The role of different thylakoid glycolipids in the function of reconstituted chloroplast ATP synthase

ATPase activity of CF₀CF₁ from spinach chloroplasts is specifically stimulated by chloroplast lipids (Pick, U., Gounaris, K., Admon, A. and Barber, J. (1984) Biochim. Biophys. Acta 765, 12–20). The association of CF₀-CF₁ with isolated lipids and their mixtures has been examined by analyzing the stimulation of ATPase and ATP-P_i exchange activities, by binding studies and by measurement of proton conductance of reconstituted proteoliposomes. Monogalactosyldiacylglycerol is the only chloroplast lipid which by itself activates ATP hydrolysis. A mild saturation of the fatty acids of the lipid partially inhibits the activation. CF₀-CF₁ has a higher binding capacity for monogalactosyldiacylglycerol (1.5 mg/mg protein) than for other thylakoid glycolipids. However, ATPase activation is not correlated with the amount of bound lipid but rather with its type. For the same amount of bound lipid, monogalactosyldiacylglycerol best activates ATP hydrolysis, while the acidic lipids phosphatidylglycerol and sulphoquinovosyldiacylglycerol inhibit ATPase activity. Optimal activation of ATP-P_i exchange requires, in addition to monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerol at a ratio of 6:3:1, respectively. Correlations between proton conductance, ATP-P_i exchange and uncoupler stimulation of ATPase activity indicate that sulphoquinovosyldiacylglycerol reduces the permeability of the proteoliposomes to protons. The results suggest that: (a) association of CF₀-CF₁ with polyunsaturated monogalactosyldiacylglycerol greatly stimulates ATPase activity; (b) reconstitution of coupled CF₀-CF₁ proteoliposomes requires a careful balance of the natural glycolipids of thylakoid membranes in similar proportions to their occurrence in chloroplasts, and (c) sulphoquinovosyldiacylglycerol may control the permeability of chloroplast membranes to protons.     

Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin

The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF₁) as well as several hydrophobic components. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yielded vesicles that catalyzed light-dependent ATP formation. Both the ³²P_i-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2′-dichloro-4′-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-β-d-glucopyranosyl-4,6′-dihydroxydihydrochalcone were also effective inhibitors of both reactions.     

Purification and Characterization of a Glycerol-Resistant CF(0)-CF(1) and CF(1)-ATPase from the Halotolerant Alga Dunaliella bardawil

The isolation of the chloroplast ATP synthase complex (CF₀-CF₁) and of CF₁ from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil α subunit migrates ahead of the β subunit and ε-migrates ahead of subunit II of CF₀ when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF₁ isolated from D. bardawil resembles the CF₁ isolated from Chladmydomonas reinhardi in that a reversible, Mg²⁺-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg²⁺-ATPase of the D. bardawil CF₁ is more resistant to glycerol inhibition than the octylglucoside-activated, Mg²⁺-ATPase of spinach CF₁ or the ethanol-activated, Mg²⁺-dependent ATPase of the C. reinhardi CF₁. Both cyclic photophosphorylation and ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ are more sensitive to high concentrations of NaCl than is the spinach complex.     

Proton efflux through the chloroplast ATP synthase (CF0 · CF1) in the presence of sulfhydryl-modifying agents

