Radiation inactivation analysis of chloroplast CF0-CF1 ATPase

Radiation inactivation technique was employed to measure the functional size of adenosine triphosphatase of spinach chloroplasts. The functional size for acid-base-induced ATP synthesis was 450 +/- 24 kilodaltons; for phenazine methosulfate-mediated ATP synthesis, 613 +/- 33 kilodaltons; and for methanol-activated ATP hydrolysis, 280 +/- 14 kilodaltons. The difference (170 +/- 57 kilodaltons) between 450 +/- 24 and 280 +/- 14 kilodaltons is explained to be the molecular mass of proton channel (coupling factor 0) across the thylakoid membrane. Our data suggest that the stoichiometry of subunits I, II, and III of coupling factor 0 is 1:2:15. Ca2+- and Mg2+-ATPase activated by methanol, heat, and trypsin digestion have a similar functional size. However, anions such as SO3(2-) and CO3(2-) increased the molecular mass for both ATPase's (except trypsin-activated Mg2+-ATPase) by 12-30%. Soluble coupling factor 1 has a larger target size than that of membrane-bound. This is interpreted as the cold effect during irradiation.     

Participation of three distinct active states of chloroplast ATPase complex CF0 X CF1 in the activation by light and dithiothreitol

Chloroplast ATPase complex (CF0 X CF1) in thylakoids is activated by illumination in the presence or absence of dithiothreitol or by incubation with both dithiothreitol and Pi in the post-illumination dark. The activation by dithiothreitol and Pi is inhibited by ADP and decreases with increasing time interval between the end of illumination and the addition of dithiothreitol and Pi. The dithiothreitol/Pi-activated ATP hydrolysis is highly sensitive to pH. The ATP hydrolysis activated by illumination in the presence of dithiothreitol decreases its sensitivity to stimulation by NH4Cl and increases its sensitivity to pH, with increasing time interval between the end of illumination and the addition of ATP. Its pH dependence approaches that of the dithiothreitol/Pi-activated ATP hydrolysis. These results suggest that in the post-illumination dark, the light/dithiothreitol-activated CFo X CF1 converts its state from the one (E2) which is sensitive to an uncoupler and relatively insensitive to pH to the one (E2i) which is insensitive to an uncoupler and highly sensitive to pH. In the post-illumination dark, the presence of both dithiothreitol and Pi brings about the activation of CFo X CF1 to E2i.     

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.     

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.     

Subunit interactions within the chloroplast ATP synthase (CF0-CF1) as deduced by specific depletion of CF0 polypeptides

The proton-linked ATP synthase (CF1-CF0) of chloroplasts consists of a catalytic component (CF1) and a membrane-embedded part (CF0) that interacts with CF1 and contains a proton channel. The subunits of CF0 which are involved in binding of CF1 were studied by examining the effect of selective depletion of subunits I, II, and IV of CF0 from the chloroplast ATP synthase on the association of the remaining CF0 subunits with CF1. Dissociated CF0 subunits were identified by sucrose density gradient centrifugation. Removal of subunit IV alone from CF0-CF1 did not cause dissociation of the other CF0 subunits from CF1. Upon removal of both subunits I and IV from CF0-CF1, subunit II also dissociated, but subunit III was still bound to CF1. Thus, at least two subunits of CF0, I and III, directly associate with CF1. Subunit II is unlikely to bind CF1 directly and may associate with subunit I. Although depletion of subunit IV does not cause dissociation of CF0 from CF1, its interaction with CF1 subunits is uncertain.     

