Evidence for solvent-induced conformational changes of the soluble Dunaliella chloroplast coupling factor 1 (CF1)

The ATPase activity of the chloroplast coupling factor 1 (CF₁) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca²⁺ -dependent activity. The Ca²⁺ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca²⁺ to Mg²⁺. Both the Ca²⁺ -dependent and Mg²⁺ -dependent ATPase activities of heat-treated Dunaliella CF₁ are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF₁. However, when assayed under identical conditions, the Ca²⁺ -dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg²⁺ -dependent activity. These data are interpreted as indicating that soluble Dunaliella CF₁ can exist in a variety of conformations, at least one of which catalyzes a Ca²⁺ -dependent ATPase and two or more of which catalyze an Mg²⁺ -dependent ATPase.     

Evidence for light-dependent reversible conformational change in spinach chloroplast CF1

We have investigated an inhibition of photophosphorylation which occurs during preillumination of isolated spinach chloroplasts. Preillumination for 4–6 min in the absence of a complete set of components required for ATP synthesis inhibits photophosphorylation to a maximum of 25–40%; no inhibition occurs if all components for phosphorylation are present from the time illumination begins. The inhibition is about 40% recoverable by imposing a dark (“rebound”) period after the preillumination. Photoinhibition is accompanied by an increased leakiness of the thylakoid membrane to protons and is prevented by the presence of FCCP during the preillumination. Several lines of evidence implicate changes in conformation of chloroplast coupling factor (CF₁) as the cause of both photoinhibition and dark rebound. Conditions which result in photoinhibition also result in a loss of Mg²⁺-dependent ATPase activity which can be elicited from chloroplasts. Both photoinhibition and dark rebound are accompanied by changes in the K_m of CF₁ for both ADP and P_i. Photoinhibition precludes further inhibition of phosphorylation by light plus N-ethylamleimide (NEM) while phosphorylating activity regained by dark rebound is sensitive to subsequent inhibition by light plus NEM. The results are consistent with the conformational coupling hypothesis in indicating that CF₁ may be able to store energy in a conformational state which can be released by the reversal of that state. The photoinhibition we observe may represent conformational changes in CF₁ which are related to conformational coupling but which lead to photoinhibition under our conditions of preillumination.     

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.     

The epsilon Subunit of the Chloroplast Coupling Factor 1 from Euglena gracilis: A Possible Role in Controlling ATPase Activity

The coupling factor from chloroplasts (CF₁) of Euglena gracilis Z strain is an active ATPase in situ, and its activity cannot be increased by treatment with trypsin or heating as is the case with the CF₁ from other sources. The smallest subunit of CF₁, the ε subunit, is supposed to be involved in controlling the ATPase activity. We have devised a simple technique for rapid and large-scale isolation of this subunit. The ε subunit from Euglena CF₁, although having only a limited inhibitory effect on Euglena CF₁, drastically inhibited the ATPase activity of heat-activated spinach CF₁. The inhibition of spinach CF₁ could be reversed by passage through Sephadex G-50 or by a second heat activation. An antibody to the ε subunit of Euglena CF₁ cross-reacted only weakly with CF₁ from spinach, Sorghum, Kalanchoë, or Anacystis nidulans, but reacted well with whole Euglena CF₁ in addition to its ε subunit. The antibody increased the ATPase activity of Euglena and Anacystis CF₁ and of unactivated or partially activated spinach CF₁. The results suggest that the function of the ε subunit in Euglena CF₁ is similar to its function in CF₁ from other sources. The data also suggest that changes induced in spinach CF₁ by activation involves modifications in subunits other than the ε one.     

Association of a thioredoxin-like protein with chloroplaast coupling factor (CF1).

The δ subunit isolated from chloroplast coupling factor (CF₁) preparations partially replaced thioredoxin in the dithiothreitol-linked activation of chloroplast fructose 1,6-bisphosphatase. The δ subunit fraction also stimulated the dithiothreitol-dependent ATPase of heated CF₁ in a manner analogous to that obseryed with each of the three thioredoxins isolated from spinach leaves (thioredoxins f, m, and c). The δ subunit used in most of these experiments was obtained from CF₁ that had been isolated by a newly devised procedure based on acid precipitation.     

