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.     

Substitutions of the Conserved Gly47 Affect the CF1 Inhibitor and Proton Gate Functions of the Chloroplast ATP Synthase ε Subunit

The conserved residue Gly47 of the chloroplast ATP synthase ε subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants εG47L, εG47R, εG47A and εG47E, respectively. All the ε variants showed lower inhibitory effects on the soluble CF1(-ε) Ca^2 -ATPase compared with wild-type ε. In reduced conditions, εG47E and εG47R had a lower inhibitory effect on the oxidized CF1(-ε) Ca^2 -ATPase compared with wild-type ε. In contrast, εG47L and εG47Aincreased the Ca^2 -ATPase activity of soluble oxidized CF1(-ε). The replacement of Gly47 significantly impaired the interaction between the subunit ε and γ in an in vitro binding assay. Further study showed that all ε variants were more effective in blocking proton leakage from the thylakoid membranes. This enhanced ATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to a level that was more efficient than that achieved by wild-type ε. These results indicate that the conserved Gly47 residue of the ε subunit is very important for maintaining the structure and function of the ε subunitand may affect the interaction between the ε subunit, β subunit of CF1 and subunit Ⅲ of CF0, therebyregulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit Ⅲ of CF0.     

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.     

The C-terminal domain of the epsilon subunit of the chloroplast ATP synthase is not required for ATP synthesis

The epsilon subunit of the ATP synthases from chloroplasts and Escherichia coli regulates the activity of the enzyme and is required for ATP synthesis. The epsilon subunit is not required for the binding of the catalytic portion of the chloroplast ATP synthase (CF1) to the membrane-embedded part (CFo). Thylakoid membranes reconstituted with CF1 lacking its epsilon subunit (CF1-epsilon) have high ATPase activity and no ATP synthesis activity, at least in part because the membranes are very leaky to protons. Either native or recombinant epsilon subunit inhibits ATPase activity and restores low proton permeability and ATP synthesis. In this paper we show that recombinant epsilon subunit from which 45 amino acids were deleted from the C-terminus is as active as full-length epsilon subunit in restoring ATP synthesis to membranes containing CF1-epsilon. However, the truncated form of the epsilon subunit was significantly less effective as an inhibitor of the ATPase activity of CF1-epsilon, both in solution and bound to thylakoid membranes. Thus, the C-terminus of the epsilon subunit is more involved in regulation of activity, by inhibiting ATP hydrolysis, than in ATP synthesis.     

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.     

The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.     

Synthesis and degradation of unassembled polypeptides of the coupling factor of photophosphorylation CF1 in 70S ribosome-deficient rye leaves

The formation of polypeptides of the coupling factor CF1 was investigated in 70S ribosome-deficient rye leaves generated by growing the plants at a non-permissive elevated temperature of 32 degrees C, in order to analyse mechanisms coordinating subunit accumulation. Antibodies were raised in rabbits against total CF1 as well as against its five individual subunits purified from chloroplast thylakoids from rye leaves. Several immunological techniques applying these antibodies (immunoprecipitation, immunoblotting, antibody affinity chromatography) were unable to detect the presence of any of the CF1 subunits in heat-treated 70S ribosome-deficient leaves. After in vivo labeling with L-[35S]methionine and subsequent immunoprecipitation, however, radioactivity was found to be incorporated into the subunits gamma and delta, but not into alpha, beta and epsilon, in 70S ribosome-deficient leaves, demonstrating the cytoplasmic synthesis of CF1-gamma and CF1-delta. Chase experiments after in vivo labeling with L-[35S]methionine indicated that the unassembled subunits gamma and delta were rapidly and preferentially degraded, while they were stabilized when integrated into the complete CF1 complex in normal green leaves from permissive growth conditions. The apparent half-times of the unassembled subunits were 2 h for CF1-gamma and 4 h for CF1-delta in 32 degrees C-grown leaves. Several other, stromal, plastid proteins of cytoplasmic origin were stable in 32 degrees C-grown leaves during the period of chase. In etiolated leaves total CF1, including all subunits, appeared to be less stable than in green leaves grown under permissive temperature conditions in light. Rapid degradation of the excess of unassembled subunits is regarded as an important mechanism ensuring a constant stoichiometry and apparently synchronous development of CF1 subunits.     

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.     

Energy-dependent conformational changes in the epsilon subunit of the chloroplast ATP synthase (CF0CF1)

Incubation of spinach chloroplast thylakoids with pyridoxal 5'-phosphate modified the epsilon subunit of ATP synthase (CF0CF1). Illumination of thylakoids stimulated the modification of one specific amino acid residue of the epsilon subunit by a factor of 3. Endoproteinase Glu-C treatment of the isolated epsilon subunit and fractionation of the peptides by high performance liquid chromatography revealed a major fluorescent peptide with the sequence GKRQKIE. Further treatment of this peptide with endoproteinase Arg-C gave a strongly fluorescent tripeptide (GXR). From the primary structure of the epsilon subunit, the specifically modified residue was deduced to be Lys-109. This suggests the energy-dependent conformational changes in the epsilon subunit which change the surroundings of Lys-109 and alter the reactivity of this residue.     

