Thylakoid Fractions Research Articles

Fractionation of thylakoid membranes into grana and stroma thylakoids.

The chloroplasts contain an extensive system of internal membranes or thylakoids in which all the light-harvesting and energy-transducing processes of the photosynthesis are located. Thylakoids are differentiated into stacked membrane regions (or grana thylakoids) and nonstacked membranes (or stroma thylakoids), each with a specialized structure and function. Both kinds of thylakoids can be separated by detergent-based methods or mechanical fragmentation such as sonication. We describe the fractionation of thylakoid membranes into grana and stroma thylakoids by treatment with the detergent digitonin and successive ultracentrifugation of the resultant vesicles. After their separation, the thylakoid fractions retain electron transport and enzymatic activities and are characterized using various parameters. The stroma thylakoids have higher chlorophyll a/chlorophyll b and protein/total chlorophyll ratios, and greater photosystem I and NADH dehydrogenase activities than the grana thylakoids. In the conditions used and on a protein basis of total thylakoids, the yield of stroma thylakoids is 5%, which is considerable taking into account that the stroma thylakoids are a minor component of total thylakoids.

Proteomics of the Chloroplast Envelope Membranes from Arabidopsis thaliana

The development of chloroplasts and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting envelope membranes. To provide the most exhaustive view of the protein repertoire of chloroplast envelope membranes, we analyzed this membrane system using proteomics. To this purpose, we first developed a procedure to prepare highly purified envelope membranes from Arabidopsis chloroplasts. We then extracted envelope proteins using different methods, i.e. chloroform/methanol extraction and alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to least hydrophobic ones. Liquid chromatography tandem mass spectrometry analyses were then performed on each envelope membrane subfraction, leading to the identification of more than 100 proteins. About 80% of the identified proteins are known to be, or are very likely, located in the chloroplast envelope. The validation of localization in the envelope of two phosphate transporters exemplifies the need for a combination of strategies to perform the most exhaustive identification of genuine chloroplast envelope proteins. Interestingly, some of the identified proteins are found to be Nalpha-acetylated, which indicates the accurate location of the N terminus of the corresponding mature protein. With regard to function, more than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are a) involved in ion and metabolite transport, b) components of the protein import machinery, and c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism, or proteins involved in responses to oxidative stress, were associated with envelope membranes. Almost one-third of the proteins we identified have no known function. The present work helps understanding chloroplast envelope metabolism at the molecular level and provides a new overview of the biochemical machinery of the chloroplast envelope membranes.

Suborganellar localisation and effect of light on Helianthus tuberosus chloroplast transglutaminases and their substrates.

The light stimulation of transglutaminase (TGase EC 2.3.2.13) activity was verified by incubating isolated chloroplasts of Helianthus tuberosus L. continuously or for alternate periods of light or dark (light/dark and dark/light). The first 10 min of incubation always represented the critical period. Light-harvesting complexes of photosystem II (LHCII) were more intensely labelled by (14)C-polyamines under light and light/dark than under dark and dark/light conditions. Chloroplasts were fractionated into thylakoid- and stroma-enriched fractions in which multiple TGase forms and substrates were found. Antibodies against TGase recognised 58- and 24-kDa bands in thylakoids and a 150-kDa band in the stroma. The latter, and its 150-kDa fraction, catalysed the conjugation of 14C-polyamines to Rubisco. In both fractions (thylakoid-pre and stroma-pre) the analysis of polyamine glutamyl derivatives showed a significant light-affected conjugation of polyamines to endogenous proteins. Alternatively, entire chloroplasts were incubated and afterwards their sub-fractions were isolated (thylakoid-post and stroma-post). The PSII and LHCII complexes were more intensely immunodetected in thylakoid-post than in thylakoid-pre, especially under dark conditions. Conversely, the conjugation of polyamines to thylakoid proteins was clearly light-stimulated in thylakoid-post, and much less in thylakoid-pre. Stroma-pre proteins were poorly polyamine-conjugated and not light-affected; on the contrary, stroma-post proteins were much more polyamine-modified and strongly light-stimulated. Thus, the light-activated conjugation depends mainly on the presence of the thylakoid fraction during the assay. The protective effect on chloroplasts under photo-damage, stress or senescence conditions attributed in the literature to free polyamines is discussed with regard to the occurrence of polyamine conjugates catalysed by TGases.

