Gamma Subunit Of Coupling Factor Research Articles

Role of the Chlamydomonas reinhardtii Coupling Factor 1 γ-Subunit Cysteine Bridge in the Regulation of ATP Synthase

The gamma-subunit of coupling factor 1 (CF1) contains a cysteine bridge that is thought to be involved in the redox control of enzymatic activity. In order to test the regulatory significance of this disulfide bond, genetic transformation experiments with Chlamydomonas reinhardtii were performed. C. reinhardtii strain atpC1 (nit1-305, cw 15, mt-), which is null for the gamma-subunit, was transformed and complemented with gamma-subunit constructs containing amino acid substitutions localized to the cysteine bridge between Cys198 and Cys204. Successful complementation was confirmed by phenotypic selection, Northern blot analysis, reverse transcription polymerase chain reaction, and cDNA sequencing. CF1 ATPase activities of the soluble enzymes were measured in the presence and absence of dithiothreitol (DTT). Mutant CF1 enzymes showed no effect of DTT although increased activity was observed for the wild-type enzyme. In vitro, phenazine methosulfate-dependent photophosphorylation assays revealed that wild-type CF1 exhibits a 2-fold stimulation in the presence of 25 mM DTT, whereas each of the mutant enzymes has activities that are DTT-independent. Growth measurements indicated that despite the absence of a regulatory disulfide/dithiol, the mutant strains grew with the same kinetics as wild type. This study provides evidence to illustrate the involvement of the gamma-subunit in the redox regulation of ATP synthesis in vivo. This work is also the first demonstration in C. reinhardtii of stable nuclear transformation using mutated genes to complement a known defect.

Alkylation of cysteine 89 of the gamma subunit of chloroplast coupling factor 1 with N-ethylmaleimide alters nucleotide interactions.

Alkylation of Cys-89 of the gamma subunit of the coupling factor portion (CF1) of the chloroplast ATP synthase by N-ethylmaleimide was previously shown to inhibit ATP synthesis and hydrolysis. The gamma subunit interacts with the inhibitory epsilon subunit. Alkylation of gamma Cys-89 could cause changes that result in the strengthening of the interactions between the two subunits which would be inhibitory. We show that the inhibition of the ATPase activity of CF1 in solution persists after removal of the epsilon subunit. Additionally, epsilon rebinds to control and alkylated CF1 at very similar CF1 to epsilon concentration ratios. Although the bound nucleotide contents of control and alkylated CF1 were similar, the rate of exchange of nucleotide bound to one site with medium nucleotide was accelerated by more than two orders of magnitude by alkylation. We show that ATP-induced release of bound 2'(3')-O-trinitrophenyl-adenosine 5'-diphosphate can be monitored conveniently by stopped-flow fluorescence. The effects of N-ethylmaleimide modification are likely to be felt over long distances. As determined by fluorescence resonance energy transfer, no nucleotide binding site so far mapped is closer than 5 nm to Cys-89. The bulky maleimide probably causes structural changes that perturb subunit and catalytic site interactions.

Characterization of the cysteinyl-containing peptides of the gamma subunit of coupling factor 1.

The cysteinyl peptides of the gamma subunit of chloroplast coupling factor 1 (CF1) have been analyzed by high performance liquid chromatography. Analysis of the reduced enzyme alkylated with 4-vinylpyridine showed that the gamma subunit contains four cysteinyl residues. Two of these residues are involved in a disulfide linkage in CF1 either in solution or bound to washed thylakoid membranes. Two free sulfhydryls, one that is readily attacked by alkylating reagents and another that is less reactive, were also detected. Each of these four cysteinyl residues is present in a separate tryptic peptide derived from the gamma subunit. These results show that 4-vinylpyridine is an excellent reagent for the analysis of cysteinyl-containing peptides and support our analyses of the roles of cysteinyl residues in the gamma subunit in ATP synthesis and hydrolysis.

Role of the gamma subunit of chloroplast coupling factor 1 in the light-dependent activation of photophosphorylation and ATPase activity by dithiothreitol.

