Spinach Chloroplast Envelope Membranes Research Articles

Catalytic Reactions of the Homogentisate Prenyl Transferase Involved in Plastoquinone-9 Biosynthesis

Homogentisate solanesyl transferase (HST) catalyzes the prenylation and decarboxylation of homogentisate to form 2-methyl-6-solanesyl-1,4-benzoquinol, the first intermediate in plastoquinone-9 biosynthesis. In vitro, HST from Spinacia oleracea L., Arabidopsis thaliana, and Chlamydomonas reinhardtii were all found to use not only solanesyl diphosphate but also short chain prenyl diphosphates of 10-20 carbon atoms as prenyl donors. Surprisingly, with these donors, prenyl transfer was largely decoupled from decarboxylation, and thus the major products were 6-prenyl-1,4-benzoquinol-2-methylcarboxylates rather than the expected 2-methyl-6-prenyl-1,4-benzoquinols. The 6-prenyl-1,4-benzoquinol-2-methylcarboxylates were not substrates for HST-catalyzed decarboxylation, and the enzyme kinetics associated with forming these products appeared quite distinct from those for 2-methyl-6-prenyl-1,4-benzoquinol formation in respect of catalytic rate, substrate K(m) value, and the pattern of inhibition by haloxydine, a molecule that appeared to act as a dead end mimic of homogentisate. These observations were reconciled into a simple model for the HST mechanism. Here, prenyl diphosphate binds to HST to form at least two alternative complexes that go on to react differently with homogentisate and prenylate it either with or without it first being decarboxylated. It is supposed that solanesyl diphosphate binds tightly and preferentially in the mode that compels prenylation with decarboxylation.

Localization of a putative ClC chloride channel in spinach chloroplasts

Seven genes seem to encode for putative ClC chloride channels (AtClC-a to AtClC-g) in Arabidopsis thaliana. Their function and localization is still largely unknown. AtClC-f shares considerable sequence similarity with putative ClC channel proteins from Synechocystis, considered to represent the precursor of chloroplasts. We show by biochemical and mass spectrometry analysis that ClC-f is located in the outer envelope membrane of spinach chloroplasts. Consistent with the plastidial localization of ClC-f, p-chlorophenoxy-acetic acid (CPA) reduces photosynthetic activity and the protein is expressed in etioplasts and chloroplasts but not in root tissue. These findings may represent a step toward the molecular identification of ion channel activities in chloroplast membranes.

Interaction of cytosolic and plastidic nitrogen metabolism in plants.

In angiosperms, the assimilation of ammonia resulting from nitrate reduction and from photorespiration depends on the operation of the plastidic GS/GOGAT cycle. The precursor for ammonia assimilation, 2-oxoglutarate, is synthesized in the mitochondria and in the cytosol. It is imported into the plastid by a 2-oxoglutarate/malate translocator (DiT1). In turn, the product of ammonia assimilation, glutamate, is exported from the plastids by a glutamate/malate translocator (DiT2). These transport processes link plastidic and cytosolic nitrogen metabolism and are essential for plant metabolism. DiT1 was purified to homogeneity from spinach chloroplast envelope membranes and identified as a protein with an apparent molecular mass of 45 kDa. Peptide sequences were obtained from the protein and the corresponding cDNA was cloned. The function of the DiT1 protein and its substrate specificity were confirmed by expression of the cDNA in yeast cells and functional reconstitution of the recombinant protein into liposomes. Recent advances in the molecular cloning of DiT2 and in the analysis of the in vivo function of DiT1 by antisense repression in transgenic tobacco plants will be discussed. In non-green tissues, the reducing equivalents required for glutamate formation by NADH-GOGAT are supplied by the oxidative pentose phosphate pathway. Glucose 6-phosphate, the immediate precursor of the oxidative pentose phosphate pathway is generated in the cytosol and imported into the plastids by the plastidic glucose 6-phosphate/phosphate translocator.

Evidence for lipid kinase activities in spinach chloroplast envelope membranes

Three spinach chloroplast envelope membrane preparations (i.e. whole, outer and inner membranes) were incubated in the presence of [γ- 32P]ATP. After lipid extraction and separation by TLC, four main phosphorylated lipids were detected by autoradiography in whole envelope preparations. These phospholipids were identified by comparing their Rf with that of lipid markers and by a deacylation procedure. They were found to be phosphatidic acid (PA) and lyso-PA, l-α-phosphatidyl-inositol 4-monophosphate (PIP) and lyso-PIP. These lipids were not equally distributed in the outer and inner envelope membranes. Chloroplast envelope membranes were verified not to be contaminated by plasma membranes. It is concluded that lipid kinase activities are associated with spinach chloroplast envelope membranes.

