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.     

Induction by different thioredoxins of ATPase activity in coupling factor 1 from spinach chloroplasts

ATPase activity of the coupling factor 1, CF1, isolated from spinach chloroplasts, was enhanced by reduction with dithiothreitol. Reduced thioredoxins from spinach chloroplasts, Escherichia coli and human lymphocytes replaced dithiothreitol as reductant and activator of the ATPase. CF1 must be in an oxidized activated state to be further activated by reduced thioredoxin. This state was obtained either by heating CF1 or removing the inhibitory intrinsic epsilon subunit from CF1. Efficiency and primary structure of the different thioredoxins were compared. The progressive addition of KCl during ATPase activation by reduced thioredoxin increases then decreases this process. We proposed that three basic amino acids corresponding to arginine 73 and lysines 82 and 96 in Escherichia coli thioredoxin play an important role in the anchorage of the thioredoxin to the negatively charged surface of the CF1 and are involved in the dual effect of KCl. The variations in the screening effect of the negative charges of the CF1 surface by K+ ions can indeed explain the changes in the anchorage of these 3 basic amino acids with concomitant variation in ATPase activity. Human thioredoxin must be 10 times more concentrated than Escherichia coli or spinach chloroplast thioredoxin to exhibit the same activation effect on the ATPase. This fact was related to the properties of a sequence equivalent to the part from amino acid 59 to 72 in Escherichia coli thioredoxin. This part which joins the two lobes of the thioredoxin is more hydrophilic and more negatively charged in human thioredoxin than in Escherichia coli or spinach chloroplast thioredoxin. Although ATPase activation was obtained at a very low concentration of the reduced spinach chloroplast thioredoxin, the thioredoxin formed only a loose complex with CF1.     

Function of tightly bound nucleotides on membrane-bound chloroplast coupling factor

The kinetic behavior of tightly bound nucleotides on chloroplast coupling factor from spinach was determined under phosphorylating and nonphosphorylating conditions. Chloroplast coupling factor 1 (CF1) was labeled with tightly bound radioactive ADP and/or ATP at two specific sites and reconstituted with thylakoid membranes depleted of CF1 by treatment with NaBr. The initial incorporation and dissociation of ADP from one of the sites requires light but occurs at the same rate under phosphorylating and non-phosphorylating conditions. The initial rate is considerably slower than the rate of ATP synthesis, but nucleotide exchange is very rapid during steady-state ATP synthesis. A direct correspondence between this nucleotide binding site and a site on soluble CF1 that hydrolyzes ATP was demonstrated. A second site binds MgATP very tightly; the MgATP does not dissociate during ATP synthesis nor does its presence alter the rate of ATP synthesis. This is analogous to the behavior found for soluble CF1 during ATP hydrolysis. These results demonstrate that the tight-binding nucleotide sites on soluble CF1 and membrane-bound coupling factor are essentially identical in terms of binding properties and kinetic behavior during ATP hydrolysis and synthesis.     

Alterations in Chloroplast Thylakoids during an in Vitro Freeze-Thaw Cycle

Plastocyanin and chloroplast coupling factor 1 (CF(1)) are released from spinach (Spinacia oleracea L.) thylakoids during a slow freezethaw cycle. CF(1) addition increases the proton uptake of thylakoids previously frozen in sucrose concentrations of 15 mm to 100 mm. Addition of CF(1) and plastocyanin restores the proton uptake of thylakoids frozen in 100 mm sucrose. Plastocyanin and CF(1) release is a manifestation, not the cause, of freeze-thaw damage.Frozen-thawed thylakoids appear to exhibit two levels of response to sucrose as measured by light-dependent proton uptake. Different levels of protection afforded by sucrose may be due, in part, to quantitative differences in CF(1) release. The results suggest at least three freeze-induced lesions in light-dependent proton uptake by thylakoids: plastocyanin release, CF(1) release, and disruption of the semi-permeability of thylakoids.     

Interactions of inorganic phosphate with spinach coupling factor 1. Effects on ATPase and ADP binding activities

Illumination of chloroplast thylakoid membranes results in both the release of adenine nucleotides from the tight nucleotide binding site(s) on chloroplast coupling factor 1 (CF1) and the activation of a light-triggered ATPase activity of CF1. Because inorganic phosphate stabilizes the light-triggered ATPase activity of CF1 in the dark, the effects of Pi on the rebinding of ADP to CF1 and on the light-triggered ATPase activity have been studied. Pi appears to be a partial noncompetitive inhibitor, with respect to ADP, of adenine nucleotide binding to the tight nucleotide binding site(s) on CF1 and induces negative cooperativity. The latter result suggests the existence of heterogeneous ADP binding sites in the presence of Pi. However, even under conditions where Pi causes a 50% reduction of tightly bound ADP, the ADP-induced dark decay of the ATPase activity is still complete. It was found that Pi inhibition of the light-induced dark binding of ADP can be reversed by the removal of the Pi. Removal of Pi also induces a small but significant ATPase activity. A model for the roles of the adenine nucleotide tight binding site(s) and Pi in the modulation of the spinach CF1 ATPase activity is proposed.     

