Substitutions of the conserved Gly47 affect the CF1 inhibitor and proton gate functions of the chloroplast ATP synthase epsilon subunit.

The conserved residue Gly47 of the chloroplast ATP synthase beta subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants epsilon G47L, epsilon G47R, epsilon G47A and epsilon G47E, respectively. All the beta variants showed lower inhibitory effects on the soluble CF1(-epsilon) Ca2+-ATPase compared with wild-type epsilon. In reduced conditions, epsilon G47E and epsilon G47R had a lower inhibitory effect on the oxidized CF1(-epsilon) Ca2+-ATPase compared with wild-type epsilon. In contrast, epsilon G47L and epsilon G47A increased the Ca2+-ATPase activity of soluble oxidized CF1(-epsilon). The replacement of Gly47 significantly impaired the interaction between the subunit epsilon and gamma in an in vitro binding assay? Further study showed that all epsilon 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 epsilon. These results indicate that the conserved Gly47 residue of the epsilon subunit is very important for maintaining the structure and function of the epsilon subunit and may affect the interaction between the epsilon subunit, beta subunit of CF1 and subunit III of CFo, thereby regulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit III of CFo.

N-terminal deletion of the gamma subunit affects the stabilization and activity of chloroplast ATP synthase.

Five truncation mutants of chloroplast ATP synthase gamma subunit from spinach (Spinacia oleracea) lacking 8, 12, 16, 20 or 60 N-terminal amino acids were generated by PCR by a mutagenesis method. The recombinant gamma genes were overexpressed in Escherichia coli and assembled with alphabeta subunits into a native complex. The wild-type (WT) alphabetagamma assembly i.e. alphabetagammaWT exhibited high (Mg2+)-dependent and (Ca2+)-dependent ATP hydrolytic activity. Deletions of eight residues of the gamma subunit N-terminus caused a decrease in rates of ATP hydrolysis to 30% of that of the alphabetaWT assembly. Furthermore, only approximately 6% of ATP hydrolytic activity was retained with the sequential deletions of gamma subunit up to 20 residues compared with the activity of the alphabetaWT assembly. The inhibitory effect of the epsilon subunit on ATP hydrolysis of these alphabetagamma assemblies varied to a large extent. These observations indicate that the N-terminus of the gamma subunit is very important, together with other regions of the gamma subunit, in stabilization of the enzyme complex or during cooperative catalysis. In addition, the in vitro binding assay showed that the gamma subunit N-terminus is not a crucial region in binding of the epsilon subunit.

Functional consequences of N- or C-terminal deletions of the delta subunit of chloroplast ATP synthase.

Mutagenesis was used to generate seven truncation mutants of the spinach (Spinacia oleracea) chloroplast ATP synthase delta subunit lacking 5, 11, 17, or 35 amino acid residues from the N-terminus or 3, 9, or 15 residues from the C-terminus. Interactions between these mutants and all other subunits of the chloroplast ATPase were investigated by a yeast two-hybrid system. The results indicate that the N-terminal deletions mainly affected interactions between the delta subunit and the other part of CF(1), but did not significantly affect interactions with the CF(0) sector. In contrast, C-terminal truncations of the delta subunit mainly affected its interaction with the CF(0) sector and caused little impairment in interactions with the other part of CF(1). The conformation of the delta subunit C-terminal domain seems to be more sensitive to the truncations, as shown by minimal expression driven by C-terminal deleted (nine residues) mutants. Further studies showed C-terminal truncations of the delta subunit greatly impaired its ability to restore cyclic photophosphorylation in NaBr vesicles, whereas N-terminal truncations had little effect on the ability of delta to plug the CF(0) channel. None of the mutants impaired ATP hydrolysis by CF(1).

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 Mg(2+)-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 ß 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.

Difference between Biological Activities and Secondary Structures of Expression Product of atpE Gene from Broad Bean and Maize Chloroplast.