The rate of photosynthetic electron transport measured in the absence of ADP and P_i is stimulated by low levels of Hg²⁺ or Ag⁺ (50% stimulation ≈ 3 Hg²⁺ or 6 Ag⁺/100 chlorophyll) to a plateau equal to the transport rate under normal phosphorylating conditions (i.e. +ADP, +P_i). Chloroplasts pretreated in the light under energizing conditions with N-ethylmaleimide show a similar stimulation of non-phosphorylating electron transport. The stimulations of non-phosphorylating electron transport by Hg²⁺, Ag⁺ and N-ethylmaleimide are reversed by the CF₁ inhibitor phlorizin, the CF₀ inhibitor triphenyltin chloride, and can be further stimulated by uncouplers such as methylamine. The Hg²⁺ and N-ethylmaleimide stimulations, but not the Ag⁺ stimulation, are completely reversed by low levels of ADP (2 μM), ATP (2 μM), and P_i (400 μM). Ag⁺, which is a potent inhibitor of ATP synthesis, has little or no effect upon phosphorylating electron transport (+ADP, +P_i). Concomitant with the stimulations of non-phosphorylating electron transport by Hg²⁺, Ag⁺ and ADP + P_i, there is a decrease in the level of membrane energization (as measured by atebrin fluorescence quenching) which is reversed when the CF₀ channel is blocked by triphenyltin. These results suggest that modification of critical CF₁ sulfhydryl residues by Hg²⁺, Ag⁺ or N-ethylmaleimide leads to the loss of intra-enzyme coupling between the transmembrane protontransferring and the ATP synthesis activities of the CF₀-CF₁ ATP synthase complex.     

Energy-linked activities in reconstituted yeast adenosine triphosphatase proteoliposome. Adenosine triphosphate formation coupled with electron flow between ascorbate and ferricyanide.

(1) Conditions are described wherein the yeast oligomycin-sensitive adenosine triphosphatase (ATPase) complex can be reconstituted together with phospholipids to yield extremely high rates of ATP-³²P_j exchange. The vesicles so formed exhibit proton uptake upon addition of Mg²⁺-ATP and a relatively slow decay of the proton gradient. (2) The stimulation of ATP-³²P_i exchange by valinomycin + K⁺ reported previously (Ryrie, I. J. (1975) Arch. Biochem. Biophys. 168, 704–711) is apparently not simply due to a diffusion potential. The findings suggest that an electroimpelled, valinomycin-dependent migration of K⁺ may occur together with the electrogenic movements of protons during ATP hydrolysis and synthesis to establish optimal energized conditions for ATP-³²P_i exchange. (3) An artificial oxidative phosphorylation system in the reconstituted vesicles is described: [³²P]ATP formation from ADP and ³²P_i is shown to be linked with electron flow between external ascorbate and internal ferricyanide where a permeable proton carrier, such as phenazine methosulfate, is used to establish a proton gradient. That the yeast ATPase is capable of net ATP synthesis has also been demonstrated in a light-dependent reaction using ATPase proteoliposomes reconstituted together with bacteriorhodopsin.     

Purification and reconstitution of the 32Pi-ATP exchange activity of bovine chromaffin granule membrane

Ghosts derived from bovine chromaffin granules have a ³²P_i-ATP exchange activity which is associated with the H⁺ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (K_m for ATP and P_i, ATP/Mg²⁺ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The ³²P_i-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a ³²P_i-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The α- and β-subunits of F₁-ATPase were major components of the preparation.     

Regulation of coupling factor in field-grown sunflower: A Redox model relating coupling factor activity to the activities of other thioredoxin-dependent chloroplast enzymes