Elevated temperature treatment induced alteration in thylakoid membrane organization and energy distribution between the two photosystems in Pisum sativum.

Two-week-old pea (Pisum sativum var. Arkal) plants were subjected to elevated temperature (38 degrees C/42 degrees C) in dark for 14-15 h. The effect of heat treatment on light-induced phosphorylation of LHCII and LHCII migration in the thylakoid membranes were investigated. The heat treatment did cause a substantial (more than two fold) increase in the extent of LHCII phosphorylation as compared to the control. Upon separation of appressed and non-appressed thylakoid fractions by digitonin treatment, the heat-treated samples showed a decrease in LHCII-related polypeptides from the grana stack (appressed region) over the control. Further, a small increase in the intensity of these (LHCII-related) bands was detected in stromal thylakoid fraction (non-appressed membranes). This suggests an enhanced extent of migration of phosphorylated LHCII from appressed to non-appressed regions due to in vivo heat treatment of pea plants. We also isolated the LHCII from control and heat treated (42 degrees C) pea seedlings. Analysis of CD spectra revealed a 5-6 nm blue shift in the 638 nm negative peak in heat treated samples suggesting alteration in the organization of Chl b in the LHCII macro-aggregates. These results suggest that in vivo heat stress not only alters the extent of migration of LHCII to stromal region, but also affects the light harvesting mechanism by LHCII associated with the grana region.

Detection and characterization of GTP-binding proteins in the chloroplast envelope of spinach (Spinacia oleracea).

Proteins binding guanosine triphosphate (GTP) have emerged as important regulators in several cellular processes in plants. To investigate any role of such proteins in chloroplast functions, we subjected envelope, stroma and thylakoid fractions isolated from spinach chloroplasts to two different GTP-binding assays. With both methods, we detected GTP-specific binding only in the envelope fraction. Two chloroplast envelope proteins with the apparent molecular weights of 30.5 and 33.5 kDa, respectively, bound [alpha-32P]GTP after SDS-PAGE followed by electroblotting onto a PVDF-membrane and renaturation. Both proteins were intrinsic proteins located in the outer chloroplast envelope. Also, when the fractions were incubated with [alpha-32P]GTP, followed by periodate oxidation and borohydride reduction to cross-link GTP to proteins, two proteins in the envelope fraction, of apparent molecular weights of 28 and 39 kDa, appeared to specifically bind GTP. When agents that stimulate heterotrimeric G-proteins, cholera toxin or the mastoparan analogue mas7, were added to isolated chloroplast envelope, the binding of radiolabelled GTP to the 39 kDa protein, a protein of the inner chloroplast envelope, was stimulated, whereas GTP-binding of the 28 kDa protein, a protein of the outer envelope, was unchanged. Mas7 also stimulated synthesis of monogalactosyl diacylglycerol in isolated chloroplast envelope. The occurrence and regulation of GTP-binding proteins in the chloroplast envelope suggests that GTP-binding proteins could be involved in communication with the extraplastidic compartment during chloroplast biogenesis and development.

Do glycerolipids display lateral heterogeneity in the thylakoid membrane?

The lateral heterogeneity of lipids in the thylakoid membrane has been questioned for over 20 yrs. It is generally believed that glycerolipids are asymmetrically distributed within the plane of the membrane. In the present investigation, we isolated several thylakoid membrane domains by using sonication followed by separation in an aqueous dextran-polyethylene glycol two-phase system. This technique, which avoids detergent treatments, allowed us to obtain stroma and grana lamellae vesicles as well as grana central core and grana margin vesicles from thylakoids. The relative distribution of the four lipid classes, i.e., monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidylglycerol, was found to be statistically identical in all four thylakoid fractions and in whole thylakoids. Similarly, the relative amount of fatty acids in each individual lipid and the eight main phosphatidylglycerol molecular species was identical in all thylakoid membrane fractions tested as well as in the intact thylakoid membrane. Based on presently available procedures for obtaining thylakoid subfractions that are unable to discriminate microdomains within the membrane, it is concluded that glycerolipids are evenly distributed within the plane of the thylakoid membrane. These data are discussed in terms of "bulk" and "specific" lipids.