Redox chains in chloroplast envelope membranes: spectroscopic evidence for the presence of electron carriers, including iron-sulfur centers.

We have shown that envelope membranes from spinach chloroplasts contain (i) semiquinone and flavosemiquinone radicals, (ii) a series of iron-containing electron-transfer centers, and (iii) flavins (mostly FAD) loosely associated with proteins. In contrast, we were unable to detect any cytochrome in spinach chloroplast envelope membranes. In addition to a high spin [1Fe]3+ type protein associated with an EPR signal at g = 4.3, we observed two iron-sulfur centers, a [4Fe-4S]1+ and a [2Fe-2S]1+, associated with features, respectively, at g = 1.921 and g = 1.935, which were detected after reduction by NADPH and NADH, respectively. The [4Fe-4S] center, but not the [2Fe-2S] center, was also reduced by dithionite or 5-deazaflavin/oxalate. An unusual Fe-S center, named X, associated with a signal at g = 2.057, was also detected, which was reduced by dithionite but not by NADH or NADPH. Extremely fast spin-relaxation rates of flavin- and quinone-free radicals suggest their close proximity to the [4Fe-4S] cluster or the high-spin [1Fe]3+ center. Envelope membranes probably contain enzymatic activities involved in the formation and reduction of semiquinone radicals (quinol oxidase, NADPH-quinone, and NADPH-semiquinone reductases). The physiological significance of our results is discussed with respect to (i) the presence of desaturase activities in envelope membranes and (ii) the mechanisms involved in the export of protons to the cytosol, which partially regulate the stromal pH during photosynthesis. The characterization of such a wide variety of electron carriers in envelope membranes opens new fields of research on the functions of this membrane system within the plant cell.

The 26- and 14-kDa phosphoproteins associated with spinach chloroplast envelope membranes are distinct membrane-bound pools of the light-harvesting complex of photosystem II and of the small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase

Two chloroplast envelope proteins from spinach (Spinacia oleracea L.) exhibiting relative molecular masses (Mrs) of 26 and 14 kDa are apparently phosphorylated by a unique Ca2+-dependent serine protein kinase. The activity of this enzyme shows the same sensitivity towards pH, Ca2+, Mg2+, H7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine] and ATP concentrations (Siegenthaler and Bovet 1993, Planta 190, 231–240). Autoradiographic analyses following two-dimensional-gel electrophoresis (isoelectric focusing and SDS-PAGE) associated with Western blotting experiments indicate that these two phosphoproteins appeared to be pools of the light-harvesting complex of photosystem II (LHCII) and of the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit, respectively. Immunoprecipitation of envelope-phosphorylated proteins, using immunoglobulins (IgG) directed to the apoprotein of LHCII and to the holoenzyme of Rubisco confirmed that LHCII and the Rubisco small subunit effectively incorporated 32P from (γ-32P)ATP in isolated envelope membranes. We propose that, in agreement with the fact that protein import is driven by ATP, the phosphorylation of LHCII and the Rubisco small subunit could take place after the processing of precursor proteins and could be an obligatory step for their internalization into chloroplasts.

Kinetic properties of monogalactosyldiacylglycerol synthase from spinach chloroplast envelope membranes.

We have investigated the functioning of monogalactosyldiacylglycerol (MGDG) synthase activity partially purified from spinach chloroplast envelope membranes, using mixed micelles containing diacylglycerol (the substrate for MGDG synthase), CHAPS (3-[(cholamidopropyl)dimethylammonio]-1- propanesulfonic acid), and phosphatidylglycerol. The presence of this anionic phospholipid was essential for optimal MGDG synthase activity because it strongly improves diacylglycerol solubilization by CHAPS. We have demonstrated that the "surface dilution" kinetic model proposed by Deems et al. (Deems, R.A., Eaton, B.R., and Dennis, E.A. (1975) J. Biol. Chem. 250, 9013-9020) is valid for MGDG synthase assayed in mixed micelles within a narrow range of CHAPS concentration. However, the experimental conditions we have set up in this study led to the description of defined equilibrium and kinetic parameters of the interaction of the envelope MGDG synthase with diacylglycerol. Two-substrate kinetic studies were performed with varied UDP-galactose molar concentrations and varied dioleoylglycerol surface concentrations. The families of reciprocal plots obtained were shown to intersect at a single point of the 1/[substrate] axis thus demonstrating that MGDG synthase is a sequential, either random or ordered, bireactant system. Therefore, MGDG synthase possesses two distinct and independent substrate-binding sites, a hydrophilic one for UDP-galactose and a hydrophobic one for diacylglycerol. The dependence of kinetic parameters on the diacylglycerol mol fraction allows a comparison of the affinity of the enzyme for a wide range of diacylglycerol molecular species. The Km values obtained were ranging between 0.0089 mol fraction (52 microM) for dilinoleoylglycerol (18:2/18:2) to 0.0666 mol fraction (416 microM) for distearoylglycerol (18:0/18:0), but the differences observed were not really related to the unsaturation of the molecule since the Km value for dilinoleoylglycerol was much lower than that (0.040 mol fraction) for dilinoleoylglycerol (18:3/18:3). The Km values for dioleoylglycerol (18:1/18:1) and for the diacylglycerol molecular species synthesized within chloroplasts, i.e. containing 18:1/16:0, were in the average range, i.e. lower than 0.030 mol fraction (around 170 microM).

