit leaving inactive but fully reconstitutable beta-less chromatophores (Gromet-Elhanan, Z., and Khanashvili, D., (1986) Methods Enzymol, 126, 528-538). A similar treatment of thoroughly washed spinach thylakoids has now been shown to release the CF1 beta subunit (CF1 beta) together with a complex containing equal amounts of CF1 alpha and CF1 beta (CF1 (alpha beta]. The purified CF1 (alpha beta) complex can reconstitute an active membrane-bound hybrid F0F1-ATPase with beta-less R. rubrum chromatophores and also catalyzes a low but very reproducible soluble MgATPase. Purified CF1 beta shows none of these activities although it can bind as efficiently as CF1 (alpha beta) to the beta-less chromatophores. By subjecting the crude spinach 2 m LiCl extract to dissociating conditions an enriched CF1 beta preparation is released. It contains traces of CF1 alpha and CF1 delta, is able to reconstitute an active hybrid F0F1-ATPase but, as the pure CF1 beta shows no soluble ATPase activity. These results indicate that trace amounts of CF1 alpha are enough for endowing CF1 beta with a reconstitutive capacity, but for exhibition of a significant soluble ATPase activity equivalent amounts of CF1 alpha and beta are required. The CF 1 (alpha beta) complex isolated and purified in this report thus represents the minimal catalytic core of the CF1-ATPase.