Association of ferredoxin-NADP oxidoreductase with the chloroplastic pyridine nucleotide dehydrogenase complex in barley leaves

Barley (Hordeum vulgare L.) leaves were used to isolate and characterize the chloroplast NAD(P)H dehydrogenase complex. The stroma fraction and the thylakoid fraction solubilized with sodium deoxycholate were analyzed by native polyacrylamide gel electrophoresis, and the enzymes detected with NADH and nitroblue tetrazolium were electroeluted. The enzymes electroeluted from band S from the stroma fraction and from bands T1 (ET1) and T2 from the thylakoid fraction solubilized with sodium deoxycholate had ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) and NAD(P)H-FeCN oxidoreductase (NAD[P]H-FeCNR) activities. Their NADPH-FeCNR activities were inhibited by 2'-monophosphoadenosine-5'-diphosphoribose and by enzyme incubation with p-chloromercuriphenylsulfonic acid (p-CMPS), NADPH, and p-CMPS plus NADPH. They presented Michaelis constant NADPH values that were similar to those of FNRs from several sources. Their NADH-FeCNR activities, however, were not inhibited by 2'-monophosphoadenosine-5'-diphosphoribose but were weakly inhibited by enzyme incubation with NADH, p-CMPS, and p-CMPS plus NADH. We found that only ET1 contained two polypeptides of 29 and 35 kD, which reacted with the antibodies raised against the mitochondrial complex I TYKY subunit and the chloroplast ndhA gene product, respectively. However, all three enzymes contained two polypeptides of 35 and 53 kD, which reacted with the antibodies raised against barley FNR and the NADH-binding 51-kD polypeptide of the mitochondrial complex I, respectively. The results suggest that ET1 is the FNR-containing thylakoidal NAD(P)H dehydrogenase complex.

Further characterization of the chloroplast grana margins: the non-detergent preparation of granal Photosystem I cannot reduce ferredoxin in the absence of NADP + reduction

The chloroplast grana margins of spinach thylakoids were isolated by sonication and aqueous-two-phase partitioning and their electron transport properties examined. Photosystem II and I electron transport activities were measured and compared to the appressed and non-appressed grana core and stroma lamellae, respectively, as well as to whole thylakoids. The results show that the PS II complexes in the grana margins are of the PS II β subtype with respect to antenna size, but are Q B reducing with respect to the acceptor side properties, while the PS I centers in the grana margins have slightly larger antennae as compared to the PS I centers in the stroma lamellae and are more like the PS I α centers located in the grana domain. The ability to reduce ferredoxin and NADP + was also tested and it was found that the grana margin membrane fraction was unable to reduce ferredoxin, even in the presence of added artificial electron donors. The stroma lamellae and whole thylakoid fractions both reduced ferredoxin at high rates. However, the grana margins could catalyze the reduction of NADP + when supplied with the necessary components (ferredoxin, ferredoxin:NADP + oxidoreductase, and an electron source). The results suggest that the PS I populations located in the margins of the grana domain are functionally different from the PS I centers located in the stroma lamellae. The data support a model whereby the PS I centers in the grana are primarily involved in non-cyclic electron transport, while the PS I centers located in the stroma lamellae are capable of participating in both cyclic and non-cyclic electron transport.