Acid-induced phosphorylation of adenosine 5'-diphosphate bound to coupling factor 1 in spinach chloroplast thylakoids.

Adenosine 5'-diphosphate, bound to coupling factor 1 (CF1) in spinach chloroplast thylakoids, is in part converted to adenosine 5'-triphosphate, upon injection of the thylakoids into strong acids in the dark. Bound phosphate serves as the phosphoryl donor for this uncoupler-insensitive conversion. Exposure of the thylakoids to heat or to urea prior to their injection into acid caused dissociation of ADP and prevents the apparent acid-induced synthesis of ATP. Conformational changes in CF1 may be elicited by acid denaturation which resemble those brought about by the proton electrochemical gradient across thylakoid membranes.     

Mechanism of phosphorylation catalyzed by chloroplast coupling factor 1. Stereochemistry

The reaction mechanism and substrate specificity of soluble chloroplast coupling factor 1 (CF1) from spinach were determined by using the purified isomers of chromium-nucleotide complexes either as substrates for the enzyme or as inhibitors of the Ca2+-dependent ATPase activity. The isolation of CrADP( [32P]Pi) formed upon the addition of the enzyme to [32P]Pi and lambda-bidentate CrADP and the observation that the lambda-bidentate CrADP epimer was 20-fold more effective in inhibiting the Ca2+-dependent ATPase activity than was the delta epimer suggest that the substrate of phosphorylation catalyzed by CF1 is the lambda-bidentate metal ADP epimer. Tridentate CrATP was hydrolyzed by soluble CF1 to CrADP(Pi) at an initial rate of 3.2 mumol (mg of CF1)-1 min-1, indicating that the tridentate metal ATP is the substrate for ATP hydrolysis. From these results a mechanism for the phosphorylation of ADP catalyzed by coupling factor 1 is proposed whereby the bidentate metal ADP isomer associates with the enzyme, phosphate inserts into the coordination sphere of the metal, and the oxygen of the beta-phosphate of ADP attacks the inorganic phosphate by an SN2 type reaction. The resulting product is the tridentate ATP ligand.     

Coupling factor activity of the purified pea mitochondrial F1-ATPase

The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose/2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation.     

Properties of the cyanobacterial coupling factor ATPase from Spirulina platensis. I. Electrophoretic characterization and reconstitution of photophosphorylation

The coupling factor ATPase (F1) from photosynthetic membranes of the cyanobacterium Spirulina platensis was purified to homogeneity by a combination of ion-exchange chromatography and sucrose density gradient centrifugation. The ATPase activity of purified Spirulina F1 is latent but can be elicited by trypsin treatment, resulting in specific activities (CaATPase) of 27-37 mumol Pi min-1 mg protein-1. On denaturing sodium dodecyl sulfate-polyacrylamide gradient gels, Spirulina F1 is resolved into five subunits with molecular weights of 53,400, 51,600, 36,000, 21,100, and 14,700, similar to the molecular weights of the subunits of spinach chloroplast coupling factor (CF1). As determined by native polyacrylamide gradient gel electrophoresis, the molecular weight of the Spirulina F1 holoenzyme was estimated to be 320,000, somewhat smaller than the estimated molecular weight of spinach CF1 (392,000). Spirulina F1 was shown to be an active coupling factor by its ability to reconstitute phenazine methosulfate-dependent cyclic photophosphorylation in membrane vesicles which had been depleted of coupling factor content by 2 M NaBr treatment. We estimate the Spirulina F1 content of membrane vesicles to be 1 F1 per 830 chlorophylls or 0.12 mol F1 mol P700(-1), based on the specific ATPase activities of the membrane vesicles and the purified Spirulina F1, the molecular weight of F1, and the P700 content of the vesicles.     

