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.     

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.     

Aspects of Subunit Interactions in the Chloroplast ATP Synthase (II. Characterization of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids)

A complex between chloroplast-coupling factor 1 (CF1) and subunit III of the membrane-spanning portion of the chloroplast ATP synthase (CF0), isolated as described in the accompanying paper (C.M. Wetzel and R.E. McCarty [1993] Plant Physiol 102: 241-249), has been further characterized. A comparison of the ATPase activities of CF1, CF1-subunit III, and the chloroplast ATP synthase (CF1-CF0) holoenzyme revealed that the properties of CF1-subunit III more closely resemble those of CF1-CF0 than those of CF1. In particular, the Ca2+-ATPase activity after reduction of the enzyme with dithiothreitol was much lower in CF1-subunit III and CF1-CF0 than in CF1, suggesting that the association of the inhibitory [epsilon] subunit is tightened by the presence of either CF0 or subunit III. Cold stability is a property of CF1-CF0 in thylakoid membranes. The ATPase activity of CF1 incubated in the cold in the presence of asolectin liposomes was lost more rapidly than that of either CF1-subunit III or CF1-CF0 incorporated into liposomes. Removal of the [epsilon] subunit from all three preparations resulted in marked stimulation of their ATPase activity. Although subunit III was also removed during depletion of the [epsilon] subunit, it is not known whether the two subunits interact directly. CF1 deficient in the [epsilon] subunit binds to liposomes containing either subunit III or CF0. Taken together, these results provide evidence that the association of CF1 and subunit III of CFo is specific and may play a role in enzyme regulation.     

叶绿体H^＋—ATP酶在平板脂双层上重组及其质子传导的研究

从菠菜(Spinacia oleracea Mill.)叶中分离获得H~ -ATP酶(CF_0-CF_1)复合体。将CF_0-CF_1重组于平板脂双层上,在电压钳位下,研究CF_0～CF_1的质子传导性能,观察到:(1)当CF_0-CF_1重组于平板脂双层上后,平板膜电阻由10～20GΩ立即下降到1GΩ左右。(2)溶液中蛋白质(CF_0-CF_1)浓度在2mg/L下可记录到单通道电流的涨落,单位电导约在5～10pS。(3)通道电流随膜两侧ΔpH变化而改变,在ΔpH为2～4时,膜电流随ΔpH增加而增大,在ΔpH为4.5时膜电流呈现回落。(4)质子传导抑制剂Dicyclohexyl-carbodiimide(DCCD)显示出迅速地且不可逆地阻断通道电流。(5)无金属离子的溶液中,跨膜(BLM)的ΔpH为3时,在0～ 150mV钳位下,镁离子比钙离子所引起的CF_0-CF_1的通道电流要大得多。以上结果不仅表明CF_0-CF_1已成功地组装于人工膜上,而且也显示出镁离子直接参与了质子传导过程。     

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.     

ATP hydrolysis catalyzed by a beta subunit preparation purified from the chloroplast energy transducing complex CF1.CF0

Washing thylakoid membranes with 1 M LiCl causes the release of the beta subunit from the chloroplast energy transducing complex (CF1.CF0) in spinach chloroplasts. This protein purifies by size exclusion chromatography as a 180-kDa aggregate and, thus, is probably composed of a trimer of beta polypeptides. The purified aggregate binds ADP to a high and a low affinity site with dissociation constants of 15 and 202 microM, respectively. Mg2+ is required for ADP to bind to both sites. Manganese binds to the protein in a cooperative manner to at least two sites with high affinity. The beta subunit preparation catalyzes Mg2+-dependent ATP hydrolysis at rates which are comparable to other subunit-deficient CF1 preparations and is increased by treatments known to activate the Mg2+-ATPase activity of CF1. However, Ca2+ is not an effective cofactor for this reaction and treatments which activate the Ca2+-ATPase of CF1 are either ineffective or inhibitory.     

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.     

Purification and reconstitution of active chloroplast F0

Chloroplast F0 (CF0) was purified from the ATP synthase by Zwittergent 3-12 treatment and DEAE-Trisacryl anion exchange chromatography. Purified CF0 contains four subunits corresponding to subunits I, II, III, and IV. CF0 mediated proton translocation across the membrane after incorporation into asolectin liposomes. The CF0-mediated proton transport was inhibited by N,N'-dicyclohexylcarbodiimide and the binding of chloroplast coupling factor 1 (CF1). Rebinding of CF1 to CF0 liposomes resulted in reconstitution of N,N'-dicyclohexylcarbodiimide and uncoupler sensitive energy-transducing activities. Like CF0 in native thylakoid membranes, purified CF0 bound CF1 as well as CF1 deficient in either the delta or epsilon subunits.     

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.     

Reconstitution of CF0F1 into liposomes using a new reconstitution procedure

The H(+)-ATPase (ATP synthase) from chloroplasts was isolated, purified and reconstituted into phosphatidylcholine/phosphatidic-acid liposomes. Liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100 and protein incorporation was studied at each step of the solubilization process. After detergent removal by SM2-Biobeads, the activities of the resulting proteoliposomes were measured indicating that the most efficient reconstitution was obtained by insertion of the protein into preformed, detergent-saturated liposomes. The conditions for the reconstitution were optimized with regard to ATP synthesis driven by an artificially generated delta pH/delta psi. An important benefit of the new reconstituted CF0F1 liposomes is the finding that the rate of ATP synthesis remains constant up to 10 s, indicating a low basal membrane permeability.     

