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.     

Energy-dependent changes in the conformation of the epsilon subunit of the chloroplast ATP synthase

Rabbit antiserum raised against the isolated native epsilon subunit of the chloroplast coupling factor 1 activated the ATPase activity of coupling factor 1 in solution by removing the epsilon subunit. Incubation of thylakoid membranes with the antiserum in the dark had no effect on photophosphorylation or on the dithiothreitol-induced Mg2+-ATPase activity. Incubation with the antiserum during illumination, however, strongly inhibited both activities and caused the membranes to become leaky to protons. The results indicate that the formation of a proton gradient across the thylakoid membrane induces a change in conformation of the epsilon subunit of the ATP synthase such that it becomes susceptible to attack and removal by the antibodies. This change may be a part of the mechanism that results in energy-dependent activation of the ATP synthase.     

C-Terminal mutations in the chloroplast ATP synthase gamma subunit impair ATP synthesis and stimulate ATP hydrolysis

Two highly conserved amino acid residues, an arginine and a glutamine, located near the C-terminal end of the gamma subunit, form a catch by hydrogen bonding with residues in an anionic loop on one of the three catalytic beta subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. The catch is considered to play a critical role in the binding change mechanism whereby binding of ATP to one catalytic site releases the catch and induces a partial rotation of the gamma subunit. This role is supported by the observation that mutation of the equivalent arginine and glutamine residues in the Escherichia coli F1 gamma subunit drastically reduced all ATP-dependent catalytic activities of the enzyme [Greene, M. D., and Frasch, W. D. (2003) J. Biol. Chem. 278, 5194-5198]. In this study, we show that simultaneous substitution of the equivalent residues in the chloroplast F1 gamma subunit, arginine 304 and glutamine 305, with alanine decreased the level of proton-coupled ATP synthesis by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATP hydrolysis activities increased by more than 3-fold as a result of these mutations; however, the sulfite-stimulated activity decreased by more than 60%. The Mg2+-dependent, but not the Ca2+-dependent, ATPase activity of the double mutant was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicating selective loss of catalytic cooperativity in the presence of Mg2+ ions. The results indicate that the catch residues are required for efficient proton coupling and for activation of multisite catalysis when MgATP is the substrate. The catch is not, however, required for CaATP-driven multisite catalysis or, therefore, for rotation of the gamma subunit.     

Regulatory role of the C-terminus of the epsilon subunit from the chloroplast ATP synthase

The ATP synthases from chloroplasts and Escherichia coli are regulated by several factors, one of which is the epsilon subunit. This small subunit is also required for ATP synthesis. Thylakoid membranes reconstituted with CF1 lacking the epsilon subunit (CF1-epsilon) exhibit no ATP synthesis and very high ATP hydrolysis. Either native or recombinant epsilon restores ATP synthesis and inhibits ATP hydrolysis. Previously, we showed that truncated epsilon, lacking the last 45 C-terminal amino acids, restored ATP synthesis to membranes reconstituted with CF1-epsilon but was not an efficient inhibitor of ATP hydrolysis. In this paper, we show that this truncated epsilon is unable to inhibit ATP hydrolysis when Mg(2+) is the divalent cation present, both for the enzyme in solution and on the thylakoid membrane. In addition, the rate of reduction of the disulfide bond of the gamma subunit by dithiothreitol is not decreased by truncated epsilon, although full-length epsilon greatly impedes reduction. Thylakoid membranes can synthesize ATP at the expense of proton gradients generated by pH transitions in the dark. Our reconstituted membranes are able to produce a limited amount of ATP under these "acid-bath" conditions, with approximately equal amounts produced by the membranes containing wild-type epsilon and those containing truncated epsilon. However, the membranes containing truncated epsilon exhibit much higher background ATP hydrolysis under the same acid-bath conditions, leading to the conclusion that, without the C-terminus of epsilon, the CF1CFo is unable to check unwanted ATP hydrolysis.     

Molecular dissection of the epsilon subunit of the chloroplast ATP synthase of spinach.

The gene encoding the epsilon subunit (atpE) of the chloroplast ATP synthase of Spinacia oleracea has been overexpressed in Escherichia coli. The recombinant protein can be solubilized in 8 M urea and directly diluted into buffer containing ethanol and glycerol to obtain epsilon that is as biologically active as epsilon purified from chloroplast-coupling factor 1 (CF1). Recombinant epsilon folded in this manner inhibits the ATPase activity of soluble and membrane-bound CF1 deficient in epsilon and restores proton impermeability to thylakoid membranes reconstituted with CF1 deficient in epsilon. Site-directed mutagenesis was used to generate truncations and single amino acid substitutions in the primary structure of epsilon. In the five mutants tested, alterations that weaken ATPase inhibition by recombinant epsilon affect its ability to restore proton impermeability to a similar extent, with one exception. Substitution of histidine-37 with arginine appears to uncouple ATPase inhibition and the restoration of proton impermeability. As in the case of E. coli, it appears that N-terminal truncations of the epsilon subunit have more profound effects than C-terminal deletions on the function of epsilon. Recombinant epsilon with six amino acids deleted from the C terminus, which is the only region of significant mismatch between the epsilon of spinach and the epsilon of Pisum sativum, inhibits ATPase activity with a reduced potency similar to that of purified pea epsilon. Four of the six amino acids are serine or threonine. These hydroxylated amino acids may be important in epsilon-CF1 interactions.     