Tightly bound sulpholipids in chloroplast CF0-CF1

Highly purified preparations of CF₀-CF₁ from chloroplasts contain a small amount of tightly bound lipids. Extraction and analysis of these lipids show that they are almost exclusively sulpholipids. The calculated amount of bound sulpholipids in spinach and in Dunaliella salina CF₀-CF₁ preparations are 5 and 20 mols/mol enzyme, respectively. Attempts to exchange the bound lipids with other lipids or with detergents have failed, indicating a very strong association with CF₀-CF₁.     

Properties of the solvent-stimulated ATPase activity of chloroplast coupling factor 1 from Chlamydomonas reinhardii

The effects of solvents on the ATPase activity of chloroplast coupling factor 1 (CF₁) isolated from wild-type Chlamydomonas reinhardii have been studied. Of the solvents examined, the following order summarizes their maximal ability to stimulate the ATPase activity of CF₁: ethanol > methanol>allyl alcohol >n-propanol > acetone≈dioxane > ethylene glycol. Glycerol inhibits the CF₁ activity at all concentrations. In the absence of organic solvents, 50% of the activity of the enzyme is irreversibly lost after a 10 min incubation at 65–70°C. Ethanol (23%) causes a 30°C drop in the temperature required for 50% inactivation. ATP partially stabilizes the CF₁ in the presence, but not in the absence, of ethanol. In the absence of organic solvents, both free Mg²⁺ and ADP inhibit the CF₁-ATPase. Mg²⁺ is a noncompetitive inhibitor with respect to MgATP, and the kinetic constants are: V, 6.3 μmol ATP hydrolyzed/mg protein per min; K_m(MgATP), 0.23 mM; K_ii(Mg²⁺), 27 μM; and K_is(Mg²⁺), 50 μM. In the presence of ethanol, double-reciprocal plots are no longer linear and have a Hill coefficient of about 1.8±0.1. V increases about 10–12-fold. The pattern of inhibition by Mg²⁺ appears to change from noncompetitive to competitive with respect to MgATP. In addition, ADP no longer inhibits the MgATPase activity of CF₁.     

Activation of the CF0-CF1, ATP synthase from spinach chloroplasts by chloroplast lipids

The interactions of CF₀-CF₁ with different lipids were studied by following the stimulation of Mg-ATPase and of P_i-ATP exchange activities of reconstituted CF₀-CF₁ proteoliposomes. The following results were obtained: (1) Both P_i-ATP exchange and Mg-ATPase activities are stimulated by lipids. Furthermore, the inhibition of Mg-ATPase by N,N′-dicyclohexylcarbodiimide is dependent on the interactions of CF₀-CF₁ with lipids. (2) A polar lipid extract of thylakoid membranes stimulates Mg-ATPase activity of CF₀-CF₁ more efficiently than phospholipids. The relative effectiveness of Mg-ATPase stimulation is: chloroplast lipids > soybean phospholipids > phosphatidylcholine/phosphatidylserine (4: 1) > phosphatidylcholine. The rate of P_i-ATP exchange in chloroplast lipids CF₀-CF₁ proteoliposomes is, however, lower than in soybean lipids CF₀-CF₁ proteoliposomes, due to their higher permeability to protons. Addition of 10% phosphatidylserine to chloroplast lipids reduces their permeability to protons and stimulates P_i-ATP exchange. (3) The kinetic mechanism of ATPase stimulation by chloroplast lipids is by decreasing the K_m (ATP) and by increasing V_max in comparison to soybean lipid proteoliposomes. This may explain the low affinity for ATP and the slow turnover rate of the purified enzyme in artificial lipids in comparison to the native enzyme in chloroplast thylakoids. (4) Chloroplast lipids lacking monogalactosyldiacylglycerols only poorly activate CF₀-CF₁. A large stimulation of P_i-ATP exchange is obtained by a mixture of 60% monogalactosyldiacylglycerol and 40% of the rest of the chloroplast lipids, but not by mixtures of monogalactosyldiacylglycerol with phospholipids. Hydrogenation of the unsaturated fatty acids of monogalactosyldiacylglycerol inhibits the activation of CF₀-CF₁. (5) The results suggest that: (a) interactions of specific chloroplast lipids with CF₀-CF₁ activates the enzyme by increasing its turnover and its affinity for ATP; (b) specific requirements for CF₀-CF₁ activation are the presence of monogalactosyldiacylglycerols together with another chloroplast lipid component and of highly unsaturated fatty acids.     