Feedback inhibition of phosphatidate phosphatase from spinach chloroplast envelope membranes by diacylglycerol.

Because the envelope phosphatidate phosphatase plays a pivotal role in chloroplast glycerolipid metabolism, we have analyzed whether diacylglycerol could be a regulatory factor of the enzyme. Using isolated envelope membranes in which the level of diacylglycerol was modified by thermolysin treatment of intact chloroplasts to destroy the galactolipid:galactolipid galactosyltransferase, we have demonstrated that phosphatidate phosphatase activity was reduced when the membrane was enriched in diacylglycerol. All 1,2-diacylglycerol molecular species assayed were demonstrated to inhibit the enzyme to about the same extent. Kinetic studies with envelope from thermolysin-treated chloroplasts were performed in the absence and presence of diacylglycerol, and diacylglycerol was shown to be a powerful competitive inhibitor of the reaction. Finally, using isolated intact spinach chloroplasts, we have demonstrated that in situ phosphatidate phosphatase activity can be modulated by the level of diacylglycerol present in the membrane. The relevance of phosphatidate phosphatase inhibition by diacylglycerol in the regulation of chloroplast glycerolipid biosynthesis is discussed.

Lipid requirement and kinetic studies of solubilized UDP-galactose:diacylglycerol galactosyltransferase activity from spinach chloroplast envelope membranes

We have demonstrated a lipid requirement for the UDPgalactose:1,2-diacylglycerol 3-beta-D-galactosyl-transferase (or monogalactosyldiacylglycerol synthase; EC 2.4.1.46), an enzyme involved in the biosynthesis of monogalactosyldiacylglycerol, solubilized from chloroplast envelope membranes and partially purified by hydroxyapatite chromatography. The enzyme fraction was highly delipidated (<0.1 mg of lipid per mg of protein), and addition of lipids extracted from chloroplast membranes was necessary to reveal the activity. Acidic glycerolipids, and especially phosphatidylglycerol, were the best activators of the enzyme. The preparation of a delipidated enzyme fraction and the development of optimal assay conditions were prerequisites for the determination of the kinetic parameters for the hydrophobic substrate of the enzyme, diacylglycerol. In addition, we have demonstrated the existence of two substrate-binding sites: a hydrophobic one for diacylglycerol and a hydrophilic one for UDP-galactose.

Protein-mediated transfer of phosphatidylcholine from liposomes to spinach chloroplast envelope membranes

We have demonstrated that an active transfer of phosphatidylcholine from liposomes towards spinach chloroplast envelope was catalyzed by a phospholipid-transfer protein purified from spinach leaves. The transfer is actually a complex process. During the first 10 min of the incubation, the exchange of phosphatidylcholine between liposomes and isolated envelope vesicles was predominant, as shown by the equilibration of phosphatidylcholine specific activity to the same level in both the liposomes and the envelope vesicles. Further incubation led to a 35% increase of the phosphatidylcholine content of envelope membranes, thus corresponding to a net transfer of phosphatidylcholine from liposomes towards envelope vesicles. After incubation of intact chloroplasts and liposomes in the presence of purified phospholipid-transfer protein, most of the radioactive phosphatidylcholine transferred to intact chloroplasts was recovered with the envelope membrane fraction. In addition, a mild phospholipase C treatment of intact chloroplasts after phosphatidylcholine transfer has demonstrated that all the radioactive phosphatidylcholine remained in the cytosolic leaflet of the outer envelope membrane and was not redistributed towards internal chloroplast membranes. Such a result, which mimics the in vivo situation, suggests that the phospholipid-transfer protein might be partly responsible (together with the apparent lack of transmembrane lipid diffusion) for the different lipid composition of the outer envelope membrane (when compared with the other plastid membranes) and for the asymmetrical distribution of phosphatidylcholine within this membrane.

