Inactivation of the chloroplast ATP synthase gamma subunit results in high non-photochemical fluorescence quenching and altered nuclear gene expression in Arabidopsis thaliana

The nuclear atpC1 gene encoding the gamma subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis in Arabidopsis thaliana. In the seedling-lethal dpa1 (deficiency of plastid ATP synthase 1) mutant, the absence of detectable amounts of the gamma subunit destabilizes the entire ATP synthase complex. The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. However, in vivo protein labeling analysis suggests that assembly of the ATP synthase alpha and beta subunits into the thylakoid membrane still occurs in dpa1. As a consequence of the destabilized ATP synthase complex, photophosphorylation is abolished even under reducing conditions. Further effects of the mutation include an increased light sensitivity of the plant and an altered photosystem II activity. At low light intensity, chlorophyll fluorescence induction kinetics is close to those found in wild type, but non-photochemical quenching strongly increases with increasing actinic light intensity resulting in steady state fluorescence levels of about 60% of the minimal dark fluorescence. Most fluorescence quenching relaxed within 3 min after dark incubation. Spectroscopic and biochemical studies have shown that a high proton gradient is responsible for most quenching. Thylakoids of illuminated dpa1 plants were swollen due to an increased proton accumulation in the lumen. Expression profiling of 3292 nuclear genes encoding mainly chloroplast proteins demonstrates that most organelle functions are down-regulated. On the contrary, the mRNA expression of some photosynthesis genes is significantly up-regulated, probably to compensate for the defect in dpa1.     

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.     

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.     

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.     

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.     

A point mutation in atpC1 raises the redox potential of the Arabidopsis chloroplast ATP synthase gamma-subunit regulatory disulfide above the range of thioredoxin modulation

The light-dependent regulation of chloroplast ATP synthase activity depends on an intricate but ill defined interplay between the proton electrochemical potential across the thylakoid membrane and thioredoxin-mediated redox modulation of a cysteine bridge located on the ATP synthase gamma-subunit. The abnormal light-dependent regulation of the chloroplast ATP synthase in the Arabidopsis thaliana cfq (coupling factor quick recovery) mutant was caused by a point mutation (G to A) in the atpC1 gene, which caused an amino acid substitution (E244K) in the vicinity of the redox modulation domain in the gamma-subunit of ATP synthase. Equilibrium redox titration revealed that this mutation made the regulatory sulfhydryl group energetically much more difficult to reduce relative to the wild type (i.e. raised the Em,7.9 by 39 mV). Enzymatic studies using isolated chloroplasts showed significantly lower light-induced ATPase and ATP synthase activity in the mutant compared with the wild type. The lower ATP synthesis capacity in turn restricted overall rates of leaf photosynthesis in the cfq mutant under low light. This work provides in situ validation of the concept that thioredoxin-dependent reduction of the gamma-subunit regulatory disulfide modulates the proton electrochemical potential energy requirement for activation of the chloroplast ATP synthase and that the activation state of the ATP synthase can limit leaf level photosynthesis.     

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.     

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.     

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.     

Resonance energy transfer between tryptophan 57 in the epsilon subunit and pyrene maleimide labeled gamma subunit of the chloroplast ATP synthase

The intrinsic fluorescence of the catalytic portion of the chloroplast ATP synthase (CF1) is quenched when cysteine 322, the penultimate amino acid of the gamma subunit, is specifically labeled with pyrene maleimide (PM). The epsilon subunit of CF1 contains the only two residues of tryptophan, which dominate the intrinsic fluorescence of unlabeled CF1. CF1 deficient in the epsilon subunit (CF1-epsilon) was reconstituted with mutant epsilon subunits in which phenylalanine replaced tryptophan at position 15 (epsilonW15F) and position 57 (epsilonW15/57F). CF1(epsilonW15F) containing a single tryptophan, epsilonW57, was labeled with PM at gammaC322. Resonance energy transfer (RET) from epsilonW57 to PM on gammaC322 occurred with an efficiency of energy transfer of 20%. RET was also observed from epsilonW57 to PM attached to the disulfide thiols of the gamma subunit (gammaC199,205) with an efficiency of approximately 45%. The R(o) (the distance at which the efficiency of energy transfer is 50%) for the epsilonW57 and PM donor/acceptor pair is 30 A, indicating that both gammaC322 and gammaC199,205 must be within 40 A of epsilonW57. These RET measurements show that both gammaC322 and gammaC199,205 are located near the base of the alpha/beta hexamer. This places the C-terminus of CF1 gamma much closer to epsilon than hypothesized based on homology to crystal structures of mitochondrial F1. These new RET measurements also allow the alignment of the predicted epsilon subunit structure. The orientation is similar to that predicted from cross-linking and mutational studies for the epsilon subunit of Escherichia coli F1.     

Nucleotide binding to the isolated beta subunit of the chloroplast ATP synthase.

