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.     

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.     

Altered expression of the chloroplast ATP synthase through site-directed mutagenesis in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The chloroplast ATP synthase gates the flow of protons out of the thylakoid lumen. In Chlamydomonas reinhardtii deletion of any of the genes for the ATP synthase subunits, or misfolding of the peptides results in photosynthetic membranes devoid of the enzyme (Lemaire and Wollman, J Biol Chem 264:675–685, 1989). This work examines the physiologic response of an algal strain in which the epsilon subunit of the chloroplast ATP synthase has been truncated. Removal of 10 amino acids from the C-terminus of the peptide results in a sharp decrease in the content of the enzyme, but does not result in its exclusion from the thylakoid membranes. The ATP synthase of this mutant strain has a higher rate of ATP hydrolysis than the wild-type enzyme. This strain of C. reinhardtii exhibits reduced growth in the light, dependence on acetate, and a low threshold for the onset of photoinhibition. The role of the ATP synthase in regulating the proton concentration of the lumen is discussed. This work was supported in part by a grant from the National Science Foundation (MCB0110232).     

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.     

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.     

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.     

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.     

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.     

Influences of energization and nucleotide binding on the reaction of Lucifer Yellow vinyl sulfone with the alpha subunits of the chloroplast ATP synthase

The catalytic portion of the chloroplast ATP synthase (CF(1)) consists of five different polypeptides in the stoichiometry alpha(3)beta(3)gammadeltaepsilon and is structurally asymmetric. Asymmetry is readily apparent in the properties of the six nucleotide binding sites and the single-copy, smaller subunits. Asymmetry is also detected in the alpha subunits by the rapid and covalent binding of Lucifer Yellow vinyl sulfone (LY) to one of the three chemically identical alpha subunits. The binding of LY to a single alpha subunit has allowed the investigation of whether asymmetry in the alpha subunits is a permanent feature of CF(1). The development of an electrochemical proton gradient across illuminated thylakoid membranes and the preincubation of CF(1) in solution with Mg(2+)-ATP were found to alter the LY distribution such that multiple alpha subunits were labeled with LY. Illumination of thylakoid membranes doubled the extent of LY labeling, and fluorescence resonance energy transfer measurements indicated that LY was bound to more than one alpha subunit. Since the change in LY distribution was inhibited by proton ionophores (uncouplers), alteration of alpha conformation by illumination is a result of the generation of a proton gradient. Preincubation of CF(1) in solution with Mg(2+)-ATP had no effect on the extent of LY labeling but resulted in multiple alpha subunits binding LY as determined by fluorescence resonance energy transfer measurements. Adenine nucleotides at substrate level concentrations inhibit the reaction of LY with the alpha subunits. No increase in LY labeling was observed when thylakoids were illuminated under conditions in which CF(1) was catalytically active.     

Molecular devices of chloroplast F(1)-ATP synthase for the regulation

In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential difference of protons (Delta(micro)H(+)) is small, e.g. at low light intensity or in the dark. To prevent this wasteful consumption of ATP, the activity of chloroplast ATP synthase is regulated as the occasion may demand. As regulation systems Delta(micro)H(+) activation, thiol modulation, tight binding of ADP and the role of the intrinsic inhibitory subunit epsilon is well documented. In this article, we discuss recent progress in understanding of the regulation system of the chloroplast ATP synthase at the molecular level.     

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.     

In vivo regulation of thylakoid proton motive force in immature leaves

In chloroplast, proton motive force (pmf) is critical for ATP synthesis and photoprotection. To prevent photoinhibition of photosynthetic apparatus, proton gradient (ΔpH) across the thylakoid membranes needs to be built up to minimize the production of reactive oxygen species (ROS) in thylakoid membranes. However, the regulation of thylakoid pmf in immature leaves is little known. In this study, we compared photosynthetic electron sinks, P700 redox state, non-photochemical quenching (NPQ), and electrochromic shift (ECS) signal in immature and mature leaves of a cultivar of Camellia. The immature leaves displayed lower linear electron flow and cyclic electron flow, but higher levels of NPQ and P700 oxidation ratio under high light. Meanwhile, we found that pmf and ΔpH were higher in the immature leaves. Furthermore, the immature leaves showed significantly lower thylakoid proton conductivity than mature leaves. These results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer. This study highlights that the activity of chloroplast ATP synthase is a key safety valve for photoprotection in immature leaves.     

Molecular evolution of the modulator of chloroplast ATP synthase: origin of the conformational change dependent regulation

Chloroplast ATP synthase synthesizes ATP by utilizing a proton gradient as an energy supply, which is generated by photosynthetic electron transport. The activity of the chloroplast ATP synthase is regulated in several specific ways to avoid futile hydrolysis of ATP under various physiological conditions. Several regulatory signals such as Delta mu H(+), tight binding of ADP and its release, thiol modulation, and inhibition by the intrinsic inhibitory subunit epsilon are sensed by this complex. In this review, we describe the function of two regulatory subunits, gamma and epsilon, of ATP synthase based on their possible conformational changes and discuss the evolutionary origin of these regulation systems.     

Function, structure and regulation of cyanobacterial and chloroplast ATP synthase

The proton-translocating ATP synthase from chloroplasts and cyanobacteria forms ATP upon photosynthetic electron transport by using the proton gradient across the thylakoid membrane. Both enzymes contain nine different subunits and from the similarity in gene organisation and the high degree of amino acid sequence homology of the subunits it appears that these ATP synthases might have a common ancestor. Both enzymes need to be activated by membrane energisation in order to perform catalytic activity but, in contrast to the chloroplast ATP synthase, that from the studied cyanobacteria (with the exception of Spirulina platensis ) shows no effect of the redox state on activation. Functionally, the cyanobacterial enzyme corresponds to the reduced form of the chloroplast ATP synthase. In the chloroplast enzyme a stretch of 9 amino acids, including two cysteines in the γ-subunit, is involved in this redox effect and this stretch is absent in cyanobacteria. With γ-mutants from the cyanobacterium Synechocystis 6803 the role of this stretch is studied. When active, both the cyanobacterial and the reduced chloroplast ATP synthase transport 4 protons per ATP synthesised and hydrolysed. This ratio may depend on the environment of the enzyme such as protein and lipid composition and pH.     

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.     

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.     

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.     

Positioning of protein-coding genes on the soybean chloroplast genome

Seven major plastid protein encoding genes were positioned on the soybean chloroplast DNA by heterologous hybridization. These include the genes for the alpha, beta and epsilon subunits of the CF₁ component of ATP synthase (atpA, atpB and atpE respectively), for subunit III of the CF₀ component of ATP synthase (atpH), for the cytochrome f (cytF), for the ‘32 Kd’ thylakoid protein (psbA), and for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL), all of which map in the large single copy region. The atpB, atpE and rbcL genes are located in the region adjacent to one of the segments of the inverted repeat. The genetic organization of the soybean chloroplast DNA is compared to that of other plastid genomes.