In leaves and intact chloroplasts, oxidation and reduction have been shown previously to regulate the ATPase activity of thylakoids. Illumination of spinach chloroplast thylakoids in the presence of dithiothreitol, which activates the ability of thylakoids to catalyze sustained ATP hydrolysis in the dark, causes increased incorporation of N-ethylmaleimide into the gamma subunit of coupling factor 1 (CF1). A disulfide bond in the gamma subunit is reduced during activation. The residues involved in this disulfide bond are the same as those in the disulfide linkage reduced during dithiothreitol activation of soluble CF1. The disulfide and dithiol forms of the gamma subunit may be separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. N-Ethylmaleimide is preferentially incorporated in the dark into the reduced form of the gamma subunit of CF1 in thylakoids previously exposed to dithiothreitol. Only a subpopulation of the CF1 in thylakoids is susceptible to dithiothreitol reduction and subsequent reaction with N-ethylmaleimide in the dark. Alkylation of the thiol groups exposed by reduction of the disulfide bond protects ATPase activity from inhibition by oxidants. At a given value of the transmembrane pH differential, photophosphorylation rates in dithiothreitol-activated thylakoids can be as much as seven to eight times those of nonactivated controls. N-Ethylmaleimide treatment of activated thylakoids in the dark prevents the loss of the stimulation of ATP synthesis on storage of the thylakoids. Photophosphorylation by intact chloroplasts lysed in assay mixtures is also activated in comparison to that by washed thylakoids. At a low ADP concentration, the rate of photophosphorylation approaches saturation as delta pH increases. These results suggest that the gamma subunit of CF1 plays an important role in regulation of ATP synthesis and hydrolysis.

Changes in activity and structure of the chloroplast proton ATPase induced by illumination of spinach leaves.

Photophosphorylating activity of chloroplasts rapidly prepared from preilluminated spinach leaves was higher than the activity of chlorplasts from leaves kept in the dark. Higher Vmax values were obtained with the former when either ADP or Pi concentrations were varied. The rate of decay of the in vivo light-activated Mg2+-ATPase was highly dependent on temperature, increasing with it. At 0 degree C it was stable for 40 min or more. The decay at 25 degrees C was prevented by 5 mM ATP or 50 mM dithioerythritol while ADP or Pi did not affect it. Gramicidin or iodosobenzoate induced a very rapid decay even at 0 degree C. Coupling factor 1 with a manifest and stable Ca2+-ATPase activity was solubilized from chloroplasts activated by light in vivo. Incubation of chloroplasts from preilluminated leaves with N-[3H]ethylmaleimide resulted in an inhibition of Ca2+-ATPase activity and in the incorporation of radioactivity into the gamma subunit of coupling factor 1 that was larger than that of chloroplasts from leaves kept in the dark. The results show that activation in vivo of the proton ATPase was manifested by higher phosphorylating and Mg2+-ATPase activities and requires both an electrochemical proton gradient and a redox change of at least one disulfide bond of its gamma subunit.

The distance between thiol groups in the gamma subunit of coupling factor 1 influences the proton permeability of thylakoid membranes.

Spinach chloroplast thylakoids treated in the light with bifunctional maleimides were previously shown to be uncoupled. The increase in proton permeability by these reagents is caused by the cross-linking of an accessible group on the gamma subunit of coupling factor 1 (CF1) to a group that becomes exposed to reaction with maleimides only when the thylakoids are energized. In this study, several bifunctional maleimides, including o-, m-, and p-phenylenebismaleimides, 2,3- and 1,5-naphthalenebismaleimides, and azophenylbismaleimide, were tested for their ability to form cross-links and to uncouple photophosphorylation. These reagents form cross-links from about 6 to 19 A. Each reagent was found to form cross-links in the light and to inhibit photophosphorylation. However, the effectiveness of these compounds as uncouplers decreased as the distance between the cross-linked groups increased, indicating that the distance between two groups on the gamma subunit of CF1 can regulate proton flux through the membrane. Monofunctional maleimides cause a light-dependent energy transfer type of inhibition of photophosphorylation. Although this inhibition was correlated to the reaction of the maleimide with a group on the gamma subunit that is exposed only in energized thylakoids, the accessible group on this subunit was also modified by the reagent. However, we show here that the accessible group plays no role in the inhibition of photophosphorylation. This group may be blocked by incubating thylakoids in the dark with methyl methanethiolsulfonate. The light-dependent inhibition of photophosphorylation by N-ethylmaleimide was unaffected by this treatment or by the subsequent removal of the methanethiol moiety from the accessible group.

Inhibition of Photosynthetic Energy Conversion by Cupric Ion

This study describes a specific Cu(2+) and light-dependent inhibition of spinach (Spinacia oleracea L.) chloroplast reactions involving coupling factor 1 function. A primary effect is an inhibition of photophosphorylation induced by illumination of Class II chloroplasts with micromolar Cu(2+) and pyocyanine in the absence of ADP, Mg(2+), and HPO(4) (2-). The inhibition, which is dependent on free Cu(2+) as indicated by protection by ethylene diamine tetraacetic acid and dithiothreitol, requires illumination (electron flow) for establishment of the specific inhibition to be noted. Protection is also afforded by uncouplers and some partial protection is provided by micromolar concentrations of ADP and ATP. The data strongly suggest that Cu(2+) causes an O(2)-independent oxidation of sulfhydryl groups on coupling factor 1, which are essential to catalytic function. This conclusion is supported by the reduction of energy-dependent (3)H-N-ethylmaleimide labeling of the gamma subunit of coupling factor 1 by the Cu(2+)-light pretreatment.

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.