Low Concentrations of Tetracycline Enhance the Photophosphorylation and P/O Ratio of Chloroplasts

This study investigated the effects of tetracycline on photophosphorylation, electron transport and P/O ratio of spinach chloroplasts. When chloroplast preparations were treated with low concentrations of tetracycline, non-cyclic and cyclic photophosphorylation activities increased, electron transport rates and P/O ratios improved, chloroplast ms-DLE also improved, and the Mg²⁺-ATPase activity of CF₁ increased in comparison to the control. These results indicate that spinach chloroplasts are sensitive to tetracycline. Next, we used the fluorescence emission spectra of CF₁ to examine the possible binding sites for tetracycline. The fluorescence emission spectra of CF₁ treated with glutaraldehyde, NEM and TNBS, which interact with CF₁ across its whole structure, at the γ subunit and at the β subunit, respectively, were compared with that of control CF₁. The peak sites of the various fluorescence emission spectra were the same, but the peak values for CF₁ treated with glutaraldehyde, NEM and TNBS were lower than that of control CF₁. The peak value of CF₁ treated with 50 μM tetracycline was very similar to that of CF₁ treated with NEM. The above results indicate that the acting site of tetracycline may be at or near the γ subunit of CF₁, and allows the creation of a model in which tetracycline binding strengthens the subunit interactions of ATP synthase, enlarges the proton motive force across the thylakoid membrane, and allows the excess proton motive force to increase ATP formation and improve the P/O ratio. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of the Activation of Membrane-Bound and Soluble CF(1) by Thioredoxin

The activation of spinach (Spinacia oleracea) chloroplast coupling factor 1 (CF₁) by thioredoxin (ThR) was characterized using membrane-bound and soluble CF₁. Light generates an electrochemical proton gradient across the thylakoid membrane, which increases the accessibility of the disulfide bond on the γ-subunit of CF₁ to reduced ThR. The proton gradient substantially accelerates the activation of CF₁ compared with thylakoids incubated in the dark with similar concentrations of dithiothreitol and ThR. The interaction of soluble CF₁ with ThR was studied using fluorescent probes. CF₁ in solution, with and without its associated ε-subunit, was labeled at Cys-322 of the γ-subunit with fluoresceinyl maleimide. ThR from Escherichia coli was labeled with eosin isothiocyanate. Labeled ThR and CF₁ showed normal activities. Fluorescence energy transfer between donor fluoresceinyl maleimide and acceptor eosin isothiocyanate, manifested by a quenching of the donor fluorescence, was detected, suggesting that ThR and CF₁ form an intermolecular complex. When the ε-subunit was absent, quenching of donor fluorescence was approximately doubled, indicating that labeled ThR could approach more closely to the γ-subunit of CF₁. The distance between the fluorescent probes on CF₁ and ThR was calculated to be approximately 65 Å when ε-subunit was present and 52 Å when ε was absent. These values are consistent with other distance measurements and energy transfer values reported previously for fluorescent probes on CF₁. Whereas the extent of quenching increased by removal of the ε-subunit, the apparent dissociation constant was unchanged. The quenching effect was reversed when the ε-subunit was added back to the titration mixture. Similarly, the addition of unlabeled ThR decreased donor quenching.     

Synergistic activation of an Mg-specific ATPase activity in chloroplast coupling factor by octylglucoside and tentoxin

(1) Octylglucoside stimulates an Mg²⁺-specific ATPase activity with CF₁ preparations from different higher plants and the alga Chlamydomonas reinhardii. (2) Tentoxin at high concentrations (10^?4–10^?3 M) in the presence of octylglucoside further stimulates the Mg²⁺-ATPase activity of CF₁ from tentoxin-sensitive species and inhibits the activity of CF₁ from tentoxin-resistant species. The extent of tentoxin stimulation and inhibition varies among species. A maximal stimulation of over 2-fold was obtained with spinach CF₁ and a maximal inhibition of 50% was obtained with C. reinhardii CF₁. In Nicotiana spp., tentoxin had only a marginal effect on the Mg²⁺-ATPase activity induced by octylglucoside.     

