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PubMed1992

Differential scanning calorimetric investigation of pea chloroplast thylakoids and thylakoid fractions.

High sensitivity differential scanning calorimetry (DSC) was employed to study the thermal denaturation of components of pea chloroplast thylakoid membranes. In contrast to previous reports utilizing spinach thylakoids, several transitions are reversible, and deconvolution of the calorimetric curves indicates nine transitions in both first and second heating scans, but overlapping transitions obscure at least three transitions in the first heating scans of control thylakoids. Glutaraldehyde fixation increases the denaturation temperature of several transitions which is consistent with a reported increase in thermal stability of thylakoid function due to fixation. Acidic pH treatment has little effect on the DSC curves, although it has been reported to have a significant effect on membrane structure. Separation of grana from stroma thylakoids indicates that components responsible for transitions centered at approximately 56, 73, 77, and 91 degrees C are predominantly or exclusively associated with grana thylakoids, whereas components responsible for transitions centered at approximately 63 and 81 degrees C are predominantly associated with stroma thylakoids. A broad transition centered at 66 degrees C is associated with grana thylakoids, whereas a sharp transition at the same temperature is due to a component associated with stroma thylakoids. Evidence obtained by washing treatments suggests the latter transition originates from the denaturation of the thylakoid ATPase (CF1). Analysis of the calorimetric enthalpy values indicates most components of the grana thylakoids denature irreversibly at high temperature, whereas components associated with the stroma thylakoids have a considerable degree of thermal reversibility.

Calorimetry, Differential Scanning↗

PubMed1985

Differentiation and development of bundle sheath and mesophyll thylakoids in maize. Thylakoid polypeptide composition, phosphorylation, and organization of photosystem II.

Photosynthetic electron flow, polypeptide pattern, presence of chlorophyll-protein complexes, and phosphorylation of thylakoid polypeptides have been investigated in differentiated mesophyll (M) and bundle sheath (B) thylakoids of the C4 plant Zea mays. The polypeptide pattern of M thylakoids and their photosynthetic electron flow are comparable to those of other green plants. B thylakoids exhibit only photosystem I (PSI) activity, contain only traces of the PSII light harvesting (LHCII) polypeptide, do not bind [3H] diuron, and lack polypeptides of the water-oxidation complex of PSII and the herbicide binding 32-kDa polypeptide, as detected by specific antibodies. However, B thylakoids possess a partially active PSII reaction center, as demonstrated by light-dependent reduction of silicomolybdate with 1,5-diphenylcarbazide (DPC) as an electron donor, and the presence of the PSII reaction center polypeptides of 44-47 kDa. Only one chlorophyll a-protein complex, corresponding to the PSI reaction center-core antenna, was detectable in B thylakoids, as opposed to chlorophyll a and chlorophyll a,b-protein complexes present in M thylakoids. The light-dependent, membrane-bound kinase activity present in M thylakoids could not be detected in B thylakoids which, nevertheless, contain a protein kinase able to phosphorylate casein. A total of 19 differences between the electrophoretic pattern of B and M thylakoid polypeptides were observed. The mRNA coding for the LHCII polypeptide is primarily, if not exclusively, localized in M cells. The development of PSII complex precedes that of PSI during the differentiation of B and M chloroplasts in expanding leaves of light-grown plants and during the greening of dark-grown etiolated seedlings. The differentiation of the maize leaf into cells programmed to form B or M chloroplasts does not require light. In light-grown plants, the differentiation of B and M thylakoids occurred progressively from the base of the leaf and was completed at 4-5 cm from the leaf base.

Cell Differentiation↗

PubMed1992

Galactolipid export from envelope to thylakoid membranes in intact chloroplasts. I. Characterization and involvement in thylakoid lipid asymmetry.

