Cross-reconstitution of the F0F1-ATP synthases of chloroplasts and Escherichia coli with special emphasis on subunit delta

F0F1-ATP synthases catalyse ATP formation from ADP and Pi by using the free energy supplied by the transmembrane electrochemical potential of the proton. The delta subunit of F1 plays an important role at the interface between the channel portion F0 and the catalytic portion F1. In chloroplasts it can plug the protonic conductance of CF0 and in Escherichia coli it is required for binding of EF1 to EF0. We wanted to know whether or not delta of one species was effective between F0 and F1 of the other species and vice versa. To this end the respective coupling membrane (thylakoids, everted vesicles from E. coli) was (partially) depleted of F1 and purified F1, F1(-delta), and delta were added in various combinations to the F1-depleted membranes. The efficiency or reconstitution was measured in thylakoids via the rate of phenazinemethosulfate-mediated cyclic photophosphorylation and in E. coli everted vesicles via the degree of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching. Addition of CF1 to partially CF1-depleted thylakoid vesicles restored photophosphorylation to the highest extent. CF1(-delta)+chloroplast delta, EF1, EF1(-delta)+E. coli delta were also effective but to lesser extent. CF1(-delta)+E. coli delta and EF1(-delta)+chloroplast delta restored photophosphorylation to a small but still significant extent. With F1-depleted everted vesicles prepared by repeated EDTA treatment of E. coli membranes, addition of CF1, CF1 (-delta)+chloroplast delta and CF1(-delta)+E. coli delta gave approximately half the extent of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching as compared to EF1 or EF1(-delta)+E. coli delta by energization of the vesicles with NADH, while Ef1(-delta)+chloroplast delta was ineffective. All 'mixed' combinations were probably reconstitutively active only by plugging the protonic leak through the exposed F0 (structural reconstitution) rather than by catalytic activity. Nevertheless, the cross-reconstitution is stunning in view of the weak sequence similarity between chloroplast delta and E. coli delta. It favors a role of delta as a conformational transducer rather than as a proton conductor between F0 and F1.     

Delta subunit of chloroplast coupling factor 1 inhibits proton leakage through coupling factor O

The ATP synthase of chloroplasts consists of a proton-conducting portion, CF0, and a catalytic portion, CF1. The smaller subunits of CF1, in particular delta, may play a key role in the coupling of proton transport to ATP synthesis. Purified subunit delta, when added to partially CF1-depleted thylakoid membranes, can restore photophosphorylation (Engelbrecht, S., and Junge, W. (1987) Eur. J. Biochem. 172, 213-218). We report here that it does so by blocking proton conduction through CF0. Thylakoids were CF1-depleted by incubation in hypoosmolar NaCl/EDTA solutions. Variation of the NaCl concentrations and of the incubation times not only changed the overall degree of CF1 depletion but also the subunit composition of solubilized CF1, namely CF1 containing delta and CF1(-delta). This was quantified by immunoelectrophoresis and by fast protein liquid chromatography. Proton conduction was measured by flash spectrophotometry by using standard electrochromic and pH-indicating absorption changes. The removal of integral CF1 was correlated with high electric conductance of thylakoid membranes, an increased extent of rapid proton leakage, and loss of ATP synthesis activity, which exceeded the percentual loss of CF1. The removal of predominantly CF1(-delta) resulted in comparatively lesser effects on the loss of ATP synthesis and on the extent and velocity of proton leakage. On the same line, addition of integral CF1 and of purified delta diminished the electric leak in CF1-depleted thylakoids. Both approaches, the controlled removal of CF1 and CF1(-delta), respectively, and addition of delta and CF1 showed that delta can act as a "stopcock" to the exposed proton channel CF0.     