Chromatographic purification of the chloroplast ATP synthase (CF0-CF1) and the role of CF0 subunit IV in proton conduction

Chromatographic procedures were developed to purify chloroplast ATP synthase (CF0-CF1) in large amounts and to resolve subunits from this enzyme. The ATP synthase thus obtained has high ATP-Pi exchange and Mg2(+)-ATPase activities upon incorporation into asolectin liposomes. The purity of this preparation was about 95%. By modifications of this chromatographic procedure, we purified subunit IV-deficient CF0-CF1, subunit IV-deficient CF0, and subunit IV. Both ATP-Pi exchange and Mg2(+)-ATPase activities were impaired by depletion of subunit IV from CF0-CF1. Partial restoration of these activities was obtained by reconstituting subunit IV-deficient CF0-CF1 with subunit IV. The impairment of these activities was likely caused by a loss in proton conductivity of CF0 upon removal of subunit IV. The dicyclohexylcarbodiimide-sensitive Mg2(+)-ATPase of subunit IV-deficient CF0-CF1 was not as sensitive to the depletion of subunit IV as ATP-Pi exchange. Nearly 90% of subunit IV could be removed, but Mg2(+)-ATPase activity was inhibited by only 40-60%. Thus subunit IV of CF0-CF1 may not participate directly in proton transfer but may have a role in organizing and/or stabilizing CF0 structure.     

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.     

Inhibition of Thylakoid ATPase by Venturicidin as an Indicator of CF1-CF0 Interaction

Venturicidin inhibits the F0 portion of membrane-located, H+-pumping ATPases. We find it meets the criteria for an energy transfer inhibitor for spinach (Spinacia oleracea) thylakoids: complete inhibition of photophosphorylation and of photophosphorylation-stimulated and basal electron flow rates, but not of electron flow under uncoupled conditions. The extent of H+ uptake in the light is stimulated by venturicidin (vtcd), as expected for a compound blocking H+ efflux through CF0. Vtcd had no effect on the nonproton pumping, methanol-stimulated ATPase of thylakoids or on soluble CF1 ATPase. Under totally uncoupled conditions (saturating NH4Cl + gramicidin), vtcd can still inhibit sulfite-stimulated thylakoid ATPase completely. The concentration of vtcd needed for inhibition of ATPase was proportional to the concentration of thylakoids present in the assay, with an apparent stoichiometry of about 10 vtcd molecules per CF1/CF0 for 50% inhibition. Vtcd raised the Km for ATP somewhat, but had a stronger effect on the Vmax with respect to ATP. Inhibition by saturating vtcd ranged from 50 to 100%, depending on the condition of the thylakoids. Grinding leaves in buffer containing 0.2 M choline chloride (known to provide superior photophosphorylation rates) helped bring on maximum vtcd inhibition; trypsin treatment or aging of thylakoids brought on vtcd-resistant ATPase. We conclude that the extent of inhibition by vtcd can be used as an indicator of the tightness of coupling between CF1 and CF0.     

Single channel H+ currents through reconstituted chloroplast ATP synthase CF0-CF1.

The purified chloroplast ATP synthase (CF(0)-CF(1)) was reconstituted into azolectin liposomes from which bilayer membranes on the tip of a glass pipette ('dip stick technique')and planar bilayer membranes were form ed. The CF(0)-CF(1) facilitated ion conductance through the bilayer membranes. Our results clearly indicated that the observed single channel currents were carried by H+ through the isolated and reconstituted chloroplast ATPase. We demonstrated that in proteoliposomes it is the whole enzyme complex CF(0)-CF(1) and not the membrane sector CF(0) alone that constitutes a voltagegated, proton-selective channel with a high conductance of 1-5 pS at pH 5.5-8.0. After removal of CF(1) from the liposomes by NaBr treatment the membrane sector CF(0) displayed various kinds of channels also permeable to monovalent cations. The open probability P(0) of the CF(0)-CF(1) channel increased considerable with increasing membrane voltage [from P(0) less than or equal to 1% (V(m) less than or equal to 120 mV) to P(0) less than or equal to 30% (120 mV less than or equal to Vm 200 mV)]. In the presence of ADP (3 microM) and P(i) (5 microM), which specifically bind to CF(1), the open probability decreased and venturicidin (1 microM), a specific inhibitor of H+ flow through CF(0) in thylakoid membranes, blocked the channel almost completely. Our results, which reveal a high channel unit conductance, and at membrane voltages less than 100 mV low open probability with concomitant mean open times in the micros timescale (less than 100 micros) for the energy coupling in the enzyme complex. At physiological membrane voltages for photophosphorylation (about 30 mV) the enzyme complex would then display a time-averaged conductance of about 1 fS.     