The galactolipid transfer from inner envelope to thylakoid membranes has been studied in intact spinach chloroplasts. Plastids, isolated from mature leaves, were dark-incubated in the presence of UDP-[14C]galactose. After various synthesis periods at 5 or 25 degrees C, intact plastids were reisolated and osmotically lysed. Thylakoid membranes were then prepared by a special procedure which removed greater than or equal to 99% of the envelope amount initially present. Under these conditions, purified thylakoids were found to contain radiolabelled MGDG and DGDG, indicating that galactolipids were exported from the inner envelope. The amounts exported were proportional to the amounts synthesized. About 55% of the MGDG and 25% of the DGDG synthesized in plastids were transferred to thylakoids, irrespectively of incubation time or temperature. The MGDG/DGDG radioactivity ratio was 7 in intact plastids and 18 in thylakoids, suggesting a preferential export of MGDG. Purified thylakoid membranes were then submitted to a lipolytic treatment designed to discriminate between the MGDG and DGDG pools belonging to the outer (stroma-facing) or to the inner monolayer. The radioactivity present in the lyso-products (corresponding to the outer pools) and in the residual parent lipids (corresponding to the inner pools) was measured. The labelled MGDG showed a transmembrane outside:inside distribution (mol%) of 50:50, which differed from the native (mass) MGDG asymmetry of 64:36. In contrast, the label and mass asymmetries of DGDG gave the same value of 15:85. These label distributions were affected neither by incubation time (from 5 to 90 min) nor by temperature (from 5 to 25 degrees C). We discuss the possibilities that transient fusions between the stroma-facing monolayers of the inner envelope and of the thylakoid membrane, and/or galactolipid transfer protein(s), together with lipid translocating activities in thylakoids, may account for the galactolipid export observed in mature spinach chloroplasts.

Biological Transport↗

PubMed1984

Thylakoid membranes: the translational site of chloroplast DNA-regulated thylakoid polypeptides.

Stromal ribosomes and those bound to thylakoid membranes were prepared from intact spinach chloroplasts which were purified on Percoll gradients. The products of read-out translation of these ribosomes supplemented with an Escherichia coli extract were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Striking similarity was found between the polypeptides labeled in the read-out translation of the chloroplastic ribosomes and those synthesized in isolated chloroplasts. Among the polypeptides translated on thylakoid-bound ribosomes, apoprotein of chlorophyll-protein complex I, alpha and beta subunits of coupling factor 1, and 32,000-Da membrane polypeptide were identified from their mobility on the polyacrylamide gel. The large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and other several stromal proteins were translated exclusively from stromal ribosomes. However, when the translation was programmed in cell-free systems from either E. coli, wheat germ, or rabbit reticulocytes by RNAs isolated separately from stroma and thylakoids, no qualitative difference was found between the products from those RNAs. These results suggest that thylakoid-bound ribosomes are the main sites of synthesis of thylakoid proteins and stromal-free ribosomes are that of stromal proteins, and that thylakoids and stroma contain mRNAs for the stromal and the thylakoid proteins, respectively, in a form not functioning in the chloroplasts.

Binding Sites↗

PubMed1993

Precursors of one integral and five lumenal thylakoid proteins are imported by isolated pea and barley thylakoids: optimisation of in vitro assays.

In vitro assays for the import of proteins by isolated pea thylakoids have been refined and optimised with respect to (a) the method of thylakoid preparation, (b) the concentration of thylakoids in the import assay, and (c) the pH and temperature of the import assay. As a result, the 23 kDa and 16 kDa proteins of the photosynthetic oxygen-evolving complex are imported with efficiencies approaching 100%; import of the third oxygen-evolving complex protein is also observed, albeit with lower efficiencies. We have also demonstrated import of three further thylakoid proteins: plastocyanin, the CFoII subunit of the ATP synthase, and the photosystem I subunit, PSI-N, using this import assay. Import of plastocyanin, PSI-N and the 33 kDa oxygen-evolving complex protein subunit requires the presence of stromal extract whereas the other three proteins are efficiently imported in the absence of added soluble proteins. Import into isolated barley thylakoids was achieved under identical assay conditions, although with somewhat lower efficiency than into pea thylakoids.

Biological Transport↗

PubMed1995

(Galacto) lipid export from envelope to thylakoid membranes in intact chloroplasts. II. A general process with a key role for the envelope in the establishment of lipid asymmetry in thylakoid membranes.