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.     

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.     

Structural analysis of the regulatory dithiol-containing domain of the chloroplast ATP synthase gamma subunit

The gamma subunit of the F1 portion of the chloroplast ATP synthase contains a critically placed dithiol that provides a redox switch converting the enzyme from a latent to an active ATPase. The switch prevents depletion of intracellular ATP pools in the dark when photophosphorylation is inactive. The dithiol is located in a special regulatory segment of about 40 amino acids that is absent from the gamma subunits of the eubacterial and mitochondrial enzymes. Site-directed mutagenesis was used to probe the relationship between the structure of the gamma regulatory segment and its function in ATPase regulation via its interaction with the inhibitory epsilon subunit. Mutations were designed using a homology model of the chloroplast gamma subunit based on the analogous structures of the bacterial and mitochondrial homologues. The mutations included (a) substituting both of the disulfide-forming cysteines (Cys199 and Cys205) for alanines, (b) deleting nine residues containing the dithiol, (c) deleting the region distal to the dithiol (residues 224-240), and (d) deleting the entire segment between residues 196 and 241 with the exception of a small spacer element, and (e) deleting pieces from a small loop segment predicted by the model to interact with the dithiol domain. Deletions within the dithiol domain and within parts of the loop segment resulted in loss of redox control of the ATPase activity of the F1 enzyme. Deleting the distal segment, the whole regulatory domain, or parts of the loop segment had the additional effect of reducing the maximum extent of inhibition obtained upon adding the epsilon subunit but did not abolish epsilon binding. The results suggest a mechanism by which the gamma and epsilon subunits interact with each other to induce the latent state of the enzyme.     

The carboxyl terminus of the epsilon subunit of the chloroplast ATP synthase is exposed during illumination

The epsilon subunit of the chloroplast ATP synthase is an inhibitor of activity of the enzyme. Recombinant forms of the epsilon subunit from spinach chloroplasts lacking the last 10, 32, or 45 amino acids were immobilized onto activated Sepharose. A polyclonal antiserum raised against the epsilon subunit was passed over these immobilized protein columns, and the purified antibodies which were not bound recognized the portions of the epsilon subunit missing from the recombinant form present on the column. The full polyclonal antiserum can strip the epsilon subunit from the ATP synthase in illuminated thylakoid membranes [Richter, M. L., and McCarty, R. E. (1987) J. Biol. Chem. 262, 15037-15040]. Exposure of illuminated thylakoid membranes to antibodies recognizing the last 32 amino acids of the epsilon subunit collapses the proton gradient and hinders ATP synthesis with similar efficiency as the full polyclonal preparation. These results indicate that antibodies against the last 32 amino acids of the epsilon subunit are capable of stripping the subunit from the ATP synthase in illuminated membranes. Neither of these effects was seen when the membranes were exposed to the antibodies in the dark. This is direct evidence that the chloroplast ATP synthase undergoes a conformational shift during its activation by the electrochemical proton gradient which specifically alters the conformation of the carboxyl-terminal domain of the epsilon subunit from protected to solvent-exposed. The relation between this shift and activation of the enzyme by the electrochemical proton gradient is discussed.     

N-terminal deletion of the gamma subunit affects the stabilization and activity of chloroplast ATP synthase

Five truncation mutants of chloroplast ATP synthase gamma subunit from spinach (Spinacia oleracea) lacking 8, 12, 16, 20 or 60 N-terminal amino acids were generated by PCR by a mutagenesis method. The recombinant gamma genes were overexpressed in Escherichia coli and assembled with alphabeta subunits into a native complex. The wild-type (WT) alphabetagamma assembly i.e. alphabetagammaWT exhibited high (Mg2+)-dependent and (Ca2+)-dependent ATP hydrolytic activity. Deletions of eight residues of the gamma subunit N-terminus caused a decrease in rates of ATP hydrolysis to 30% of that of the alphabetaWT assembly. Furthermore, only approximately 6% of ATP hydrolytic activity was retained with the sequential deletions of gamma subunit up to 20 residues compared with the activity of the alphabetaWT assembly. The inhibitory effect of the epsilon subunit on ATP hydrolysis of these alphabetagamma assemblies varied to a large extent. These observations indicate that the N-terminus of the gamma subunit is very important, together with other regions of the gamma subunit, in stabilization of the enzyme complex or during cooperative catalysis. In addition, the in vitro binding assay showed that the gamma subunit N-terminus is not a crucial region in binding of the epsilon subunit.     