Localization of galactolipid: galactolipid galactosyltransferase and acyltransferase in outer envelope membrane of spinach chloroplasts

We have measured the localization within spinach chloroplast envelope membranes of two galactolipidmanipulating enzymes, the galactolipid:galactolipid galactosyltransferase and the galactolipid:galactolipid acyltransferase. Both are localized on the outer envelope membrane. This situation differs strikingly from the localization of the enzymes involved in monogalactosyldiacylglycerol synthesis, on the inner envelope membrane.

Spinach chloroplasts: localization of enzymes involved in galactolipid metabolism

A shortened procedure for the separation of spinach chloroplast envelope membranes is presented, resulting in high yields of enriched inner and outer envelopes. Fractionated envelopes were tested for activities in galactolipid synthesis by measuring incorporation of UDPGal and by using a specific assay for galactolipid: galactolipid galactosyltransferase (GGGT); additionally, acyl-CoA synthetase activities were tested. UDPGal incorporation and GGGT were found predominantly in enriched inner envelopes, acyl-CoA synthetase was predominantly in enriched outer envelopes. These observations point to a localization of both UPDGal: diacylglycerol galactosyltransferase (UDGT) and GGGT in the inner membrane. Alternatively, chloroplasts were incubated with [ 14C]acetate under conditions permitting fatty acid synthesis in the presence and absence of UDP[ 3H]Gal. After incubation, envelope fractions were isolated and analyzed for labeled lipids. Analysis agreed with localization of UDGT in the inner envelope. However, labeled products of GGGT were found both in enriched inner and outer envelopes. Furthermore, inactivation of GGGT by the proteinase thermolysin indicates a location on the cytosolic face of the chloroplast. These apparently contradictory results concerning GGGT might be explained by the involvement of contact sites between inner and outer envelopes or by lipid transformation during membrane fractionation.

Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts.

We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes.

Purification and some properties of an Mg 2+-, Ca 2+- and calmodulin-stimulated ATPase from spinach chloroplast envelope membranes

Spinach chloroplasts display an ATPase activity which is associated with the envelope. This envelope-bound activity is stimulated by Ca 2+, Mg 2+ and calmodulin (Nguyen, T.D. and Siegenthaler, P.A. (1983) FEBS Lett. 164, 67–70). The Triton X-100-solubilized enzyme was retained specifically on a calmodulin-Sepharose affinity column in the presence of calcium. The fractions eluted by EGTA contained two proteins characterized by p I values of 7.3 and 6.0 (isoelectric focusing). Both proteins, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis), were resolved into a single polypeptide having and identical apparent M rmr of 65 000. This suggests that the two initial proteins might be isoelectric variants. However, the amount of the enzyme fraction obtained by the calmodulin-Sepharose column was small and the ATPase activity was very labile. A linear glycerol gradient allowed the recovery of a greater amount of the enzyme which was, however, only partially purified, but the activity of which was much more stable. Electrophoresis of the ATPase-containing fractions in a native polyacrylamide gradient gel permitted the separation of a 260 kDa protein which was resolved by SDS-polyacrylamide gel electrophoresis into a single polypeptide of 65 kDa. Thus, the chloroplast envelope-bound ATPase might be a tetramer (260 kDa) consisting of 4 identical monomers (65 kDa). The purified ATPase had properties similar to that of the envelope-bound enzyme. The K m value for ATP was 0.45 mM. The activity was stimulated by Ca 2+ and Mg 2+, and further enhanced by calmodulin. The physiological significance of the chloroplast envelope-bound ATPase is discussed.

Localization of polypeptides to the cytosolic side of the outer envelope membrane of spinach chloroplasts.

Nonpenetrating proteolytic enzymes (such as thermolysin) were used to probe the cytosolic surface of the outer envelope membrane from spinach chloroplasts. Up to 20 different envelope polypeptides were susceptible to a mild digestion of isolated intact chloroplasts by thermolysin. Most of the thermolysin-sensitive envelope polypeptides were not extracted by a mixture of chloroform/methanol (2:1, v/v). A clear exception was E10 which is hydrophobic and, in addition, is an integral membrane polypeptide. Using antibodies to envelope polypeptides sensitive (E10 and E24) and insensitive (E30 and E37) to thermolysin, we demonstrated that only antibodies to E10 and E24, but not antibodies to E30 and E37, induced agglutination of intact chloroplasts. In addition, immunofluorescence experiments demonstrated that only antibodies to E10 and E24, but not antibodies to E30 and E37, gave a green fluorescence at the outer surface of intact chloroplasts. These experiments demonstrate that E10 and E24, and probably all the thermolysin-sensitive envelope polypeptides, are accessible from the cytosolic side of the outer membrane of the chloroplast envelope.