Spin label EPR study of lipid solvation of supramolecular photosynthetic protein complexes in thylakoids

Lipid-protein association in the chloroplast membrane and its various thylakoid fractions from higher plants, namely pea and maize, rich in Photosystem I (PSI) and Photosystem II (PSII), respectively, were studied using EPR spectroscopy of spin-labelled lipid molecules. All the EPR spectra consisted of two spectral components corresponding to bulk fluid lipids and solvation lipids motionally restricted at the hydrophobic surface of membrane proteins. Spin-labelled stearic acid and phosphatidylglycerol exhibited marked selectivity towards the supramolecular protein complexes of both PSI and PSII although to different extent. In addition, lipid-protein titration experiments are described for partially delipidated PSII-enriched membrane fractions of pea chloroplasts, incorporating unlabelled egg phosphatidylcholine prior to or after the incorporation of spin-labelled lipids. Two sets of solvation sites were resolved by timed labelling experiments and a significant result of these studies was that a well-defined population of solvation sites (approx. 100 mol lipids/820 kDa protein) was rapidly exchanged by laterally diffusing membrane lipids, while other solvation sites (approx. 50 mol lipids/820 kDa protein) were exchanged much slower or not exchanged at all.

State transitions, light-harvesting antenna phosphorylation and light-harvesting antenna migration in vivo in the higher plant Spirodela oligorrhiza

Plants can balance the relative levels of excitation energy reaching the two photosystems of photosynthesis via state transitions. This process was investigated in vivo using the aquatic higher plant Spirodela oligorrhiza. State transitions were followed by 77 K chlorophyll a (Chl a) fluorescence and phosphorylation of the Chl a b light harvesting complex (LHCII). A response spectrum for the state transition indicated that light absorbed predominantly by Chl b led to state 2 and light absorbed predominantly by Chl a resulted in state 1. The kinetics of LHCII phosphorylation ( t 1 2 = 4 min ) during a state 1 to state 2 transition were similar to the rise in fluorescence at 77 K from Photosystem I (PS I) relative to Photosystem II (PS II) ( t 1 2 = 3 min ). As well, for the transition from state 2 to state 1, the kinetics of LHCII dephosphorylation ( t 1 2 = 13 min ) and the rate of loss of fluorescence from PS I relative to PS II ( t 1 2 = 10 min ) were comparable. The phosphatase inhibitor, NaF, suppressed both LHCII dephosphorylation and the decrease in the PS I/PS II fluorescence emission ratio, thus showing prevention of a transition to state 1 under PS I illumination. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), which indirectly inactivates the LHCII kinase, triggered a transition to state 1 in PS II light. Upon exposure to PS II light, phosphorylated LHCII was observed to appear in granal thylakoid fractions prior to its appearance in stromal thylakoid fractions, indicating vectoral movement between the two membrane compartments. Determinations of the absolute yield of PS II and PS I fluorescence showed a complementary decrease and increase in PS II and PS I emission, respectively, in state 2 compared to state 1. Our results strongly support the hypothesis that LHCII phosphorylation and antenna migration between the photosystems are key components of the state transition mechanism in vivo.

Effect of growth arrest on carotene accumulation and photosynthesis in Dunaliella

The halotolerant green alga Dunaliella bardawil is known to accumulate β-carotene in response to stress factors such as high light intensity, high salt concentrations and nutrient limitation. In this report, the accumulation of β-carotene was studied in cells from nitrate-limited chemostat cultures, in comparison with those of D. salina, a strain that does not accumulate β-carotene under stress conditions. D. bardawil responded to growth arrest by accumulating β-carotene and, to a lesser degree, lutein and zeaxanthin. A substantial fraction of β-carotene and all the lutein and zeaxanthin was associated with the thylakoid fraction. The accumulation of carotenoids in D. bardawil occurred only in the light, but the light intensities were far below those where the photosynthetic rate is maximal. After growth arrest, the amount of chlorophyll (Chi) decreased in both strains. However, in D. bardawil Chi a decreased to a lesser extent in comparison with Chi b, which resulted in an increased Chi alb ratio. The maximum photosynthetic capacity declined rapidly in both strains after growth arrest. In contrast, the photosynthetic efficiency showed a temporary increase in D. bardawil and a decrease in D. salina. This increase did not occur when carotenogenesis was inhibited by diphenylamine, implying a causal relationship between enhanced carotenogenesis and increase of photosynthetic efficiency. The possible involvement of stress-accumulated carotenoids in photosynthetic activity is discussed.