Subunit delta of H(+)-ATPases: at the interface between proton flow and ATP synthesis

The ATP synthases in photophosphorylation and respiration are of the F-type with a membrane-bound proton channel, F0, and an extrinsic catalytic portion, F1. The properties of one particular subunit, delta (in chloroplasts and Escherichia coli) and OSCP (in mitochondria), are reviewed and the role of this subunit at the interface between F0 and F1 is discussed. Delta and OSCP from the three sources have in common the molecular mass (approximately 20 kDa), an elongated shape (axial ratio in solution about 3:1), one high-affinity binding site to F1 (Kd approximately 100 nM) plus probably one or two further low-affinity sites. When isolated delta is added to CF1-depleted thylakoid membranes, it can block proton flow through exposed CF0 channels, as do CF1 or CF1(-delta)+ delta. This identifies delta as part of the proton conductor or, alternatively, conformational energy transducer between F0 (proton flow) and F1 (ATP). Hybrid constructs as CF1(-delta)+ E. coli delta and EF1(-delta)+ chloroplast delta diminish proton flow through CF0.CF1(-delta) + E. coli delta does the same on EF0. Impairment of proton leaks either through CF0 or through EF0 causes "structural reconstitution' of ATP synthesis by remaining intact F0F1. Functional reconstitution (ATP synthesis by fully reconstructed F0F1), however, is absolutely dependent on the presence of subunit delta and is therefore observed only with CF1 or CF1(-delta) + chloroplast delta on CF0 and EF1 or EF1(-delta) + E. coli delta on EF0. The effect of hybrid constructs on F0 channels is surprising in view of the limited sequence homology between chloroplast and E. coli delta (36% conserved residues including conservative replacements). An analysis of the distribution of the conserved residues at present does not allow us to discriminate between the postulated conformational or proton-conductive roles of subunit delta.     

Reconstitution of photophosphorylation in EDTA-treated thylakoids by added chloroplast coupling factor 1 (ATPase) and chloroplast coupling factor 1 lacking the delta subunit. Structural or functional?

Upon EDTA treatment thylakoids lose the chloroplast coupling factor 1 (CF1) part of their ATP synthase, CF0CF1, this exposes the proton channel, CF0. The previously established ability of the CF1 subunit delta to block the proton leak through CF0 prompted us to study (a) the ability of complete CF1 and, for comparison, CF1 lacking the delta subunit to block proton leakage and thereby to reconstitute structurally some photophosphorylation activity of the remaining CF0CF1 molecules and (b) their ability to form functional enzymes (functional reconstitution). In order to discriminate between activities caused by added CF1 or CF1(-delta) and remaining CF0CF1, the former were inhibited by chemical modification of subunit beta by N,N'-dicyclohexyl carbodiimide (DCCD) and the latter by tentoxin. We found that added CF1 acted both structurally and functionally while added DCCD-treated CF1 (DCCD-CF1) acted only structurally. In contrast to previous observations, CF1(-delta) and DCCD-CF1(-delta) also acted structurally although the reduction of proton leakage was smaller than with DCCD-CF1. Hence there was no functional reconstitution without subunit delta present. Previous studies indicated that only a small fraction of exposed CF0 is highly conducting and that this small fraction is distinguished by its high affinity for added CF1. The results of this study point rather to a wider distribution of CF0 conductance states and binding affinities.     

Aspects of Subunit Interactions in the Chloroplast ATP Synthase (I. Isolation of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids)

A chloroplast ATP synthase complex (CF1 [chloroplast-coupling factor 1]-CF0 [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF0 subunits except subunit III (also known as the proteolipid subunit) was purified to study the interaction between CF1 and subunit III. Subunit III has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit inter-actions involving subunit III will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CF1-CF0 preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit III in the complex was positively identified by amino acid analysis and N-terminal sequencing. The association of subunit III with CF1 was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF1 was removed from the CF1-III complex by ethylenediaminetetracetate treatment. The subunit III-proteoliposomes were competent to rebind purified CF1. These results indicate that subunit III directly interacts with CF1 in spinach thylakoids.     

Inhibition of Thylakoid ATPase by Venturicidin as an Indicator of CF1-CF0 Interaction

Venturicidin inhibits the F0 portion of membrane-located, H+-pumping ATPases. We find it meets the criteria for an energy transfer inhibitor for spinach (Spinacia oleracea) thylakoids: complete inhibition of photophosphorylation and of photophosphorylation-stimulated and basal electron flow rates, but not of electron flow under uncoupled conditions. The extent of H+ uptake in the light is stimulated by venturicidin (vtcd), as expected for a compound blocking H+ efflux through CF0. Vtcd had no effect on the nonproton pumping, methanol-stimulated ATPase of thylakoids or on soluble CF1 ATPase. Under totally uncoupled conditions (saturating NH4Cl + gramicidin), vtcd can still inhibit sulfite-stimulated thylakoid ATPase completely. The concentration of vtcd needed for inhibition of ATPase was proportional to the concentration of thylakoids present in the assay, with an apparent stoichiometry of about 10 vtcd molecules per CF1/CF0 for 50% inhibition. Vtcd raised the Km for ATP somewhat, but had a stronger effect on the Vmax with respect to ATP. Inhibition by saturating vtcd ranged from 50 to 100%, depending on the condition of the thylakoids. Grinding leaves in buffer containing 0.2 M choline chloride (known to provide superior photophosphorylation rates) helped bring on maximum vtcd inhibition; trypsin treatment or aging of thylakoids brought on vtcd-resistant ATPase. We conclude that the extent of inhibition by vtcd can be used as an indicator of the tightness of coupling between CF1 and CF0.     