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.     

Electron microscopy and single molecule averaging of subunit-deficient F1-ATPases from Escherichia coli and spinach chloroplasts

The morphology of F1-ATPases lacking one or more small subunits has been investigated by minimal-beam electron microscopy of close-packed monolayers of molecules. Computer-based rotational analyses of single molecules were performed on reconstituted 3-subunit F1-ATPase (-delta epsilon) from Escherichia coli and both 3-subunit (-delta epsilon) and 4-subunit (-delta) F1-ATPase from chloroplasts. Optical diffraction measurements of close-packed arrays revealed maximal dimensions of 122 +/- 4 A and 129 +/- 9 A for 3-subunit ECF1 and 4-subunit CF1, respectively. Molecules which displayed either hollow or solid hexagonal morphologies were observed in all preparations. Averaged reconstructions were obtained from molecules with hollow morphologies in 3-subunit preparations and demonstrated strong hexagonal symmetry in projection with a central, stain-filled cavity. The average reconstruction obtained from molecules with the solid morphology in 4-subunit CF1 preparations, was also strongly hexagonal with six peripheral units ringed about a central subunit. Differences between hollow and solid morphologies cannot be attributed solely to the presence or absence of the delta and epsilon subunits; therefore, the two image types may represent staining variants of a common structure. Overall, the reconstructions are consistent with an alpha 3 beta 3 gamma stoichiometry for the coupling factors from both E. coli and chloroplasts.     

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.     

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.     

The T3/T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits.

Clonally distributed (clonotypic) antigen receptors on human T lymphocytes (alpha and beta chains) are associated with three invariable T3 polypeptide chains (T3 gamma, delta and epsilon), together forming the T3/T cell receptor complex. Monoclonal antibodies prepared against the two 20-kd T3 polypeptide chains demonstrated that T3-delta and T3-epsilon are distinct polypeptide chains. Only one monoclonal antibody (anti-T3-delta chain) reacted with the T cell surface as judged by indirect immunofluorescence, and by its mitogenicity for quiescent peripheral blood lymphocytes. Immunohistological staining and immunoprecipitation experiments showed that both the T3-delta and T3-epsilon chains are T cell-specific. As seen with the anti-alpha/beta chain reagent WT-31, anti-T3-delta chain monoclonal antibodies stained medullary thymocytes more intensely than cortical thymocytes, whereas the difference between the staining of cortical and medullary thymocytes was generally not apparent with anti-T3-epsilon chain antibodies. Because of this specificity and their ability to react with both the denatured and the native forms of each polypeptide chain, these new monoclonal reagents will be useful tools in studies of the biosynthesis and cell surface expression of the T3/T cell receptor complex during normal and malignant thymic differentiation.     

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.     

Chloroplast ATP synthase contains one single copy of subunit delta that is indispensable for photophosphorylation

F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit delta, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast delta, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the literature, we reinvestigated two further aspects related to subunit delta, namely (a) its stoichiometry in CF0CF1 and (b) whether or not delta is required for photophosphorylation. By rocket immunoelectrophoresis of thylakoid membranes and calibration against purified delta, we confirmed a stoichiometry of one delta per CF0CF1. In CF1-depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit delta but, surprisingly, also by CF1 (-delta). We found that the latter was attributable to a contamination of CF1 (-delta) preparations with integral CF1. To lesser extent CF1 (-delta) acted by complementary rebinding to CF0 channels that were closed because they contained delta [CF0(+delta)]. This added catalytic capacity to proton-tight thylakoid vesicles. The ability of subunit delta to control proton flow through CF0 and the absolute requirement for delta in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: delta may be part of the coupling site between electrochemical, conformational and chemical events in this 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)     

Identification of multiple PKC isoforms in Swiss 3T3 cells: differential down-regulation by phorbol ester.

The expression of members of the Ca2+ and phospholipid-dependent protein kinase (PKC) family were studied in murine Swiss 3T3 cells. In addition to PKC-alpha, the presence of immunoreactive PKC-delta, -epsilon, and zeta was detected. Treatment with 500 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) led to the down-regulation of alpha, delta, and epsilon isoforms, but not that of zeta. Higher concentrations of TPA similarly had no effect on the level of PKC-zeta. In contrast to PKC-alpha, the membrane localization of PKC-delta, -epsilon, and -zeta was not enhanced by extraction in Ca(2+)-containing buffers, whereas acute TPA treatment increased membrane association of PKC-alpha, -delta, and -epsilon but not that of PKC-zeta.