Characterization of high-light-induced increases in xanthophyll cycle pigment and lutein contents in photoautotrophic cell cultures

The effect of prolonged high light treatment on xanthophyll pigment content was analysed in photoautotrophic cell cultures of Chenopodium rubrum and Marchantia polymorpha. Photoinhibitory effects of high light treatment were indicated by a reduction in F v/ F m (dark), which was accompanied by a partial de-epoxidation of the xanthophyll cycle pigment pool. Xanthophyll cycle pigments were found in grana thylakoids and stroma thylakoids and the epoxidation states were comparable in both thylakoid membrane subfractions. During high light treatment, the contents of the xanthophyll cycle pigments and lutein increased by de novo synthesis of these pigments. Increases were observed in grana thylakoids and stroma thylakoids. In the stroma thylakoid fraction, an enrichment of the 28 kDa apoprotein of light-harvesting complex IIb (LHCIIb) was found after high light treatment, but in unfractionated thylakoid membranes marked increases in the apoproteins of the dominant pigment-protein complexes could not be detected. In addition, the stoichiometry of pigment changes did not correspond to that of isolated pigment-protein complexes from untreated plants. Therefore we conclude that the ratio of xanthophyll cycle pigments to pigment-protein complexes was increased after prolonged light stress.

Newly Imported Rieske Iron-Sulfur Protein Associates with Both Cpn60 and Hsp70 in the Chloroplast Stroma.

The precursor of the Rieske FeS protein, a thylakoid membrane protein, was imported by isolated pea chloroplasts, and the mature protein was shown to be integrated into the cytochrome bf complex of the thylakoid membranes. Insertion into the thylakoid membrane was sensitive to the ionophores nigericin and valinomycin, suggesting a requirement for a proton motive force. A considerable proportion of the imported Rieske protein was detected in the stromal fraction of the chloroplasts, and this increased when membrane insertion was blocked with ionophores. Electrophoresis of the stromal fraction under nondenaturing conditions resolved two distinct complexes containing the Rieske protein. One of these complexes was identified as an association of the Rieske protein with the chaperonin Cpn60 complex by its electrophoretic mobility, Mg-ATP-dependent dissociation, and immunoprecipitation with anti-Cpn60 antibodies. Coimmunoprecipitation of imported Rieske protein with anti-heat shock protein 70 (Hsp70) antibodies indicated that the Rieske protein was also associated, in an ATP-dissociable form, with a chloroplast Hsp70 homolog. Immunoprecipitation analysis of an import time course detected the highest amounts of the Cpn60-Rieske protein complex early in the time course, whereas highest amounts of the Hsp70-Rieske protein complex were formed much later. The disappearance of the Cpn60-Rieske protein complex correlated with increased amounts of the Rieske protein in the thylakoid fraction.