The transfer of organelle of newly synthesized lipid molecules from inner envelope to thylakoid membranes, as well as their subsequent transbilayer distribution in these membranes, have been studied in intact chloroplasts isolated from young and mature spinach, young pea and mature lettuce leaves, using a recently developed methodology (Rawyler, A., Meylan, M. and Siegenthaler, P.A. (1992) Biochim. Biophys. Acta 1104, 331-341). Three radiolabelled precursors were used. UDP-[14C]galactose allowed to follow the fate of mono- and digalactosyldiacylglycerol (MGDG and DGDG) made from polyunsaturated, preexisting diacylglycerol (DAG), whereas [14C]acetate and [14C]glycerol 3-phosphate were used to follow the fate of MGDG and phosphatidylglycerol (PG), respectively, after de novo synthesis. MGDG, DGDG and PG molecules assembled at the envelope level were found to be exportable to thylakoids in amounts strictly proportional to the amounts synthesized, provided that the necessary substrates were not limiting. Lipid export was class-selective; under our conditions, as much as 50-80% of the MGDG, 87% of the PG and 20-30% of the DGDG synthesized were exported to thylakoids. However, within the MGDG class labelled from [14C]acetate, there was hardly any selectivity in the export of its various molecular species. For MGDG, the proportionality coefficient, which reflects the efficiency of the export process, was higher in chloroplasts from young than from mature leaves, and higher in spinach than in pea and lettuce. Temperature affected the efficiency of galactolipid export in a class-dependent way. MGDG synthesis and export had similar Q10 values of about 4 in young and 3 in mature spinach leaves, while the Q10 of DGDG export was higher than that of its synthesis. In most cases, the transmembrane distribution of labelled lipids in thylakoids was found to match closely the corresponding distribution of mass, regardless of plant age and species and of incubation time and temperature. In some cases however, small but significant differences occurred between the label and the mass transbilayer distributions of MGDG (labelled molecules more inwardly oriented), DGDG and PG (more outwardly oriented). We propose a general model in which the thylakoid lipid asymmetry is primarily preestablished in the chloroplast envelope by the topography of its lipid-synthesizing enzymes, together with the occurrence of relatively fast lateral diffusion and translocation rates of the newly synthesized lipids. Transient fusions between inner envelope and thylakoid membranes would allow lipid export by lateral diffusion and build the observed lipid asymmetry in the latter.

Age Factors↗

PubMed1989

ADP-ribosylation of thylakoid membrane polypeptides by cholera toxin is correlated with inhibition of thylakoid GTPase activity and protein phosphorylation.

Incubation of pea thylakoid membranes with [32P]-NAD+ in the presence of cholera toxin resulted in the [32P]-ADP-ribosylation of a 60 kDa thylakoid membrane polypeptide. When ATP was included in the incubation mixture, a 29 kDa polypeptide was also labelled. In the absence of electron transfer cofactors or inhibitors, the extent of labelling depended on whether the membranes were preincubated in the light or dark and also on the developmental stage of the leaves used for thylakoid isolation. Irrespective of the latter, the strongest labelling was observed when DCMU was present in the light. After pretreatment of the thylakoid membranes with cholera toxin plus NAD+ under the same conditions, light-stimulated GTPase activity and protein phosphorylation were inhibited. The extent of inhibition for both processes appeared to be correlated with the amount of [32P]-ADP-ribosylation found when [32P]-NAD+ was included in the pretreatment mixture. The data presented are fully consistent with the 60 and 29 kDa polypeptides functioning as thylakoid membrane associated guanine nucleotide binding regulatory proteins.

Adenosine Diphosphate Ribose↗

PubMed2003

Cytochrome c is reduced mainly by plastoquinol and not by superoxide in thylakoid membranes at low and medium light intensities: its specific interaction with thylakoid membrane lipids.

We have found that, at low light intensity (5-10 micromol photons x m(-2) x s(-1)), photoreduction of cyt (cytochrome) c by isolated thylakoids was not inhibited by dinitrophenylether of iodonitrothymol, an inhibitor of the cyt b6- f complex, and the inhibition was only partial at medium light intensity (50-200 micromol photons x m(-2) x s(-1)). The photoreduction was not significantly influenced by superoxide dismutase. The conclusion that cyt c could be reduced directly by the plastoquinone pool was confirmed by the observation that plastoquinol-9 reduced cyt c efficiently when it was incorporated into liposome membranes prepared from thylakoid membrane lipids. It was shown that the cyt is specifically bound to thylakoid lipid liposomes owing to the presence of negatively charged lipids, phosphatidylglycerol and sulphoquinovosyldiacylglycerol, and the reduction was stimulated by the presence of monogalactosyldiacylglycerol, an inverted micelles-forming lipid, in the membranes, where the cyt c reduction by plastoquinol probably takes place. The results obtained are also discussed in terms of reliability of the method of cyt c photoreduction for determining superoxide production by illuminated thylakoids.