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.     

Proton flux through the chloroplast ATP synthase is altered by cleavage of its gamma subunit

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the gamma subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the gamma subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the epsilon subunit. Our results suggest that overcoming inhibition by the epsilon subunit costs energy.     

Proton flux through the chloroplast ATP synthase is altered by cleavage of its gamma subunit

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the γ subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the γ subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the ε subunit. Our results suggest that overcoming inhibition by the ε subunit costs energy.     

Recombinant production and purification of the subunit c of chloroplast ATP synthase

The unique immunoglobulin idiotype expressed on the surface of B lymphoma cells can be used as an effective antigen in tumor-specific vaccines when fused to immunostimulatory proteins and cytokines. A DNA vaccine encoding for an idiotype antibody single chain Fv (scFv) fragment fused to the Tetanus Toxin Fragment C (TTFrC) has been shown to induce protective anti-tumor responses. Protein-based strategies may be more desirable since they provide greater control over dosage, duration of exposure, and in vivo distribution of the vaccine. However, production of fusion protein vaccines containing complex disulfide bonded idiotype antibodies and antibody-derived fragments is challenging. We use an Escherichia coli-based cell-free protein synthesis platform as well as high-level expression of E. coli inclusion bodies followed by refolding for the rapid generation of an antibody fragment – TTFrC fusion protein vaccine. Vaccine proteins produced using both methods were shown to elicit anti-tumor humoral responses as well as protect from tumor challenge in an established B cell lymphoma mouse model. The development of technologies for the rapid production of effective patient-specific tumor idiotype-based fusion protein vaccines provides opportunities for clinical application.     

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.     

Effects of sequential deletions of residues from the N- or C-terminus on the functions of epsilon subunit of the chloroplast ATP synthase

Ten truncated mutants of chloroplast ATP synthase epsilon subunit from spinach (Spinacia oleracea), which had sequentially lost 1-5 amino acid residues from the N-terminus and 6-10 residues from the C-terminus, were generated by PCR. These mutants were overexpressed in Escherichia coli, reconstituted with soluble and membrane-bound CF(1), and the ATPase activity and proton conductance of thylakoid membrane were examined. Deletions of as few as 3 amino acid residues from the N-terminus or 6 residues from the C-terminus of epsilon subunit significantly affected their ATPase-inhibitory activity in solution. Deletion of 5 residues from the N-terminus abolished its abilities to inhibit ATPase activity and to restore proton impermeability. Considering the consequence of interaction of epsilon and gamma subunit in the enzyme functions, the special interactions between the epsilon variants and the gamma subunit were detected in the yeast two-hybrid system and in vitro binding assay. In addition, the structures of these mutants were modeled through the SWISS-MODEL Protein Modeling Server. These results suggested that in chloroplast ATP synthase, both the N-terminus and C-terminus of the epsilon subunit show importance in regulation of the ATPase activity. Furthermore, the N-terminus of the epsilon subunit is more important for its interaction with gamma and some CF(o) subunits, and crucial for the blocking of proton leakage. Compared with the epsilon subunit from E. coli [Jounouchi, M., Takeyama, M., Noumi, T., Moriyama, Y., Maeda, M., and Futai, M. (1992) Arch. Biochem. Biophys. 292, 87-94; Kuki, M., Noumi, T., Maeda, M., Amemura, A., and Futai, M. (1988) J. Biol. Chem. 263, 4335-4340], the chloroplast epsilon subunit is more sensitive to N-terminal or C-terminal truncations.     

The 20 C-terminal amino acid residues of the chloroplast ATP synthase gamma subunit are not essential for activity.

It has been suggested that the last seven to nine amino acid residues at the C terminus of the gamma subunit of the ATP synthase act as a spindle for rotation of the gamma subunit with respect to the alpha beta subunits during catalysis (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). To test this hypothesis we selectively deleted C-terminal residues from the chloroplast gamma subunit, two at a time starting at the sixth residue from the end and finishing at the 20th residue from the end. The mutant gamma genes were overexpressed in Escherichia coli and assembled with a native alpha3beta3 complex. All the mutant forms of gamma assembled as effectively as the wild-type gamma. Deletion of the terminal 6 residues of gamma resulted in a significant increase (>50%) in the Ca-dependent ATPase activity when compared with the wild-type assembly. The increased activity persisted even after deletion of the C-terminal 14 residues, well beyond the seven residues proposed to form the spindle. Further deletions resulted in a decreased activity to approximately 19% of that of the wild-type enzyme after deleting all 20 C-terminal residues. The results indicate that the tip of the gammaC terminus is not essential for catalysis and raise questions about the role of the C terminus as a spindle for rotation.