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with ?antisense? rbcS

Tobacco (Nicotiana tabacum L.) plants transformed with ‘antisense’ rbcS to decrease the expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) have been used to investigate the contribution of Rubisco to the control of photosynthesis in plants growing at different irradiances. Tobacco plants were grown in controlled-climate chambers under ambient CO2 at 20°C at 100, 300 and 750 μmol·m−2·s−1 irradiance, and at 28°C at 100, 300 and 1000 μmol·m−2·s−1 irradiance. (i) Measurement of photosynthesis under ambient conditions showed that the flux control coefficient of Rubisco (C infRubisco supA ) was very low (0.01–0.03) at low growth irradiance, and still fairly low (0.24–0.27) at higher irradiance. (ii) Short-term changes in the irradiance used to measure photosynthesis showed that C infRubisco supA increases as incident irradiance rises, (iii) When low-light (100 μmol·m−2·s−1)-grown plants are exposed to high (750–1000 μmol·m−2·s−1) irradiance, Rubisco is almost totally limiting for photosynthesis in wild types. However, when high-light-grown leaves (750–1000 μmol·m−2·s−1) are suddenly exposed to high and saturating irradiance (1500–2000 μmol·m−2·s−1), C infRubisco supA remained relatively low (0.23–0.33), showing that in saturating light Rubisco only exerts partial control over the light-saturated rate of photosynthesis in “sun” leaves; apparently additional factors are co-limiting photosynthetic performance, (iv) Growth of plants at high irradiance led to a small decrease in the percentage of total protein found in the insoluble (thylakoid fraction), and a decrease of chlorophyll, relative to protein or structural leaf dry weight. As a consequence of this change, high-irradiance-grown leaves illuminated at growth irradiance avoided an inbalance between the “light” reactions and Rubisco; this was shown by the low value of C infRubisco supA (see above) and by measurements showing that non-photochemical quenching was low, photochemical quenching high, and NADP-malate dehydrogenase activation was low at the growth irradiance. In contrast, when a leaf adapted to low irradiance was illuminated at a higher irradiance, Rubisco exerted more control, non-photochemical quenching was higher, photochemical quenching was lower, and NADP-malate dehydrogenase activation was higher than in a leaf which had grown at that irradiance. We conclude that changes in leaf composition allow the leaf to avoid a one-sided limitation by Rubisco and, hence, overexcitation and overreduction of the thylakoids in high-irradiance growth conditions, (v) ‘Antisense’ plants with less Rubisco contained a higher content of insoluble (thylakoid) protein and chlorophyll, compared to total protein or structural leaf dry weight. They also showed a higher rate of photosynthesis than the wild type, when measured at an irradiance below that at which the plant had grown. We propose that N-allocation in low light is not optimal in tobacco and that genetic manipulation to decrease Rubisco may, in some circumstances, increase photosynthetic performance in low light.

Protochlorophyllide and Chlorophyllide in Reformed Prolamellar Bodies and Thylakoids of Irradiated Dark-grown Wheat ( Trificum aestivum L.)

Summary Reformed prolamellar bodies and thylakoids were isolated from dark-grown wheat leaves irradiated with white light (4.4 · W m −2 ) for 4 h and redarkened for 16 h. In order to follow the synthesis of chlorophyllous pigments in the dark, the leaves were cut and incubated with 14 C-5-aminolevulinic acid during the 16 h dark period. Prolamellar bodies and thylakoids were isolated from both intact and cut leaves in continuous sucrose density gradients. The relative amount of phototransformable protochlorophyllide, i.e. protochlorophyllide associated with the active site of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33), was ten times higher in the reformed prolamellar bodies than in the thylakoids, as judged from the 657/633 nm fluorescence emission ratios at −196 °C of the two membrane fractions. The pigments were separated by HPLC. Both membrane fractions contained non-labelled chlorophyllide and chlorophyll most likely synthesized during the 4 h of irradiation. The labelled pigments that accumulated during the 16 h dark period were protochlorophyllide and chlorophyllide, and were both present in the reformed prolamellar body fraction. The only labelled pigment in the thylakoid fraction was protochlorophyllide. The presence of labelled chlorophyllide indicates its synthesis and incorporation into reformed prolamellar bodies during the dark period. The possibility of thylakoid components as constituents of reformed prolamellar bodies is discussed.

Biochemical Characterization of Photosystem II Antenna Polypeptides in Grana and Stroma Membranes of Spinach