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.     

Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase. The role of the delta subunit for proton conduction through CF0

Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the delta & epsilon subunits: CF1(-delta) and CF1(-epsilon). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the beta subunit and in separate peaks CF1(-delta) and CF1(-epsilon). Elution from Mono Q in the presence of tenside yielded a beta delta fragment, CF1(-delta) and CF1(-delta epsilon). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-delta) and CF1(-delta epsilon) were active with cross-reactivity between spinach, pea and maize. delta-containing CF1 always reconstituted higher activities than delta-deficient CF1. The beta delta fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while beta and DCCD-inhibited CF1(-delta) were not. These results support the notion that subunit delta can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082].     

CF0, the proton channel of chloroplast ATP synthase. After removal of CF1 it appears in two forms with highly different proton conductance

The discharge of the flash-induced transmembrane voltage through the exposed proton channel, CF0, of the chloroplast ATP synthase, CF0CF1 was investigated. EDTA treatment of thylakoid membranes exposed approximately 50% of total CF0 by removal of the CF1 counterparts. This greatly accelerated the decay of the transmembrane voltage, as was apparent from electrochromic-absorption changes of intrinsic pigments and by pH-indicating-absorption changes of added dyes. Two decay processes were discernible, one rapid with a typical half-decay time of 2 ms, and a slower one with a half-decay time variable between 20-100 ms. Both were sensitive to CF0 inhibitors, but only the rapid decay process was also inhibited by added CF1. CF1 was effective in surprisingly small amounts, which were significantly lower than those previously removed by EDTA treatment. This finding corroborated our previous conclusion that the rapid decay of the transmembrane voltage was attributable to only a few high-conductance channels among many CF0 molecules, typically in the order of one channel/CF1-depleted EDTA vesicle. Inhibition of photophosphorylation in control thylakoids was measured as function of the concentration of CF0 inhibitors. It was compared with the inhibition of proton conduction through exposed CF0 in EDTA vesicles. Photophosphorylation and proton conduction by the high-conductance form of CF0 were inhibited by the same low inhibitor concentrations. This suggested that the high-conducting form of CF0 with a time-averaged single-channel conductance of 1 pS [Lill, H., Althoff, G. & Junge, W. (1987) J. Membrane Biol. 98, 69-78] represented the proton channel in the integral enzyme, which acted as a low-impedance access from the thylakoid lumen to the coupling site in CF0CF1. The slow decay process was attributed to a majority of low-conductance CF0 channels, i.e. about 50 molecules/vesicle. The conductance of these channels was more than 100-fold lower and they did not compete with the very few highly conducting channels for rebinding of added CF1. The low proton conduction of the majority of exposed CF0 molecules, possibly due to a structural rearrangement, may be protecting the thylakoid membrane against rapid energy dissipation caused by accidental loss of CF1. It may also explain the low single-channel conductance of bacterial F0 reported in the literature.     

The chloroplast ATP synthase in Chlamydomonas reinhardtii. I. Characterization of its nine constitutive subunits

We have characterized the subunit composition of the chloroplast ATP synthase from Chlamydomonas reinhardtii by means of a comparison of the polypeptide deficiencies in a mutant defective in photophosphorylation, with the polypeptide content in purified coupling factor (CF)1 and CF1.CF0 complexes. We could distinguish nine subunits in the enzyme, four of which were CF0 subunits. Further characterization of these subunits was undertaken by immunoblotting experiments, [14C]dicyclohexylcarbodiimide binding and analysis of their site of translation. In particular, we were able to show the presence of an as yet unidentified delta subunit in CF1 from C. reinhardtii. We have identified a 70-kDa peripheral membrane protein in the thylakoid membranes of C. reinhardtii, which is immunologically related to the beta subunit of CF1. We discuss its conceivable ATPase function with respect to the Ca2+-dependent ATPase activity previously reported in the thylakoid membranes from C. reinhardtii.     