The beta subunit isolated from the chloroplast ATP synthase F1 (CF1) has a single dissociable nucleotide binding site, consistent with the proposed function of this subunit in nucleotide binding and catalysis. The beta subunit bound the nucleotide analogs trinitrophenyl-ATP (TNP-ATP) or trinitrophenyl-ADP (TNP-ADP) with nearly equal affinities (Kd = 1-2 microM) but did not bind trinitrophenyl-AMP. Both ATP and ADP effectively competed with TNP-ATP for binding. Other nucleoside triphosphates were also able to compete with TNP-ATP for binding to beta; their order of effectiveness (ATP greater than GTP, ITP greater than CTP) mimicked the normal substrate specificity of CF1. The single nucleotide binding site on the isolated beta subunit very closely resembles the low affinity catalytic site (site 3) of CF1 (Bruist, M.F., and Hammes, G. G. (1981) Biochemistry 20, 6298-6305), suggesting that tight nucleotide binding by other sites on the enzyme involves other CF1 subunits in addition to the beta subunit. The results are inconsistent with an earlier report (Frasch, W.D., Green, J., Caguial, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069), which suggested more than one nucleotide binding site per beta subunit. Binding of nucleotides to the isolated beta subunit was eliminated by a brief heat treatment (40 degrees C for 10 min) of the protein. A small change in the circular dichroism spectrum of beta accompanied the heat treatment indicating that a localized (rather than global) change in the folding of beta, involving at least part of the nucleotide binding domain, had occurred. Also accompanying the loss of nucleotide binding was a loss of the reconstitutive capacity of the beta subunit. ATP protected against the effects of the heat treatment.     

Second stalk of ATP synthase. Cross-linking of gamma subunit in F1 to truncated Fob subunit prevents ATP hydrolysis

ATP synthase consists of two portions, F(1) and F(o), connected by two stalks: a central rotor stalk containing gamma and epsilon subunits and a peripheral, second stalk formed by delta and two copies of F(o)b subunits. The second stalk is expected to keep the stator subunits from spinning along with the rotor. We isolated a TF(1)-b'(2) complex (alpha(3)beta(3)gammadeltaepsilonb'(2)) of a thermophilic Bacillus PS3, in which b' was a truncated cytoplasmic fragment of F(o)b subunit, and introduced a cysteine at its N terminus (bc'). Association of b'(2) or bc'(2) with TF(1) did not have significant effect on ATPase activity. A disulfide bond between the introduced cysteine of bc' and cysteine 109 of gamma subunit was readily formed, and this cross-link caused inactivation of ATPase. This implies that F(o)b subunit bound to stator subunits of F(1) with enough strength to resist rotation of gamma subunit and to prevent catalysis. Contrary to this apparent tight binding, some detergents such as lauryldodecylamine oxide tend to cause release of b'(2) from TF(1).     

Structural mapping of cysteine-63 of the chloroplast ATP synthase beta subunit.

The single sulfhydryl residue (cysteine-63) of the beta subunit of the chloroplast ATP synthase F1 (CF1) was accessible to labeling reagents only after removal of the beta subunit from the enzyme complex. This suggests that cysteine-63 may be located at an interface between the beta and the alpha subunits of CF1, although alternative explanations such as a conformational change in beta brought about by its release from CF1 cannot be ruled out. Cysteine-63 was specifically labeled with [(diethylamino)methylcoumarinyl]-maleimide, and the distance between this site and trinitrophenyl-ADP at the nucleotide binding site on beta was mapped using fluorescence resonance energy transfer. Cysteine-63 is located in a hydrophobic pocket, 42 A away from the nucleotide binding site on beta.     

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.     

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.     

Inverse regulation of rotation of F1-ATPase by the mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase

In F1-ATPase, the rotation of the central axis subunit gamma relative to the surrounding alpha3beta3 subunits is coupled to ATP hydrolysis. We previously reported that the introduced regulatory region of the gamma subunit of chloroplast F1-ATPase can modulate rotation of the gamma subunit of the thermophilic bacterial F1-ATPase (Bald, D., Noji, H., Yoshida, M., Hirono-Hara, Y., and Hisabori, T. (2001) J. Biol. Chem. 276, 39505-39507). The attenuated enzyme activity of this chimeric enzyme under oxidizing conditions was characterized by frequent and long pauses of rotation of gamma. In this study, we report an inverse regulation of the gamma subunit rotation in the newly engineered F1-chimeric complex whose three negatively charged residues Glu210-Asp211-Glu212 adjacent to two cysteine residues of the regulatory region derived from chloroplast F1-ATPase gamma were deleted. ATP hydrolysis activity of the mutant complex was stimulated up to 2-fold by the formation of the disulfide bond at the regulatory region by oxidation. We successfully observed inverse redox switching of rotation of gamma using this mutant complex. The complex exhibited long and frequent pauses in its gamma rotation when reduced, but the rotation rates between pauses remained unaltered. Hence, the suppression or activation of the redox-sensitive F1-ATPase can be explained in terms of the change in the rotation behavior at a single molecule level. These results obtained by the single molecule analysis of the redox regulation provide further insights into the regulation mechanism of the rotary enzyme.