In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential difference of protons (Delta(micro)H(+)) is small, e.g. at low light intensity or in the dark. To prevent this wasteful consumption of ATP, the activity of chloroplast ATP synthase is regulated as the occasion may demand. As regulation systems Delta(micro)H(+) activation, thiol modulation, tight binding of ADP and the role of the intrinsic inhibitory subunit epsilon is well documented. In this article, we discuss recent progress in understanding of the regulation system of the chloroplast ATP synthase at the molecular level.

Chloroplast ATP synthase is a thiol-modulated enzyme whose DeltamuH(+)-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys(199) and Cys(205) on the gamma subunit. In solubilized chloroplast coupling factor 1 (CF(1)), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity. To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu(210)-Asp-Glu(212) close to the two cysteine residues and also on the following region from Leu(213) to Ile(230), and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant gamma subunits were reconstituted with the alpha and beta subunits from F(1) of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant gamma subunit in which Glu(210) to Glu(212) had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation. This complex was insensitive to the inhibitory CF(1)-epsilon subunit when the mutant gamma subunit was oxidized. In contrast, the deletion of Glu(212) to Ile(230) converted the complex from a modulated state into a highly active state.

Thylakoid energization by light causes destabilization of CF0CF1 so that the peripheral CF1 sector is more readily detached from the membrane by intermediate concentrations of the chaotropic salt NaSCN. Here we have investigated the correlation between the proton gradient-induced change of CF0CF1 interaction and CF0CF1 activation. The results indicate a close relationship between the two phenomena. The effect is most probably due to reduction of the electrostatic interaction between the two subcomplexes CF0 and CF1 as a consequence of protonations in the interface region.

The structure of thylakoid membrane-bound chloroplast coupling factor CF1 was studied by limited proteolysis followed by sodium dodecylsulfate polyacrylamide gel electrophoresis and N-terminal sequence analysis. The N-terminal fragment of the alpha-subunit was shown to have an exposed area including the peptide bond R21-E22. The cleavage of this peptide bond caused the alphaK24-V25 bond to be exposed to the outside. In the N-terminal fragment of the beta-subunit, the L14-E15 bond was identified and found to be subject to trypsinolysis. Also, the alphaR140-S141, alphaG160-R161, and betaG102-G103 bonds were accessible to the proteolytic attack. In general, the beta-subunit of membrane-bound CF1 is more sensitive to proteolysis than that of solubilized CF1. The products of proteolysis of the alpha-subunit did not contain the polypeptides typical of the reaction of cleavage of the alphaE17-G18 and alphaE22-V23 bonds in isolated CF1. These results suggest a significant structural difference between soluble and membrane-bound CF1. A number of peptide bonds, alphaG160-R161 in particular, were shown to be shielded from proteolytic attack by papain in illuminated thylakoid membranes, probably as a result of membrane energization. In contrast, the light-induced reduction of the gamma-subunit caused an increase in the accessibility of some peptide bonds to this protease, including the alphaG160-R161 bond.

Plastids of diatoms and other chromophytic algae have four surrounding membranes. In contrast to plastids of green algae, higher plants and red algae chromophytic cells are thought to have evolved by secondary endocytobiosis, i.e. by uptake of a eukaryotic photosynthetic organism by a eukaryotic host cell. This review gives a brief summary of the current views about the origin of diatom plastids and discusses possible mechanisms the cells might employ to transport nucleus‐encoded plastid proteins into these organelles.

We have generated the mutation T168S in the beta subunit of the chloroplast ATP synthase complex of Chlamydomonas reinhardtii by site directed mutagenesis and chloroplast transformation. CF1 and the alpha3beta3gamma complex of this mutant strain were isolated and their enzymatic activities were characterized and compared to those of the corresponding wild type complexes. Without activation the mutant CF1 exhibits MgATPase activity with at least 10 times higher rates than the wild type enzyme. The MgATPase activity could be stimulated to some extent by methanol, but less by ethanol and octylglucoside. The alpha3beta3gamma complex had an even higher MgATPase activity, which was only slightly enhanced by ethanol or methanol. The ATPase activities of the mutant complexes, like those of the wild type complexes, displayed a sharp concentration optimum for Mg2+. Free ADP inhibited neither the mutant nor the wild type ATPase significantly. Azide, which strongly inhibited the ATPase activity of the wild type enzyme, inhibited the mutant enzyme only at an about 30 times higher concentration suggesting that the mutation T168S prevents trapping of a tightly bound MgADP by a catalytic site that regulates chloroplast ATPase activity. The mutant cells grew photoautotrophically at a growth rate of about 50%. Similar to the wild type the cells survived on minimal medium in the dark. Under heterotrophic conditions with acetate as energy and carbon source the mutant cells grew much faster than the wild type cells, but the chlorophyll content per cell decreased dramatically.