Purified subunit delta of chloroplast coupling factor CF1 reconstitutes photophosphorylation in partially CF1-depleted membranes

The ATP synthase of chloroplasts consists of the proton channel, CF0, and the catalytic part, CF1, which carries nucleotide-binding sites on subunits alpha and beta. The still poorly understood interaction between CF0 and the catalytic sites on CF1 is mediated by the smaller subunits gamma, delta and epsilon of CF1. We investigated the ability of purified delta to block proton leakage through CF0 channels after their exposure by removal of the CF1 counterpart. Thylakoids were partially depleted of CF1 by EDTA treatment. This increased their proton permeability and thereby reduced the rate of photophosphorylation. Subunit delta was isolated and purified by FPLC [Engelbrecht, S. and Junge, W. (1987) FEBS Lett. 219, 321-325]. Addition of delta to EDTA-treated thylakoids reconstituted high rates of phenazine-methosulfate-mediated photophosphorylation. Since delta does not interact with nucleotides by itself, the reconstitution was due to a reduction of the proton leakage through open CF0 channels. The molar ratio of purified delta over exposed CF0, which started to elicit this effect, was 3:1. However, if delta was added together with purified CF1 lacking delta, in a 1:1 molar ratio, the relative amount over exposed CF0 was as low as 0.06. This corroborated our previous conclusion [Lill, H., Engelbrecht, S., Sch?nknecht, G. and Junge, W. (1986) Eur. J. Biochem. 160, 627-634] that only a very small fraction of exposed CF0 was actually proton-conducting but with a very high unit conductance. CF1 including delta was apparently rebound preferentially to open CF0 channels. Although the ability of delta to control proton conduction through CF0 was evident, it remains to be established whether delta acts as a gated proton valve or as a conformational transducer in the integral CF0CF1 ATPase.     

Role of the gamma subunit of chloroplast coupling factor 1 in the light-dependent activation of photophosphorylation and ATPase activity by dithiothreitol

In leaves and intact chloroplasts, oxidation and reduction have been shown previously to regulate the ATPase activity of thylakoids. Illumination of spinach chloroplast thylakoids in the presence of dithiothreitol, which activates the ability of thylakoids to catalyze sustained ATP hydrolysis in the dark, causes increased incorporation of N-ethylmaleimide into the gamma subunit of coupling factor 1 (CF1). A disulfide bond in the gamma subunit is reduced during activation. The residues involved in this disulfide bond are the same as those in the disulfide linkage reduced during dithiothreitol activation of soluble CF1. The disulfide and dithiol forms of the gamma subunit may be separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. N-Ethylmaleimide is preferentially incorporated in the dark into the reduced form of the gamma subunit of CF1 in thylakoids previously exposed to dithiothreitol. Only a subpopulation of the CF1 in thylakoids is susceptible to dithiothreitol reduction and subsequent reaction with N-ethylmaleimide in the dark. Alkylation of the thiol groups exposed by reduction of the disulfide bond protects ATPase activity from inhibition by oxidants. At a given value of the transmembrane pH differential, photophosphorylation rates in dithiothreitol-activated thylakoids can be as much as seven to eight times those of nonactivated controls. N-Ethylmaleimide treatment of activated thylakoids in the dark prevents the loss of the stimulation of ATP synthesis on storage of the thylakoids. Photophosphorylation by intact chloroplasts lysed in assay mixtures is also activated in comparison to that by washed thylakoids. At a low ADP concentration, the rate of photophosphorylation approaches saturation as delta pH increases. These results suggest that the gamma subunit of CF1 plays an important role in regulation of ATP synthesis and hydrolysis.     