Salt treatment induces frost hardiness in leaves and isolated thylakoids from spinach

Frost hardiness of spinach (Spinacia oleracea L.) leaves was increased by high concentrations of NaCl in the hydroponic culture medium. Freezing damage was determined by measurement of slow chlorophyll fluorescence quenching after freezing of leaves. Both the osmolality of the leaf sap and forst hardiness of the leaves were linearly correlated with the salt concentration in the hydroponic culture medium. Freezing damage occurred, irrespective of the extent of frost hardening, when dehydration of cells during extracellular ice formation decreased cellular volume to approximately 14% of the volume of unfrozen cells. The resistance of isolated, washed thylakoids against mechanical and chemical damage by freezing was investigated. Chemical damage by freezing caused by salt accumulation was measured as release of chloroplast coupling factor (CF1; EC 3.6.1.3), and mechanical damage was measured as release of the lumenal protein plastocyanin from the membranes during an in-vitro freeze-thaw cycle. Isolated thylakoids from salt-treated frost-hardy spinach and those from plants hardened under natural conditions did not exhibit improved tolerance against chemical freezing stress exerted by high salt concentrations. They were, however, more hardy than thylakoids from unhardened control leaves against mechanical damage by freezing.Abbreviation CF1 peripheral part of chloroplast coupling factor ATPase     

Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5'-triphosphate Synthesis in Chloroplasts

Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.     

Light-dependent cleavage of the gamma subunit of coupling factor 1 by trypsin causes activation of Mg2+-ATPase activity and uncoupling of photophosphorylation in spinach chloroplasts

Trypsin treatment of spinach chloroplast thylakoids in the light but not in the dark, results in a highly active Mg2+-ATPase and an uncoupling of photophosphorylation. These light-dependent effects are due to a modification of coupling factor 1 (CF1). CF1 purified from thylakoids treated with trypsin in the light contained a clipped beta subunit and a partially clipped gamma subunit, whereas that from thylakoids treated in the dark with trypsin contained only the clipped beta subunit. CF1 containing this modified gamma subunit also retained a high level of Ca2+-ATPase activity in solution. These results suggest that the gamma subunit becomes highly sensitive to trypsin only when the CF1 is in an active conformation. A similar hypersensitivity to proteases of the gamma subunit in highly purified CF1 is seen only after the enzyme is activated (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). The conversion of the enzyme to its active form, both on the membrane and in solution, therefore, seems to involve conformational changes that expose the gamma subunit to proteolysis.     

Nucleotide sequences of three soybean chloroplast tRNAsLeu and re-examination of bean chloroplast tRNA2Leu sequence.

The nucleotide sequences of soybean chloroplast tRNAsLeu were determined using post-labeling techniques. Comparison of the primary structures of soybean chloroplast tRNAsLeu with their bean, maize and spinach counterparts only show few base differences. Contrary to previously published results (1) a re-examination of bean tRNALeu sequence shows that this tRNA resembles soybean and maize tRNA2Leu in structure.     

Membrane rupture is the common cause of damage to chloroplast membranes in leaves injured by freezing or excessive wilting

The effects of freezing and desiccation of spinach leaves (Spinacia oleracea L. cv Yates) on the thylakoid membranes were assessed using antibodies specific for thylakoid membrane proteins. The peripheral part of the chloroplast coupling factor ATPase (CF1) was used as a molecular marker for chemical membrane damage by chaotropic solutes. Plastocyanin, a soluble protein localized inside the closed thylakoid membrane system, was a marker for damage by mechanical membrane rupture. After freezing and wilting of leaves which resulted in damage, very little CF1 was detached from the membranes, whereas almost all plastocyanin was released from the thylakoids. It is suggested that in vivo dehydration both by freezing and desiccation results in membrane rupture rather than in the dissociation of peripheral thylakoid membrane proteins.     

Evidence for a catalytic function of the coupling factor 1 protein reconstituted with chloroplast thylakoid membranes.

The effects of tentoxin on the ATPase activities of coupling factor 1 proteins (CF1) and photophosphorylation with isolated chloroplasts and chloroplasts reconstituted with coupling factor proteins have been examined. 1. The calcium-dependent ATPase activities of coupling factors isolated from spinach, lettuce and Nicotiana otophora are completely inhibited by tentoxin. The ATPase activities of coupling factors isolated from Nicotiana tabacum and Nicotiana knightiana are not affected by tentoxin. 2. Phenazine methosulfate-catalyzed cyclic photophosphorylation with chloroplasts isolated from spinach, lettuce and N. otophora is completely inhibited by tentoxin, whereas chloroplasts isolated from N. knightiana and N. tabacum are relatively insensitive to tentoxin. 3. Spinach chloroplasts, partially depleted in CF1, can be reconstituted with coupling factors isolated from a wide variety of plants including lettuce, radish, N. tabacum, N. knightiana and N. otophora. 4. Spinach chloroplasts reconstituted with spinach, lettuce and N. otophora CF1 retain their sensitivity to tentoxin; however, when reconstituted with N. knightiana and N. tabacum coupling factor proteins, a significant fraction of the reconstituted rate remains tentoxin insensitive. These data are interpreted as evidence that coupling factors that reconstitute with spinach thylakoid membranes have both a catalytic and structural function.     