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.     

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.     

Conformational change of the chloroplast ATP synthase on the enzyme activation process detected by the trypsin sensitivity of the gamma subunit

Delta mu H(+) is known to stimulate the enzyme activity of chloroplast ATP synthase in addition to its important role as energy supply for ATP synthesis. In the present study, we focused on the relationship between the proton translocation via the membrane sector of ATP synthase, F(o), and the conformational change of the central stalk subunit gamma. The conformational change of CF(1) mainly at the gamma subunit was induced by the proton flow via F(o) in the absence of substrates. The effects of inhibitors on CF(o) or CF(1) for this conformational change were also examined. The observed conformational change was partially suppressed by ADP binding. From these results, we propose the Delta mu H(+)-dependent conformational change of CF(1) on the enzyme activation process, which is affected by both ADP binding to the catalytic sites and proton flow via F(o) portion.     

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.     

Interactions between beta D372 and gamma subunit N-terminus residues gamma K9 and gamma S12 are important to catalytic activity catalyzed by Escherichia coli F1F0-ATP synthase

Substitution of Escherichia coli F(1)F(0) ATP synthase residues betaD372 or gammaS12 with groups that are unable to form a hydrogen bond at this location decreased ATP synthase-dependent cell growth by 2 orders of magnitude, eliminated the ability of F(1)F(0) to catalyze ATPase-dependent proton pumping in inverted E. coli membranes, caused a 15-20% decrease in the coupling efficiency of the membranes as measured by the extent of succinate-dependent acridine orange fluorescence quenching, but increased soluble F(1)-ATPase activity by about 10%. Substitution of gammaK9 to eliminate the ability to form a salt bridge with betaD372 decreased soluble F(1)-ATPase activity and ATPase-driven proton pumping by 2-fold but had no effect on the proton gradient induced by addition of succinate. Mutations to eliminate the potential to form intersubunit hydrogen bonds and salt bridges between other less highly conserved residues on the gamma subunit N-terminus and the beta subunits had little effect on ATPase or ATP synthase activities. These results suggest that the betaD372-gammaK9 salt bridge contributes significantly to the rate-limiting step in ATP hydrolysis of soluble F(1) while the betaD372-gammaS12 hydrogen bond may serve as a component of an escapement mechanism for ATP synthesis in which alphabetagamma intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven gamma subunit rotation.     

Depletion of stromal P(i) induces high 'energy-dependent' antenna exciton quenching (q(E)) by decreasing proton conductivity at CF(O)-CF(1) ATP synthase.

This work tests two models to account for the effects of depletion of stromal inorganic phosphate (P(i)), which results in down-regulation of light capture via the exciton quenching (q(E)) mechanism and has been proposed to act in feedback regulation of the light reactions. In both models, antenna down-regulation is activated by acidification of the lumen, despite the fact that linear electron flow (LEF) (and associated proton flux) is decreased upon P(i) depletion. In one model, an imbalance of ATP or NADPH activates cyclic electron transfer around photosystem I (CEF1), increasing proton influx to the lumen. In the second, the effective conductivity of the CF(O)-CF(1) ATP synthase to protons (g(H)(+)) is decreased, retarding proton efflux from the lumen. Sequestering of P(i) by mannose infiltration increased sensitivities of q(E) and pmf to LEF. The effects were attributable to decreases in g(H)(+), but not to CEF1 and were largely reversed by subsequent P(i) feeding. Rapid recovery of g(H)(+) in the dark suggested that dark-labile metabolic pools are responsible for regulation of the ATP synthase. Overall, these results support models where accumulation of Benson-Calvin cycle intermediates or lowering of stromal P(i) below its K(M)at the ATP synthase, retards proton efflux from the lumen, leading to build-up of pmf and subsequent down-regulation of photosynthetic light capture.     

Differential effects of triphenyltin and 8-azido-ATP on the ATP synthesis, ATP-Pi exchange, and ATP hydrolysis in liposomes containing ATP synthase and bacteriorhodopsin

The ATP hydrolysis activity of purified ATP synthase reconstituted in liposomes was inhibited by triphenyltin in a manner different from that of other thiol-specific reagents. In liposomes containing ATP synthase and bacteriorhodopsin, ATP hydrolysis and ATP-Pi exchange were inhibited by triphenyltin to a greater extent than the ATP synthesis, in contrast to what was found with an F1-specific inhibitor, 8-azido-ATP. The possibility is discussed that ATP hydrolysis and ATP synthesis are differently coupled to proton conduction through F0.     

Singlet oxygen inhibits ATPase and proton translocation activity of the thylakoid ATP synthase CF1CFo

Singlet oxygen (¹O₂) produced in plants during photosynthesis has a strong damaging effect not only on both photosystems but also on the whole photosynthetic machinery. This is also applicable for the adenosine triphosphate (ATP) synthase. Here we describe the impact of ¹O₂ generated by the photosensitizer Rose Bengal on the ATP hydrolysis and ATP-driven proton translocation activity of CF1CFo. Both activities were reduced dramatically within 1 min of exposure. Interestingly, it is shown that oxidized thylakoid ATP synthase is more susceptible to ¹O₂ than CF1CFo in its reduced state, a new insight on the mechanism of ¹O₂ interaction with the γ subunit.     

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.