A light-dependent dicyclohexylcarbodiimide-sensitive Ca-ATPase activity in chloroplasts which is not coupled to proton translocation

The early observation of light-dependent Ca-ATPase activity in chloroplast thylakoids [Avron, M. (1962) J. Biol. Chem. 237, 2011-2017] has been reinvestigated. It is demonstrated that in contrast to light-triggered Mg-ATP activity, Ca-ATPase activity is strictly dependent on delta microH+, the transthylakoid membrane electrochemical potential gradient, since (a) there is an absolute requirement for continuous illumination; (b) electron-transport mediators that catalyze proton uptake, like phenazinemethosulphate, methylviologen of ferricyanide, are essential and (c) uncouplers inhibit the activity. The Ca-ATPase activity is essentially unaffected by dithiols, but is inhibited by CF0-CF1 inhibitors including tentoxin, dicyclohexylcarbodiimide and antisera to CF1. Addition of Ca-ATP to thylakoids does not induce delta pH or delta psi (the electrical potential gradient) formation either in the light or following preillumination with dithiols, demonstrating that it is not coupled to proton translocation. It is also demonstrated that Ca-ATP or Ca-ADP does not induce a proton leak through CF0-CF1. It is concluded that the Ca-ATPase activity in chloroplast thylakoid reflects a partial reaction of ATP synthesis catalyzed by CF0-CF1, which is internally uncoupled from proton translocation but is dependent on energization by a transmembrane delta microH+.     

The dithiothreitol-stimulated dissociation of the chloroplast coupling factor 1 epsilon-subunit is reversible

The chloroplast coupling factor 1 complex (CF1) contains an epsilon-subunit which inhibits the CF1 ATPase activity. Chloroform treatment of Chlamydomonas reinhardtii thylakoid membranes solubilizes only forms of the enzyme which apparently lack the delta-subunit. Four interrelated observations are described in this paper. (1) The dithiothreitol- (DTT) induced ATPase activation of CF1(-delta) and the DTT-induced formation of a physically resolvable CF1(-delta,epsilon) from the CF1(-delta) precursor are compared. The similar time-courses of these two phenomena suggest that the dissociation of the epsilon-subunit is an obligatory process in the DTT-induced ATPase activation of soluble CF1. (2) The reversible dissociation of the epsilon-subunit of the CF1 is demonstrated by the exchange of subunits between distinguishable oligomers. 35S-labelled chloroplast coupling factor 1 lacking the delta and epsilon subunits [CF1(-delta,epsilon)] was added to a solution of non-radioactive coupling factor 1 lacking only the delta subunit [CF1(-delta)]. After separation of the two enzyme forms, via high resolution anion-exchange chromatography, radioactivity was detected in the chromatographic fractions containing CF1(-delta). (3) epsilon-deficient CF1 can be resolved from DTT pretreated epsilon-containing CF1 for several days after the removal of DTT. On the other hand, brief incubation of the DTT pretreated epsilon-containing CF1 with low concentrations of o-iodosobenzoate results in chromatographs containing only the peak of epsilon-containing CF1. A simple explanation for this phenomenon is that reduction of CF1 with DTT increases the apparent dissociation constant for the epsilon-subunit to an estimated 3.5 x 10(-8) M (+/- 1.0 x 10(-8) M) from a value of less than or equal to 5 x 10(-11) M for the oxidized enzyme. (4) ATPase activity data show that oxidation of the epsilon-deficient enzyme does not completely inhibit its manifest activity, but oxidation of DTT pre-treated CF1 which contains the epsilon-subunit completely inhibits manifest activity. A simple model is proposed for the influence of the oxidation state of the soluble enzyme on the distribution of ATPase-inactive and ATPase-active subunit configurations.     

Purification and reconstitution of active chloroplast F0

Chloroplast F0 (CF0) was purified from the ATP synthase by Zwittergent 3-12 treatment and DEAE-Trisacryl anion exchange chromatography. Purified CF0 contains four subunits corresponding to subunits I, II, III, and IV. CF0 mediated proton translocation across the membrane after incorporation into asolectin liposomes. The CF0-mediated proton transport was inhibited by N,N'-dicyclohexylcarbodiimide and the binding of chloroplast coupling factor 1 (CF1). Rebinding of CF1 to CF0 liposomes resulted in reconstitution of N,N'-dicyclohexylcarbodiimide and uncoupler sensitive energy-transducing activities. Like CF0 in native thylakoid membranes, purified CF0 bound CF1 as well as CF1 deficient in either the delta or epsilon subunits.     