A time lag in the onset of ATP-Pi exchange catalyzed by purified ATP-synthase (CF0-CF1) proteoliposomes and by chloroplasts

The time course of ATP-Pi exchange which is catalyzed by the isolated chloroplast ATP synthase in phospholipid vesicles was studied. The following observations were made. (i) The onset of 32Pi incorporation into ATP lags behind ATP hydrolysis. The lag lasts for about 2 min at 37 degrees C and is followed by a steady-state rate which is constant for more than 30 min. Under the same experimental conditions, ATP hydrolysis shows an initial burst followed by a constant, slower rate. (ii) The initial lag is independent of Mg-ATP concentration in the range 0.2-5 mM and of the presence of ADP. In contrast, the steady-state rate of ATP-Pi exchange has an apparent Km of 0.3 mM for Mg-ATP and is stimulated by ADP. (iii) Increasing the temperature from 30 to 45 degrees C decreases the lag from 6 min to zero. The steady-state rate of ATP-Pi exchange is affected to a much smaller extent by the temperature in this range. (iv) The lag is insensitive to valinomycin or tetraphenylboron, while the steady-state rate is partially inhibited. Nigericin and protonophores affect both the lag and steady-state rate. (v) ATP-induced membrane potential formation, as followed by oxonol VI, does not correlate with the lag in its kinetics and temperature dependence. ATP-induced pH gradient formation could not be detected in the proteoliposome system. (vi) Light-triggered ATP-Pi exchange in chloroplasts shows essentially the same time course as the proteoliposome system, but the lag lasts for only about 20 s at room temperature and is unaffected by a preexisting proton gradient. These results suggest that the initial lag in ATP-Pi exchange does not reflect the time required for the buildup of a protomotive force (delta - mu H+) nor the time required to produce ADP. It is suggested, therefore, that the lag reflects an internal autocatalytic conformational change in the ATP-synthase complex which is initiated by ATP hydrolysis and which converts the enzyme from an "exclusive ATPase state" to a "reversible ATP-synthase state". This slow transition is not directly coupled to a trans-membrane pH or potential gradient.     

Correlation between the ATP synthetic active state and the ATP hydrolytic active state in chloroplast ATP synthase-ATPase complex CF0 . CF1

Illumination of chloroplast thylakoids activates ATP synthase-ATPase complex CF0 . CF1. The time course of ATP synthesis is linear if ADP and Pi are added before or simultaneously with illumination. ATP synthesis initiated by adding the substrates in the light exhibits a curvilinear time course with a low initial rate (Vi). Vi, but not the rate at a steady state, decreases with increasing preillumination time with a half-time of 2 s. Coincident with this decrease in Vi, activation of ATP hydrolysis takes place. In the postillumination dark, restoration of Vi is observed: Vi increases with increasing time intervals between the end of illumination and the addition of the substrates with simultaneous reillumination (half-time of 3 s). Coincident with this restoration of Vi, inactivation of ATP hydrolysis takes place. Such an increase in Vi in the postillumination dark is not observed in thylakoids pretreated with N-ethylmaleimide. These results suggest the following: in the light, the ATP synthetically active, but ATP hydrolytically inactive state (Es) converts to the ATP hydrolytically active, but ATP synthetically inactive (or less active) state (Eh) in the absence of ADP and Pi. The N-ethylmaleimide pretreatment inhibits this process. In the postillumination dark, the reverse conversion takes place.     