Simultaneous, non-invasive measurements were made of the rate of photosynthetic CO₂ fixation and the state of activation of the chloroplast CF₁CF₀-ATP synthase (CF) in field-grown sunflower (Helianthus annuus L.) during the dark-to-light transition at sunrise. CO₂ fixation showed a linear response with light intensity from zero to about 500–700 E m^-2 s^-1. However, at light intensities of only 5–22 E m^-2 s^-1, the energetic threshold for activation of the CF was found to be significantly lowered (as compared to the pre-dawn state), presumably through reduction of the regulatory sulfhdryl groups of the -subunit of the CF. When these studies were extended to chamber-grown plants, it was found that as little as 5 seconds of illumination at 4 E m^-2 s^-1 caused apparently full CF reduction. It is clear, therefore, that the catalytic activation of CF is not rate limiting to the induction of carbon assimilation under field conditions during a natural dark-to-light transition at sunrise. A model, based on the redox properties of the regulatory sulfhydryls, was developed to examine the significance of sulfhydryl midpoint potential in explaining the differences in light sensitivity and oxidation and reduction kinetics, between the CF and other thioredoxin-modulated chloroplast enzymes. Computer simulations of the light-induced regulation of three representative thioredoxin-modulated enzymes are presented.Abbreviations CF chloroplast CF₁-CF₀ ATP synthease or coupling factor - E_a active form of CF - E_a ⁰ active, oxidized form of CF - E_a ^r active, reduced form of CF - E_i inactive form of CF - E_i ⁰ inactive, oxidized form of CF - E_i ^r inactive, reduced form of CF - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin oxidoreductase - G6PDH glucose-6-phosphate dehydrogenase - MDH NADP-malate dehydrogenase - pmf protonmotive force - pmf_T threshold pmf required to activate CF - pmf_T ⁰ threshold pmf required to active the oxidized form of CF - pmf_T ^r threshold pmf required to activate the reduced form of CF - TR thioredoxin     

Delayed light studies on photosynthetic energy conversion. VIII. Evidence from millisecond emission of chloroplasts for two adenylate binding sites on membrane-bound coupling factor,CF1

Effects of adenylates on chloroplast delayed light emission, at millisecond dark times, are inverse to the previously characterized effects of adenylates on electron transport rates. Either ADP alone or ATP alone increase intensity of delayed light, while ADP plus P_i decrease it. ADP alone requires the presence of an electron acceptor to have this effect on delayed light, but ATP does not.All three adenylate effects are abolished by uncoupling with gramicidin, by partial removal of photophosphorylation coupling factor (CF₁) with EDTA, and by antibody to CF₁. Readdition of CF₁ re-established the adenylate effects in EDTA-stripped membranes. The three adenylate effects are differentially sensitive to pH, and pH differentially affected their abolition by antibody to CF₁. The two adenylate effects shown in the absence of P_i are exhibited at lower adenylate concentrations than the ADP plus P_i effect, and are also less sensitive to phloridzin.These results are discussed in terms of probable adenylate effects on membrane-bound chloroplast coupling factor, CF₁. At least two ADP binding sites would differ with respect to adenylate concentration for half maximal binding; pH of optimal binding capacity; phloridzin sensitivity; and functional regulation of electron transport, proton uptake, and energy storage within the membrane as measured by delayed light emission. It remains unclear whether the high affinity ADP binding site is identical to a high affinity ATP binding site on CF₁.     

The effect of chloroplast coupling factor ATP synthetase (CF1 · CF0) reconstitution on fluidity properties of isolated thylakoid lipid vesicles

The reconstitution of chloroplast coupling factor ATP synthetase (CF₁ · CF₀) with thylakoid lipids by cholate dialysis produced vesicles that displayed higher steady-state anisotropy (r_s) values for both 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenyl hexatriene fluorescence than the pure lipid alone. This is interpreted as meaning that the insertion of protein into the lipid bilayer brings about an increase in the ordering of acyl chains. This ordering effect became more obvious as the protein-to-lipid ratio was increased. Time-resolved decay analyses of DPH fluorescence anisotropy confirmed the conclusion drawn from the steady-state measurements, but further indicated that the dynamic motion of the probe was also slightly restricted after CF₁ · CF₀ incorporation. The restriction of DPH motion and the change in the half-angle for its cone of rotation was observed at relatively low protein-to-lipid ratios as compared with other reconstituted or biological membranes, suggesting that perhaps lipid-protein interactions occur with the inserted CF₁ · CF₀ complex.     