The photosystem (PS) II antenna system comprises several biochemically and spectroscopically distinct complexes, including light-harvesting complex II (LHCII), chlorophyll-protein complex (CP) 29, CP26, and CP24. LHCII, the most abundant of these, is both structurally and functionally diverse. The photosynthetic apparatus is laterally segregated within the thylakoid membrane into PSI-rich and PSII-rich domains, and the distribution of antenna complexes between these domains has implications for antenna function. We report a detailed analysis of the differences in the polypeptide composition of LHCII, CP29, and CP26 complexes associated with grana and stroma thylakoid fractions from spinach (Spinacia oleracea L.), making use of a very high-resolution denaturing gel system, coupled with immunoblots using monospecific antibodies to identify specific antenna components. We first show that the polypeptide composition of the PSII antenna system is more complex than previously thought. We resolved at least five type I LHCII apoproteins and two to three type II LHCII apoproteins. We also resolved at least two apoproteins each for CP29 and CP26. In state 1-adapted grana and stroma thylakoid membranes, the spectrum of LHCII apoproteins is surprisingly similar. However, in addition to overall quantitative differences, we saw subtle but reproducible qualitative differences in the spectrum of LHCII apoproteins in grana and stroma membrane domains, including two forms of the major type II apoprotein. The implications of these findings for models of PSII antenna function in spinach are discussed.

Chloroplast envelope proteins are encoded by the chloroplast genome of Chlamydomonas reinhardtii.

To characterize envelope proteins encoded by the chloroplast genome, envelopes were isolated from Chlamydomonas reinhardtii cells labeled with [35S] sulfate while blocking synthesis by cytoplasmic ribosomes. One and two-dimensional gel electrophoresis of envelopes and fluorography revealed four highly labeled proteins. Two with masses of 29 and 30 kDa and pI 5.5 were absent from the stroma and thylakoid fractions, while the others at 54 kDa, pI 5.2 and 61 kDa, pI 5.4 were detected there in smaller amounts. The 29- and 30-kDa proteins were associated with outer envelope membranes separated from inner envelope membranes after chloroplast lysis in hypertonic solution. A 32-kDa protein not labeled by [35S]sulfate was found exclusively in the inner membrane fraction, suggesting the existence of a phosphate translocator in C. reinhardtii. To identify envelope proteins exposed on the chloroplast surface, isolated active chloroplasts were surface-labeled with 125I and lactoperoxidase. The 54-kDa, pI 5.2 protein as well as a protein corresponding to either of the 29- or 30-kDa proteins described above were among the labeled components. These results show that envelope proteins of C. reinhardtii are encoded by the chloroplast genome and two are located on the outer envelope membranes.

In Vivo and In Vitro Protein Phosphorylation Studies on Ochromonas danica, an Alga with a Chlorophyll a/c/Fucoxanthin Binding Protein

The phosphorylation of thylakoid membranes in the Chromophyte alga Ochromonas danica was studied in whole cells and in vitro. Protein kinase activity was observed in the thylakoid fraction, and several membrane-bound polypeptides were found to be phosphorylated. The thylakoid protein kinase demonstrated several unusual regulatory properties. Both the polypeptides that were phosphorylated and the rate of protein phosphorylation were independent of illumination. Protein kinase activity was also unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron. The kinase activity was inhibited under strong reducing conditions. Whole cells labeled with (32)PO(4) (3-) were converted to light states I and II by pre-illumination favoring photosystem I or photosystem II, respectively. Analysis of the phosphoproteins from cells in state I and state II showed that no changes in phosphorylation accompanied the change in energy redistribution.

Protein Methylation in Pea Chloroplasts

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with [(3)H-methyl]-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. One is a polypeptide with a molecular mass of 64 kD, a second has an M(r) of 48 kD, and the third has a molecular mass of less than 10 kD. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide, having a molecular mass of 24 kD, is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. One methyl-linkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile [(3)H]methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the [(3)H]methyl group.

Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledons

Cotyledons were excised from imbibed watermelon seeds, grown for 4 days in darkness on water or 10 microM benzyladenine (BA) and then tested for the presence of the light-harvesting chlorophyll a/b protein (LHCP) and its mRNA. LHCP was assayed immunologically by western blotting of SDS gels: the protein was present in plastids, but it was not recovered with the thylakoid fraction. Antibodies directed against LHCP precipitated a 32 kDa polypeptide from translation products of poly(A) RNA of cotyledons only if these had been grown on BA. Taken together the data suggest that in absence of light cytokinins are necessary for the maintenance of a detectable level of LHCP-mRNA as well as for synthesis of the protein.