The chloroplast atpE genes from broad bean and maize were overexpressed in E.coli, respectively. After proper refolding and purification, two types of epsilon-subunit proteins with biological activity were obtained. When reconstituted with CF(1)(-epsilon)from different chloroplast ATPase, the effects of the reconstructed epsilon-subunit protein of maize CF(1) to the Ca(2+)-ATPase activity of epsilon-deficient CF(1) and to the proton leakage through CF(0) were markedly stronger than that of broad bean CF(1). It was also observed that the enhancing effect of maize epsilon-subunit protein to the chloroplast photophosphorylation activity was greater than that of broad bean epsilon-subunit protein. The results indicate that (1)the inhibition of epsilon-subunit is closely related to the its affinity with other parts of CF(1) (2)both functions of epsilon-subunit as inhibitor of ATPase and as proton pathway are closely linked. The circular dichroism was performed to show the differences in their secondary structure of these epsilon-subunit proteins based on the deduced primary sequences. Unconstrained analysis of the CD spectrum for the maize epsilon-subunit protein gave 22.6% alpha-helix, 30.6% beta-sheet, 9.3% beta-turn, and 37.7% unordered structure and 31.4%alpha-helix, 22.3% beta-sheet, 13.8% beta-turn, and 32.4% unordered structure for the epsilon-subunit from broad bean.

Cloning, Sequencing and Expression of the atpE Gene of Broad Bean Chloroplast.

The ninth KpnI fragment (about 4.0 kb) of broad bean chloroplast DNA contains the entire chloroplast atpE gene and atpB gene. This fragment was inserted into the pUC18 polylinker region, yielding plasmid pUK1. Southern hybridization using atpE gene from maize chloroplast as probe revealed that atpE gene encoding epsilon subunit of broad bean chloroplast was localized in 0.9 kb ClaI fragment of pUK1. The whole sequence of atpE gene of broad bean chloroplast was determined by using primer F and primer R of the polylinker region of pBluescript KS (+) DNA. The entire atpE gene of broad bean chloroplast was inserted into the polylinker region of vector pJLA505 to form recombinant plasmids pJLA505-atpE. The expression plasmid was transformed into E. coli DH5alpha cells which were then induced at 42 degrees. By analysis with SDS-PAGE, the product of interest accounted for more than 38% of total E. coli cell proteins. Identification of the expressed product by Western blot analysis demonstrated that it can react specifically with anti-epsilon serum of maize chloroplast CF(1). The expressed product aggregated to form insoluble inclusion bodies and was purified. The purified product had the same function as that of the native epsilon subunit.

Site-directed Mutagenesis of epsilon Subunit of ATP Synthase of Maize Chloroplast.

The effects of some residues in maize chloroplast epsilon subunit on its activity have been studied by site-directed mutagenesis. After replacing Thr-42 of epsilon subunit with Cys, Arg, Ile or Pro, and forming the mutant epsilonT42C, mutant epsilonT42R, mutant epsilonT42I and mutant epsilonT42P respectively, it was found that the mutant epsilonT42P protein could no longer be expressed, but expression of other epsilon subunit mutants was similar to that of wild type. Comparing the inhibitory potency of different mutants of epsilon subunit with that of the wild type, it was found that the inhibitory effects of epsilon subunit mutants epsilonT42C and epsilonT42R on ATPase activity were slightly higher than that of wild type, but the epsilonT42I protein strongly inhibited the Ca(2+)-ATPase activity.

The Characteristics of DCPIPH(2) right curved arrow MV Electron Transport in Rb. sphaeroides 601.

The absorption spectrum and fluorescence emission spectrum of RS601 were found to keep the typical characteristics of those of the purple nonsulfide bacteria Rb. sphaeroides. Under illumination, methyl viologen was reduced by RS601 chromatophores in the presence of DCPIPH(2) as the electron donor, setting up a standard noncyclic electron transport. o-phenanthroline with I(50) of 1.0 mM inhibited the DCPIPH(2) right curved arrow MV electron transport. Antimycin A did not inhibit the DCPIPH(2) right curved arrow MV electron transport and had no I(50). The results suggested that the exact site where methyl viologen accepted electron should locate between the secondary electron acceptor, Q(B), and cyt b, but not at the Q(A) binding site as indicated before. The difference of electron transport between reduced sides of reaction centers of Rb. sphaeroides and Rs. rubrum was discussed.