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     

Interaction of Sulfite with the Noncatalytic and Catalytic Sites of Chloroplast Coupling Factor CF1

The interaction between sulfite, an efficient Mg²⁺-dependent F₁-ATPase activator, and chloroplast CF₁-ATPase was studied. The sulfite anion was shown to inhibit ADP and ATP binding to the noncatalytic sites of CF₁. The stimulating activity of sulfite persists when all noncatalytic sites are nucleotide-occupied. Phosphate, a competing candidate for binding to CF₁ catalytic sites, suppresses this activity. These results support the suggestion that the stimulation of Mg²⁺-dependent ATPase activity of CF₁ is caused by sulfite binding to its catalytic sites.     

Distribution of chloroplast coupling factor (CF1) particles on plastid membranes during development

Samples of internal membrane systems separated from lysates of intact plastids from dark grown Avena sativa L. (vars, Cooba and Mostyn) and Hordeum vulgare L. (vars, Himalaya and Deba Abed) given different periods of illumination before isolation were assayed for trypsin-activated Ca²⁺-dependent ATPase activities and also examined in the electron microscope after treatment in the manner described by Oleszko and Moudinanakis (1974) which assists the visualization of the chloroplast coupling factor (CF₁) particles. Concentrations of membrane-attached CF₁ particles were not observed on the membrane surfaces of the prolamellar bodies (PLBs) proper but only on the attached extruded lamellar membranes. Increasing lengths of illumination followed by plastid isolation and subsequent membrane separation had the effect of progressively increasing the mean distance between these individual lamellar-attached CF₁ particles. Measurements of trypsin-activated Ca²⁺-dependent ATPase activities during similar developmental regimes indicated that functions associated with CE₁ particles are relative constant and largely independent of the period of illumination if the values were expressed on a per plastid basis indicating that assembly of CF₁ particles may take place in either etioplasts, etiochloroplasts or mature chloroplasts.Abbreviations PLB prolamellar body - EDTA ethylene-diaminetetra-acetic acid - CF₁ chloroplast coupling factor particles - ATPase adenosine triphosphatase     

Characterization of CF1 from the Diatom Odontella sinensis

The CF₁ moiety of the chloroplast ATPase of the diatom Odontella sinensis was solubilized from isolated thylakoids by chloroform extraction. Further purification was achieved by HPLC on a Superose-6 column. The resulting four-subunit complex was identified as CF₁ lacking subunit δ. The larger two subunits, α and β, showed cross-reactivity with antisera raised against the homologous subunits of spinach-CF₁. Western blot analysis further revealed that — contrary to other ATPases — migration in SDS-PAGE of α was faster than migration of β, suggesting a deletion of 40 to 50 amino acids in subunit α of Odontella. The assumed deletion does not involve the N-terminal side of the protein, as was established by protein sequencing. The N-terminal sequences of subunits α and γ showed highest homologies with the equivalent subunits of blue-green algae. According to SDS-PAGE, the apparent molecular weights of the four Odontella subunits were 53.2 (β), 51.2 (α), 39.3 (γ) and 16.2 (ε) kD. ATPase activity of isolated Odontella-CF₁ could be induced by trypsin or octylglucoside, and to a lesser extent by sulfite or by alcohols such as methanol or ethanol.     

Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach

The effects of nano-TiO₂ (rutile) on the photochemical reaction of chloroplasts of spinach were studied. The results showed that when spinach was treated with 0.25% nano-TiO₂, the Hill reaction, such as the reduction rate of FeCy, and the rate of evolution oxygen of chloroplasts was accelerated and noncyclic photophosphorylation (nc-PSP) activity of chloroplasts was higher than cyclic photophosphorylation (c-PSP) activity, the chloroplast coupling was improved and activities of Mg²⁺-ATPase and chloroplast coupling factor I (CF₁)-ATPase on the thylakoid membranes were obviously activated. It suggested that photosynthesis promoted by nano-TiO₂ might be related to activation of photochemical reaction of chloroplasts of spinach.     