Phosphorylation of chloroplast ribulose bisphosphate carboxylase/oxygenase small subunit by an envelope-bound protein kinase in situ.

A new protein kinase of the cAMP independent type was found to be bound to the outer envelope membrane of spinach chloroplasts. While stimulated by Mg2+ and inhibited by ADP, the enzyme showed no response to conventional protein substrates and was essentially independent of pH in the physiological (pH 7 to 8) range. The new protein kinase phosphorylated the mature form of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase and, to a lesser extent, an unidentified 24-kDa polypeptide, both of which were bound to the outer envelope membrane. The results suggest that phosphorylation of cytoplasmically synthesized protein constituents of chloroplasts is involved in their transport through the chloroplast envelope membrane barrier.

Super-ultrastructure of the envelope membranes of spinach chloroplasts.

Super-ultrastructure of the envelope membranes of spinach chloroplasts.

Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts

Envelope membranes of spinach chloroplasts contain appreciable activities of the carotenogenic enzymes phytoene synthase (formation of phytoene by condensation of two molecules geranylgeranyl pyrophosphate) and phytoene dehydrogenase (formation of lycopene from phytoene), plus a phosphatase activity. These results were obtained by coincubation experiments using isolated envelope membranes and either a phytoene-forming in vitro system (from [1-(14)C]isopentenyl pyrophosphate) or [(14)C]geranylgeranyl pyrophosphate or a geranylgeranyl-pyrophosphate-forming in vitro system (from [1-(14)C]isopentenyl pyrophosphate). Within thylakoids carotenogenic enzymes could not be detected. It is concluded that the chloroplast envelope is at least a principal site of the membrane-bound steps of carotenoid biosynthesis in chloroplasts.

Dicarboxylate transport across the inner membrane of the chloroplast envelope

The uptake of radioactively labeled dicarboxylates into the sorbitol-impermeable 3H 2O space (the space surrounded by the inner envelope membrane) of spinach chloroplasts has been studied by means of silicone layer filtering centrifugation. 1. 1. Malate, aspartate and a number of other dicarboxylates are rapidly transported across the envelope leading to an accumulation in the chloroplasts. This uptake proceeds mainly by a counterexchange with the dicarboxylates present there. 2. 2. The dicarboxylate transport shows saturation characteristics allowing the determination of K m and V. 3. 3. All dicarboxylates transported act as competitive inhibitors of the transport. 4. 4. The activation energy of the transport as determined from the temperature dependency is evaluated to be 7 kcal/mol. 5. 5. The rate of dicarboxylate transport is influenced by illumination, the countertransported molecules and the pH in the medium. These changes effect the transport velocity, whereas the corresponding K m values are not altered. 6. 6. It is discussed whether there is more than one carrier involved in dicarboxylate transport in spinach chloroplasts.

Adenylate kinase bound to the envelope membranes of spinach chloroplasts

The activity of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in both the forward (2ADP → ATP + AMP) and backward (ATP + AMP → 2ADP) reactions was found to be associated with the envelope membranes which were isolated from spinach chloroplasts. Sonication and repeated washing in a medium of high ionic strength were unable to release the enzymes from the envelope membranes. Adenylate kinase bound to the envelope is stable in the cold and inactivated by heat and acid treatments. The enzyme requires magnesium ion as an activator. The pH-activity profile of the forward reaction catalyzed by membrane-bound adenylate kinase gave a maximal activity at pH 8.5. The apparent Michaelis constant, K m, value for ADP in the forward reaction was estimated to be 1.3 ± 0.2 × 10 −4 m. A Lineweaver-Burk plot of the forward reaction gave a straight line when the reciprocal of the reaction rate was plotted versus the reciprocal, and not the square of the reciprocal, of the concentration of substrate ADP. This favors the view that the adenylate kinase bound to the chloroplast envelope has a single or equivalent binding site of Mg-ADP −. The probable involvement of adenylate kinase bound to the chloroplast envelope in controlling the energy pool and adenylate translocation in chloroplasts is suggested.