Investigation of quercetin binding sites on chloroplast coupling factor 1.

The quercetin binding sites on spinach chloroplast coupling factor 1 (CF1) have been investigated using direct and competitive binding, stopped-flow, temperature-jump, and fluorescence resonance energy transfer measurements. It was found that 8-anilino-1-naphthalensulfonic acid (ANS) competes with quercetin binding at two sites on the solubilized enzyme which are distinct from the two tight nucleotide binding sites and the 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reactive site. The bimolecular association of quercetin with CF1 is too fast to measure directly and is followed by two slower conformational changes. The distances from the tight nucleotide sites to the quercetin-ANS sites were estimated as 40-48 A by fluorescence resonance energy transfer using 1,N6-ethenoadenosine diphosphate and 1,N6-ethenoadenylyl imidodiphosphate as donors and quercetin as the acceptor. The distance from the quercetin-ANS site to the NBD-C1 reactive site was found to be about 30 A using ANS as a donor and NBD-C1 reacted with a tyrosine group on CF1 as the energy acceptor. A model is proposed for the relative location of these sites on CF1.     

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.     

Antigenic relationships between chloroplast and cytosolic fructose-1,6-bisphosphatases.

Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea (Pisum sativum L. cv Lincoln) and spinach (Spinacia oleracea L. cv Winter Giant) did not cross-react by double immunodiffusion and western blotting with either of the antisera raised against the chloroplast enzyme of both species; similarly, pea and spinach chloroplast FBPases did not react with the spinach cytosolic FBPase antiserum. On the other hand, spinach and pea chloroplast FBPases showed strong cross-reactions against the antisera to chloroplast FBPases, in the same way that the pea and spinach cytosolic enzymes displayed good cross-reactions against the antiserum to spinach cytosolic FBPase. Crude extracts from spinach and pea leaves, as well as the corresponding purified chloroplast enzymes, showed by western blotting only one band (44 and 43 kD, respectively) in reaction with either of the antisera against the chloroplast enzymes. A unique fraction of molecular mass 38 kD appeared when either of the crude extracts or the purified spinach cytosolic FBPase were analyzed against the spinach cytosolic FBPase antiserum. These molecular sizes are in accordance with those reported for the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast and cytosolic FBPases underwent increasing inactivation when increasing concentrations of chloroplast or cytosolic anti-FBPase immunoglobulin G (IgG), respectively, were added to the reaction mixture. However, inactivations were not observed when the photosynthetic enzyme was incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative results obtained by enzyme-linked immunosorbent assays (ELISA) showed 77% common antigenic determinants between the two chloroplast enzymes when tested against the spinach photosynthetic FBPase antiserum, which shifted to 64% when assayed against the pea antiserum. In contrast, common antigenic determinats between the spinach cytosolic FBPase and the two chloroplast enzymes were less than 10% when the ELISA test was carried out with either of the photosynthetic FBPase antisera, and only 5% when the assay was performed with the antiserum to the spinach cytosolic FBPase. These results were supported by sequencing data: the deduced amino acid sequence of a chloroplast FBPase clone isolated from a pea cDNA library indicated a 39,253 molecular weight protein, with a homology of 85% with the spinach chloroplast FBPase but only 48.5% with the cytosolic enzyme from spinach.     

Chloroplast DNA rearrangements are more frequent when a large inverted repeat sequence is lost

We examined the arrangement of sequences common to seven angiosperm chloroplast genomes. The chloroplast DNAs of spinach, petunia and cucumber are essentially colinear. They share with the corn chloroplast genome a large inversion of approximately 50 kb relative to the genomes of three legumes--mung bean, pea and broad bean. There is one additional rearrangement, a second, smaller inversion within the 50 kb inversion, which is specific to the corn genome. These two changes are the only detectable rearrangements that have occurred during the evolution of the species examined (corn, spinach, petunia, cucumber and mung bean) whose chloroplast genomes contain a large inverted repeat sequence of 22-25 kb. In contrast, we find extensive sequence rearrangements in comparing the pea and broad bean genomes, both of which have deleted one entire segment of the inverted repeat, and also in comparing each of these to the mung bean genome. Thus there is a relatively stable arrangement of sequences in those genomes with the inverted repeat and a much more dynamic arrangement in those that have lost it. We discuss several explanations for this correlation, including the possibility that the inverted repeat may play a direct role in maintaining a conserved arrangement of chloroplast DNA sequences.