Photophosphorylation and the chemiosmotic perspective

Photophosphorylation was discovered in chloroplasts by D. Arnon and coworkers, and in bacterial ‘chromatophores’ (intercytoplasmic membranes) by A. Frenkel. Initial low rates were amplified by adding electron-carrying compounds such as FMN, later shown to support the ‘pseudocyclic’ electron flow. ATP synthesis, and coupling to electron flow, was detected accompanying linear electron flow from H₂O to either NADP⁺ or ferricyanide. Another pattern of electron flow supporting photophosphorylation was that of a cycle around Photosystem I (PS I). Isolation and analysis of the ATP synthase showed, as with mitochondrial and bacterial analogues, an intrinsic membrane complex (CF₀) and an extrinsic complex (CF₁). CF₁ is a latent ATPase, activated additively by the high-energy state of the thylakoids, and by reduction of a disulfide bond on the gamma subunit. Once reduced, ATP synthesis occurs at lower energy levels. The search for an ‘intermediate’ linking electron flow and ATP synthesis led to the discovery of post-illumination ATP synthesis by thylakoids, where turnover occurs in the dark. Once interpreted by P.Mitchell's chemiosmotic hypothesis, this led to the discovery of light-driven proton uptake into the thylakoid lumen, with accompanying Cl⁻ intake and Mg²⁺ and K⁺ output. Chemiosmosis was confirmed in several ways, including ATP synthesis in the dark due to an acid-to-base transition of thylakoids, and photophosphorylation accomplished in artificial lipid vesicles containing both the proton-pumping bacterial rhodopsin and a mitochondrial ATPase complex. The now generally accepted chemiosmotic interpretation is able to clarify some other aspects of photosynthesis as well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enzymes of plastid ribosome-deficient mutants chloroplast ATPase (CF1)

Summary The mutant line albostrians ofHordeum vulgare L. was investigated by a variety of methods to detect the presence of the chloroplast coupling factor of photophosphorylation (CF₁). The plastids in white leaves of the line albostrians lack ribosomes and are therefore not able to synthesize proteins.Plastid membranes were isolated from light-grown and dark-grown leaves of the wild-type and of the plastid ribosome-deficient mutant. CF₁ could be removed from chloroplast membranes of the wild-type by treatment with EDTA. However, no ATPase activity was found in EDTA extracts of the mutant, and no trace of CF₁ could be detected in this extract by reaction with antiserum against CF₁ in two-dimensional immunoelectrophoresis. Eight isoenzymes of ATPase were found in the fraction of soluble proteins of green wild-type leaves. No CF₁ is detected in this fraction isolated from mutant leaves, but four of the ATPases (not identical with CF₁) are reduced in activity or lacking. Visualization of CF₁ upon internal membranes of etioplasts of the wild-type was made possible by a special electron microscope procedure. CF₁ particles were found in large numbers attached to the pro-thylakoid membranes. CF₁ particles could not be observed upon the membranes from plastids prepared from the mutant.Since no trace of CF₁ could be detected in the plastids of the mutant by various methods, it is concluded that the absence of CF₁ in this mutant is a direct effect of the deficiency in the capacity of the plastid to synthesize proteins.This is part XVII of a series on: Structure and function of the genetic information in plastids.     

Effect of methanol on spinach thylakoid ATPase

Methanol at 35% ($\text{v}\text{v}$) overcomes the latency of spinach thylakoid ATPase. Activation is immediate and reversible involving changes in the V_max, not the K_m of the enzyme, MgATP is a much better substrate than CaATP; free Mg²⁺ noncompetitively inhibits activity. This inhibition can be overcome by the addition of Na₂SO₃. While both MgATP and MgGTP act as substrates, free ATP and GTP both inhibit activity. ADP and MgADP are also inhibitory. Insensitivity to certain inhibitors indicates that methanol neither induces the same conformational changes in CF₁ as illumination does, nor does it lead to coupling between H⁺ movement through CF₀ and ATP hydrolysis. Methanol activation provides a much improved method for assaying thylakoid ATPase.