We have studied the change of the catalytic activity of chimeric complexes that were formed by chloroplast coupling factor 1 (CF1) -gamma, alpha and beta subunits of thermophilic bacterial F-1 after formation or reduction of the disulfide bridge of different gamma subunits modified by oligonucleotide-directed mutagenesis techniques. For this purpose, three mutant gamma subunits were produced: gamma(Delta 194-230), here 37 amino acids from Pro-194 to Ile-230 are deleted, gamma(C199A), Cys-199 is changed to Ala, and gamma(Delta 200-204), amino acids from Asp-200 to Lys-204 are deleted. All of the chimeric subunit complexes produced from each of these mutant CF1-gamma subunits and alpha and beta subunits from thermophilic bacterial F-1 lost the sensitivity against thiol reagents when compared with the complex containing wild-type CF1-gamma. The pH optimum (pH 8.5-9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations, These indicate that the introduction of the mutations did not change the main features of ATPase activity of the chimeric complex,However, the interaction between gamma subunit and epsilon subunit was strongly influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex that contained gamma(Delta 200-204) or gamma(C199A) was inhibited by the addition of recombinant epsilon subunit from CF1 similarly to complexes containing the reduced wild-type gamma subunit, the recombinant epsilon subunit did not inhibit the ATPase of the complex, which contained the oxidized form of gamma subunit, Therefore the affinity of the epsilon subunit to the gamma subunit may be dependent on the state of the gamma subunit or the epsilon subunit may bind to the oxidized form of gamma subunit in a mode that does not inhibit the activity. The ATPase activity of the complex that contains gamma Delta 194-230 was not efficiently inhibited by epsilon subunit, These results show that the formation or reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region that directly affects the interaction of this subunit with the adjacent epsilon subunit.

We produced atpA and atpB deletion mutants of C. reinhardtii cw15 in order to investigate the role of certain domains of the α and β subunits of the chloroplast ATP synthase (CF0CF1) by site-directed mutagenesis. The deletion mutants were obtained by transformation with constructs containing the aadA cassette inserted into the atpA or atpB genes in place of the corresponding open reading frames. Homoplasmic strains were obtained by continued selection under heterotrophic growth conditions in the presence of spectinomycin. The deletion mutants could be complemented by transformation with the wild-type atpA or atpB genes, respectively. In both deletion mutants none of the subunits of CF1 and of CF0 were detected by electrophoretic analysis. Since the mother strain cw15 as well as the deletion mutants are cell wall-deficient, we were able to prepare photosynthetically active thylakoids and to study the photosynthetic characteristics of cw15 thylakoids and mutant thylakoids lacking the ATP synthase. Electron transport measurements showed that the PSII and PSI activities were not affected in the deletion mutants. Thylakoids from the deletion mutants were able to maintain a significantly higher light-induced proton gradient than thylakoids from cw15, confirming the absence of a functional proton channel in these deletion mutants. When substrates of photophosphorylation were added, the transmembrane proton gradient in wild-type thylakoids decreased and ATP was formed, while in the deletion mutant thylakoids the proton gradient was not affected and no ATP was formed. The phosphorylation/ΔpH relationship in C. reinhardtii thylakoids was shifted to lower ΔpH values by reduction with dithiothreitol, indicating that the C. reinhardtii enzyme is also similar to the higher plant chloroplast ATP synthase in this respect.

The participation of the amino acid β83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by site-directed mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid β83 was changed from Glu to Asp (βE83D) and to Lys (βE83K), and the highly conserved tetrapeptide βT82-E83-G84-L85 (ΔTEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15ΔatpB and FUD50 with the mutated atpB genes. The transformants containing the βE83D and βE83K mutant genes grew well photoautotrophically. The ΔTEGL transformant did not grow photoautotrophically, and no CF1 subunits were detected by immunostaining of Western blots using CF1 specific antibodies. The rates of ATP synthesis at clamped ΔpH with thylakoids isolated from cw15 and the two mutants, βE83D and βE83K, were similar. However, only the phosphorylation activity of the mutant βE83D was inhibited by tentoxin with 50% inhibition attained at 4 μM. These results confirm that amino acid β83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF1s isolated from cw15, βE83D, and βE83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF1 from the βE83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the βE83K-CF1 than that of CF1 from the other two strains. Moreover, the optimal activity of the βE83K-CF1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the “crown” domain affects the active conformation of the CF1-ATPase.

The activation characteristics of the F1Fo-ATP synthase (where F1 and Fo are the hydrophilic and membrane-bound parts respectively of the enzyme) from Synechocystis 6803 wild-type and a Synechocystis 6803 mutant with a chloroplast-like insertion in the gamma subunit have been studied. Activation of the ATP synthase in wild-type and mutant membrane vesicles was performed by acid-base transition-induced generation of a proton motive force (Delta mu H+). Since the mutant containing the regulatory segment of the chloroplast gamma subunit showed thiol-modulation (typical of the chloroplast enzyme), this segment is indeed involved in the regulation of enzyme activation. It is shown that the ATP synthase from Synechocystis 6803 wild type corresponds functionally to the reduced form of the chloroplast ATP synthase, in view of the low Delta mu H+ required for activation of the enzyme and the high stability of the active state. Both the cyanobacterial wild-type and mutant ATP synthases can be activated by methanol, which apparently does not require the presence of the gamma subunit regulatory segment.

The kinetics of thiol modulation of the chloroplast H+-ATPase (CF0CF1) in membrana were analyzed by employing thioredoxins that were kept reduced by 0.1 mmdithiothreitol. The kinetics of thiol modulation depend on the extent of the proton gradient. The process is an exponential function of the thioredoxin concentration and reaction time and can be described by an irreversible second order reaction. The results indicate that the formation of the complex between thioredoxin and CF0CF1 is slow compared with the subsequent reduction step. Furthermore we have compared the efficiencies of theEscherichia coli thioredoxin Trx and the two chloroplast thioredoxins Tr-m and Tr-f. The second order rate constants are 0.057 (Tr-f), 0.024 (Trx), and 0.010 s−1μm−1 (Tr-m) suggesting that Tr-f rather than Tr-m is the physiological reductant for the chloroplast ATPase. The often employed artificial reductant dithiothreitol exhibits a second order rate constant in thiol modulation of 1.02·10−6s−1 μm−1.