Differential inhibition of Pi-ATP exchange in relation to ATP synthesis and hydrolysis by modification of chloroplast thylakoid membranes with glutaraldehyde

Limited modification of thylakoid membranes with glutaraldehyde inhibits the P_i-ATP exchange reaction much more than ATP synthesis or hydrolysis. More extensive modification of the membranes results in the inhibition of all activities of the ATP synthetase, but does not affect electron transport. Limited modification also does not have much effect on the tight binding of [³H]ADP or the ΔpH supported by ATP hydrolysis. The modification affects the catalytic process itself and not the activation of the latent enzyme. Cross-linking between thylakoid polypeptides is observed only after extensive treatment with glutaraldehyde, while limited modification does not result in cross-linking between polypeptides. The differential inhibition of the P_i-ATP exchange relative to ATP hydrolysis can be explained by the decrease in only one of the kinetic rate constants involved in these reactions. However, the relative insensitivity of photophosphorylation to the modification suggests that different enzyme conformations may participate in phosphorylation (light) and ATP hydrolysis or P_i-ATP exchange (dark).     

Chloroplast genes encoding subunits of the H+-ATPase complex of Chlamydomonas reinhardtii are rearranged compared to higher plants: sequence of the atpE gene and location of the atpF and atpI genes

Summary The chloroplast gene for the epsilon subunit (atpE) of the CF₁/CF₀ ATPase in the green alga Chlamydomonas reinhardtii has been localized and sequenced. In contrast to higher plants, the atpE gene does not lie at the 3 end of the beta subunit (atpB) gene in the chloroplast genome of C. reinhardtii, but is located at a position 92 kb away in the other single copy region. The uninterrupted open reading frame for the atpE gene is 423 bp, and the epsilon subunit exhibits 43% derived amino acid homology to that from spinach. Codon usage for the atpE gene follows the restricted pattern seen in other C. reinhardtii chloroplast genes.The genes for the CF₀ subunits I (atpF) and IV (atpI) of the ATPase complex have also been mapped on the chloroplast genome of C. reinhardtii. The six chloroplast ATPase genes in C. reinhardtii are dispersed individually between the two single copy regions of the chloroplast genome, an organization strikingly different from the highly conserved arrangement in two operon-like units seen in chloroplast genomes of higher plants.Abbreviations bp base pairs - CF₁ chloroplast coupling factor 1 - CF₀ chloroplast coupling factor 0 - F₁ coupling factor 1 - F₀ coupling factor 0 - kb kilobase pairs     

Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 Å subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 Å CF₁ are attached by stalks to an aggregate of CF₀.The CF₁-CF₀ complex was incorporated into phospholipid vesicles; freezefracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; these averaged 96 Å in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF₀, was isolated from membranes by butanol extraction and was incorporated rated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 Å in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF₀ and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.     

Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF₁F_O-ATP synthase) of plants is integrated into the thylakoid membrane via its F_O-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H⁺-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F₁ sector.Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF₁F_O-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å.     

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.     

Malate regulation of Mg2+-ATPase of chloroplast coupling factor 1

The regulatory effects of malate on chloroplast Mg²⁺-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF₁ Mg²⁺-ATPase activity. The subunit of CF₁ may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg²⁺-ATPase was also controlled by the Mg²⁺/ATP ratio in the reaction medium. The enhancing effect of malate on Mg²⁺-ATPase activity depended on the presence of high concentrations of Mg²⁺ in the reaction mixture. Kinetic study showed that malate raised the V_max of catalysis without affecting the K_m for Mg²⁺ ATP. The experiments imply that the stimulation of Mg²⁺-ATPase by malate is probably correlated with the P_i binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF₁ chloroplast coupling factor 1 - CF₁ (-) and CF₁ (-) CF₁ deficient in the and subunit - MF₁ mitochondria coupling factor 1 - NEM N-ethylmaleimide - PMS phenazine methosulfate - OG n-octyl--d-glucopyranoside     

Decay of membrane potential under phosphorylating conditions in chloroplasts with in vivo activated ATPase