Functional Consequences of Deletions of the N Terminus of the [epsilon] Subunit of the Chloroplast ATP Synthase

The [epsilon] subunit of the chloroplast ATP synthase functions in part to prevent wasteful ATP hydrolysis by the enzyme. In addition, [epsilon] together with the remainder of the catalytic portion of the synthase (CF1) is required to block the nonproductive leak of protons through the membrane-embedded component of the synthase (CFO). Mutant [epsilon] subunits of the spinach (Spinacia oleracea) chloroplast ATP synthase that lack 5, 11, or 20 amino acids from their N termini ([epsilon]-[delta]5N, [epsilon]-[delta]11N, and [epsilon]-[delta]20N, respectively), were overexpressed as inclusion bodies. Using a procedure that resulted in the folding of full-length, recombinant [epsilon] in a biologically active form, none of these truncated forms resulted in [epsilon] that inhibited the ATPase activity of CF1 deficient in [epsilon], CF1(-[epsilon]). Yet, the [epsilon]-[delta]5N and [epsilon]-[delta]11N peptides significantly inhibited the ATPase activity of CF1(-[epsilon]) bound to CFO in NaBr-treated thylakoids. Although full-length [epsilon] rapidly inhibited the ATPase activity of CF1(-[epsilon]) in solution or bound to CFO, an extended period was required for the truncated forms to inhibit membrane-bound CF1(-[epsilon]). Despite the fact that [epsilon]-[delta]5N significantly inhibited the ATPase activity of CF1(-[epsilon]) bound to CFO, it did not block the proton conductance through CFO in NaBr-treated thylakoids reconstituted with CF1(-[epsilon]). Based on selective proteolysis and the binding of 8-anilino-1-naphthalene sulfonic acid, each of the truncated peptides gained significant secondary structure after folding. These results strongly suggest (a) that the N terminus of [epsilon] is important in its binding to CF1, (b) that CF0 stabilizes [epsilon] binding to the entire ATP synthase, and (c) that the N terminus may play some role in the regulation of proton flux through CFO.     

Modifications of the gamma subunit of chloroplast coupling factor 1 alter interactions with the inhibitory epsilon subunit.

The treatment of chloroplast coupling factor 1 (CF1) with dithiothreitol or with trypsin modifies the gamma subunit. Reduction of the gamma subunit disulfide bond in CF1 in solution with dithiothreitol enhances the dissociation of epsilon (Duhe, R. J., and Selman, B. R. (1990) Biochim. Biophys. Acta 1017, 70-78). The Ca(2+)-ATPase activity of either oxidized or reduced CF1 increases as the enzyme is diluted. Added epsilon subunit inhibits the Ca(2+)-ATPase activity of both forms of the diluted CF1, suggesting that epsilon dissociation is the cause of activation by dilution. Half-maximal activation occurred at much higher concentrations of the reduced CF1, indicating that reduction decreases the affinity for epsilon about 20-fold. Immunoblotting techniques show that there is only one epsilon subunit/CF1 in intact chloroplasts, in thylakoid membranes, and in solution. No epsilon is released from CF1 in thylakoids under conditions of ATP synthesis. The gamma subunit of CF1 in illuminated thylakoids is specifically cleaved by trypsin. CF1 purified from thylakoids treated with trypsin in the light is deficient in epsilon subunit, and has a high rate of ATP hydrolysis. Added epsilon neither inhibits the ATPase activity of, nor binds tightly to the cleaved enzyme.     

叶绿体atpE基因表达产物的生化特性

在细菌中表达的叶绿体ａｔｐＥ基因产物ε亚基蛋白对不同方式激活的叶绿体ＡＴ－Ｐａｓｅ均有抑制作用,而其抗血清则促进ＡＴ－Ｐａｓｅ活力。Ｅ．ｃｏｌｉ中表达的ε亚基蛋白在光合磷酸化反应中对循环和非循环光合磷酸化都有促进作用,其抗血清对循环光合磷酸化有抑制作用,而对非循环光合磷酸化则起促进作用。     

Rotation of the c subunit oligomer in EF(0)EF(1) mutant cD61N

ATP synthases (F(0)F(1)-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F(0), to ATP synthesis within the peripheral portion, F(1). The coupling most probably occurs through the rotation of a central rotor (subunits c(10)epsilon gamma) relative to the stator (subunits ab(2)delta(alpha beta)(3)). The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab(2). In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation. However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons. Using detergent-solubilized and immobilized EF(0)F(1) and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme. This observation excluded slippage among subunit gamma, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.     

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)