Stimulation mechanism of soluble ATPase from chloroplasts (CF1)]

Effects of various compounds on Mg-dependent ATPase activity of chloroplast coupling factor--CF1--were studied. It was shown that the stimulating effect of compounds is increased with the increase in their hydrophobicity. Under given experimental conditions all compounds under study readily accept and donate protons. The maximal efficiency is reached when pH of the medium is close to the pK value of conjugated acid. It is assumed that the stimulating effects of compounds on Mg-dependent chloroplast ATPase consist in the increase of the rate of the limiting step of enzyme induced proton translocation coupled to the catalytic step of ATP hydrolysis.     

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-Conductivity of CF0-CF1 Reconstructed into Planar Lipid Bilayer

The proton conductable ATP synthase (CF0-CF1) is the key enzyme of energy conversion in the membrane of bacteria, mitochondria and chloroplast. In spite of a large body of studies, the structure and molecular mechanism of ATP synthases are still elusive. In order to learn the mechanism of ATP synthases, the authors used voltage-olamp technique to study the effect of different conditions on the proton conductance of F0-F1 into planar lipid bilayer membrane. The results obtained were as follows: (1) When CF0-CF1 was reconstructed into planar lipid bilayer membrane, the resistance decreased by 10 times. (2) Channel-like current was recorded at the low concentration of CF0-CFl(protein 2 mg/L) in the solution. (3) In metal ion-free solution, the channel currents changed with the trans-membrane proton gradient (ApH). Under holding potential from 0 to + 150 mV, the stimulation of △pH on channel current increased with a rise in the ApH from 2 to 4, the stimulation of 4.5 △pH on channel current was weaker than that of △pH 4.0. (4) The proton conduetance inhibitor, dicyclohexylcarbodiimide (DCCD), showed a rapid and irreversible inhibition effect on the channel current. (5) In metal ion-free solution (10 mmol/L Tris-HC1), when the ApH across the black lipid membrane (BLM) maintained at 3.0, the addition of Mg2 + caused a alger channel current of CF0-CF1 than the addition of Ca2+ , with holding potential from 0 to + 150 mV. The results indicated that reconstruction of CF0-CF1 was successful and Mg2 + was directly involved in the proton conductance pathways.     

Energy-dependent conformational changes in the epsilon subunit of the chloroplast ATP synthase (CF0CF1)

Incubation of spinach chloroplast thylakoids with pyridoxal 5'-phosphate modified the epsilon subunit of ATP synthase (CF0CF1). Illumination of thylakoids stimulated the modification of one specific amino acid residue of the epsilon subunit by a factor of 3. Endoproteinase Glu-C treatment of the isolated epsilon subunit and fractionation of the peptides by high performance liquid chromatography revealed a major fluorescent peptide with the sequence GKRQKIE. Further treatment of this peptide with endoproteinase Arg-C gave a strongly fluorescent tripeptide (GXR). From the primary structure of the epsilon subunit, the specifically modified residue was deduced to be Lys-109. This suggests the energy-dependent conformational changes in the epsilon subunit which change the surroundings of Lys-109 and alter the reactivity of this residue.     

Proton 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 Pi is stimulated by low levels of Hg2+ or Ag+ (50% stimulation approximately or equal to 3 Hg2+ or 6 Ag+/100 chlorophyll) to a plateau equal to the transport rate under normal phosphorylating conditions (i.e. +ADP, +Pi). 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-ethylmalemide stimulations, but not the Ag+ stimulation, are completely reversed by low levels of ADP (2 microM), ATP (2 microM), AND Pi (400 microM). Ag+, which is a potent inhibitor of ATP synthesis, has little or no effect upon phosphorylating electron transport (+ADP, +Pi). Concomitant with the stimulations of non-phosphorylating electron transport by Hg2+, Ag+ and ADP + Pi, 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-ethylmalemide leads to the loss of intra-enzyme coupling between the transmembrane proton-transferring and the ATP synthesis activities of the CF0-CF1 ATP synthase complex.