Chloroplasts↗

PubMed1991

Transport of proteins into chloroplasts. Delineation of envelope "transit" and thylakoid "transfer" signals within the pre-sequences of three imported thylakoid lumen proteins.

The targeting of cytosolically synthesized proteins into the thylakoid lumen is mediated by an aminoterminal pre-sequence consisting of an "envelope transit" and a "thylakoid transfer" signal in tandem. We have investigated the structural characteristics of several thylakoid transfer signals by determining the intermediate sites at which the stromal processing peptidase cleaves to remove the transit sequences. Using this approach we have found that the thylakoid transfer signals of Silene pratensis plastocyanin, 23-kDa oxygen-evolving complex protein from wheat, and 33-kDa oxygen-evolving complex protein from wheat, are 25, 39, and 48 residues in length, respectively. All of the transfer signals contain hydrophobic core sequences and a "-3,-1" motif reminiscent of those found in signal sequences, but the amino-terminal regions of the transfer signals of the 23- and 33-kDa proteins are both longer and more highly charged. The net charge of each amino-terminal region of the transfer sequences is +1, including the amino-terminal amino group. In each case, the stromal processing peptidase cleaves immediately after a positively charged residue, but otherwise the cleavage sites exhibit no common elements of either primary or secondary structure.

Amino Acid Sequence↗

PubMed1989

An imported thylakoid protein accumulates in the stroma when insertion into thylakoids is inhibited.

The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP) that is imported into chloroplasts and assembled into thylakoid membranes. Under appropriate conditions, either pLHCP or LHCP will integrate into isolated thylakoids. We have identified two situations that inhibit integration in this assay. Ionophores and uncouplers inhibited integration up to 70%. Carboxyl-terminal truncations of pLHCP also interfered with integration. A 22-residue truncation reduced integration to about 25% of control, whereas a 93 residue truncation completely abolished it. When pLHCP was imported into chloroplasts in the presence of uncouplers or when truncated forms of pLHCP were used, significant amounts of the imported proteins failed to insert into thylakoids and instead accumulated in the aqueous stroma. Accumulation of stromal LHCP occurred at uncoupler concentrations required to dissipate the trans-thylakoid proton electrochemical gradient and was enhanced at reduced levels of ATP. The latter effect may be a secondary consequence of a reduction in ATP-dependent degradation within the stroma. These results indicate that the stroma is an intermediate location in the LHCP assembly pathway and provide the first evidence for a soluble intermediate during biogenesis of a chloroplast membrane protein.

Adenosine Triphosphate↗

PubMed1994

Import of barley photosystem I subunit N into the thylakoid lumen is mediated by a bipartite presequence lacking an intermediate processing site. Role of the delta pH in translocation across the thylakoid membrane.

Translocation across the thylakoid membrane of the recently identified photosystem I polypeptide, PSI-N, has been analyzed in pea (Pisum sativum) and barley (Hordeum vulgare). PSI-N from barley is synthesized in the cytosol with a bipartite presequence similar in structural terms to those of other cytosolically synthesized proteins routed to the thylakoid lumen. In vitro reconstitution assays demonstrate that translocation into thylakoids is absolutely dependent on the transthylakoidal delta pH, but that nucleotide triphosphates are not required; the translocation mechanism is thus similar in these respects to those utilized by the 23- and 16-kDa proteins of the oxygen-evolving complex. The translocation of PSI-N is unique in that the presequence of PSI-N does not contain an intermediate cleavage site for the stromal processing peptidase; important experiments using intact chloroplasts depleted of a delta pH by nigericin treatment demonstrate the accumulation of the full precursor protein in the stroma. Translocation across the thylakoid membrane can take place in the absence of stromal factors, although the presence of stromal extracts leads to a consistent but slight enhancement of translocation efficiency. We also show that efficient translocation of the 33-kDa protein of the oxygen-evolving complex can take place in the complete absence of a delta pH, in apparent contradiction with earlier findings; the translocation of this protein is thus similar in several respects to that of plastocyanin. The data indicate the operation of two very different types of translocation mechanism, with PSI-N exhibiting additional separate characteristics.

Chloroplasts↗

PubMed1998

Implications of a developmental-stage-dependent thylakoid-bound protease in the stabilization of the light-harvesting pigment-protein complex serving photosystem II during thylakoid biogenesis in red kidney bean

Intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II (LHCII) that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.

Journal Article↗

PubMed1980