(1) The relation between the membrane potential and phosphorylation was studied in chloroplasts rapidly prepared from illuminated spinach leaves (light chloroplasts) and from dark-adapted leaves (dark chloroplasts). Light chloroplasts had a higher ATP hydrolysis activity than dark chloroplasts. (2) In the presence of ADP or ATP, a rapidly decaying phase of the field-indicating 518 nm absorbance change with a half-time of 15 ms became apparent in addition to the slow phase with a half-time of more than 300 ms in either type of chloroplast. Under these conditions, light chloroplasts showed a larger rapid phase than dark chloroplasts. (3) The rapid phase was suppressed by dicyclohexylcarbodiimide and was assumed to reflect the dissipation of membrane potential due to proton movements inside the CF₁-CF₀ ATP synthetase. (4) A model for the proton movement in ATP synthetase is proposed.     

Detection of Sendai virus fusion with human erythrocytes by fluorescence photobleaching recovery

The subunit stoichiometry of the ATP synthetase (CF₁-CF₀) immunoprecipitated from Triton X-100 extracts of chloroplast thylakoid membranes was determined to be α₃, β₃, γ, δ, ? (CF₁) and I_0.3, II_0.6–0.9, III₄₍₆₎ (CF₀). Antibodies against the polypeptides α, β, γ, δ, I, II and ? combined specifically with the isolated subunits as analysed by the protein blotting method. Applying this technique, antibodies against the CF₁ subunits were found to form complexes with the corresponding polypeptides of thylakoids, whereas those against I (M_r 20 000) and II (M_r 17 000) combined with M_r 26 000 and M_r 24 500 membrane polypeptides, respectively. The M_r 26 000 polypeptide was identified as the major subunits of the light-harvesting chlorophyll a/b-protein (LHCP) complex and the M_r 24 500 component seems to be functionally connected with this complex. From the results it is concluded that the chloroplast ATP synthetase consists of the subunit of the α, β, γ, δ, ? and III (proteolipid only and that proteolytically altered LHCP polypeptides bind artifically to the protein complex during isolation.     

Effects of nitrofen on chloroplast coupling factor-dependent reactions

Nucleotides induce a conformational change in the proteins of the CF₀-CF₁ complex. They give rise to reduced proton permeability of the thylakoid membranes. This reaction is paralleled by an enhanced yield of the steady-state proton uptake and a reduced nonphosphorylating electron-transport rate. Nitrofen acts as an energy-transfer inhibitor. It inhibits the rate of nucleotide exchange on CF₁ both at ‘loose’ and ‘tight’ binding sites. During illumination the percentage of nucleotide-free CF₀-CF₁ complex seems to be enhanced in the presence of nitrofen. This results in a prevention of the described ADP effects on proton uptake and electron transport. These similar effects of nitrofen on loose and tight nucleotide-binding sites correspond with the idea that both types are different states of identical sites.     

Concentration Gradient Effects of Sodium and Lithium Ions and Deuterium Isotope Effects on the Activities of H-ATP Synthase from Chloroplasts

We explored the concentration gradient effects of the sodium and lithium ions and the deuterium isotope's effects on the activities of H⁺-ATP synthase from chloroplasts (CF₀F₁). We found that the sodium concentration gradient can drive the ATP synthesis reaction of CF₀F₁. In contrast, the lithium ion can be an efficient enzyme-inhibitor by blocking the entrance channel of the ion translocation pathway in CF₀. In the presence of sodium or lithium ions and with the application of a membrane potential, unexpected enzyme behaviors of CF₀F₁ were evident. To account for these observations, we propose that both of the sodium and lithium ions could undergo localized hydrolysis reactions in the chemical environment of the ion channel of CF₀. The protons generated locally could proceed to complete the ion translocation process in the ATP synthesis reaction of CF₀F₁. Experimental and theoretical deuterium isotope effects of the localized hydrolysis on the activities of CF₀F₁, and the energetics of these related reactions, support this proposed mechanism. Our experimental observations could be understood in the framework of the well-established ion translocation models for the H⁺-ATP synthase from Escherichia coli, and the Na⁺-ATP synthase from Propionigenium modestum and Ilyobacter tartaricus.