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PubMed2004

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.

Cell Fractionation↗

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↗

PubMed2004

Deletion of the chloroplast-localized Thylakoid formation1 gene product in Arabidopsis leads to deficient thylakoid formation and variegated leaves.

Development of thylakoid membranes depends upon the transport of membrane vesicles from the chloroplast inner envelope and subsequent fusion of vesicles within the interior of the plastid. The Arabidopsis (Arabidopsis thaliana) Thylakoid formation1 (Thf1) gene product is shown here to control an important step required for the normal organization of these vesicles into mature thylakoid stacks and ultimately for leaf development. The Arabidopsis Thf1 gene encodes an imported chloroplast protein, as shown by in vitro import and localization of a Thf1-green fluorescent protein fusion product in transgenic plants. This gene is conserved in oxygenic photoautotrophs ranging from cyanobacteria to flowering land plants. Transcript levels for Thf1 are induced in the light and decrease under dark conditions, paralleling profiles of light-regulated nuclear genes involved in chloroplast function. Disruption of the Thf1 gene via T-DNA insertion results in plants that are severely stunted with variegated leaf patterns. Nongreen sectors of variegated leaves lacking Thf1 expression contain plastids that accumulate membrane vesicles on the interior and lack organized thylakoid structures. Green sectors of Thf1-disrupted leaves contain some chloroplasts that form organized thylakoid membranes, indicating that an inefficient compensatory mechanism supports thylakoid formation in the absence of Thf1. Genetic complementation of a Thf1 knockout line confirms the role of this gene in chloroplast and leaf development. Transgenic plants expressing the Thf1 gene in antisense orientation are stunted with altered thylakoid organization, especially in young seedlings. The data indicate that the Thf1 gene product plays a crucial role in a dynamic process of vesicle-mediated thylakoid membrane biogenesis.

Amino Acid Sequence↗

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↗

PubMed1988

Development of Oat Prothylakoids into Thylakoids during Greening Does Not Change Transmembrane Galactolipid Asymmetry but Preserves the Thylakoid Bilayer.

The lipase from Rhizopus arrhizus and the lipolytic acyl hydrolase from potato tubers have been used to determine the transmembrane distribution of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) in prothylakoids and thylakoids from oat (Avena sativa). Both galactolipids were found to be asymmetrically distributed. The molar outside/inside distribution was 70 +/- 8/30 +/- 8 for MGDG and 10 +/- 4/90 +/- 4 for DGDG in the prothylakoid membrane. Mature thylakoids presented a similar distribution, i.e. 63 +/- 4/37 +/- 4 for MGDG and 12 +/- 3/88 +/- 3 for DGDG. This distribution has been assessed under a variety of different conditions, namely (a) in media favoring thylakoid stacking or unstacking and inducing various membrane surface potentials, (b) in the presence of defatted bovine serum albumin which removed free fatty acids and partially lyso-galactolipids, (c) under various temperature conditions which resulted in different hydrolysis rates and degrees of fluidity of the membrane, and (d) in the presence of different enzyme concentrations which influenced the hydrolysis rate. The above distribution was found to be independent of the type of conditions used. Nonbilayer forming/bilayer forming lipid ratios suggest that both monolayers of the prothylakoid and the inner monolayer of oat thylakoid membranes should display lamellar structures (e.g. ratios <2.5). In contrast the outer monolayer of the thylakoid membrane should display non-lamellar configurations (e.g. ratio >2.5). Thus, it is concluded that the incorporation of chlorophyll-protein complexes into the nascent thylakoid membrane modifies neither the galactolipid nor the phospholipid transmembrane distribution. However, these complexes appear to be crucial to preserve a bilayer configuration to the greening membrane which, otherwise, would adopt nonlamellar structures. The possible origin of galactolipid transversal asymmetry which appears very early during the biogenesis of oat thylakoid membranes is discussed.

Journal Article↗

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↗

PubMed2006

HCF164 receives reducing equivalents from stromal thioredoxin across the thylakoid membrane and mediates reduction of target proteins in the thylakoid lumen.

HCF164 is a membrane-anchored thioredoxin-like protein known to be indispensable for assembly of cytochrome b6 f in the thylakoid membranes. In this study, we report the finding that chloroplast stroma m-type thioredoxin is the source of reducing equivalents for reduction of HCF164 in the thylakoid lumen, providing strong evidence that higher plant chloroplasts possess a trans-membrane reducing equivalent transfer system similar to that found in bacteria. To probe the function of HCF164 in the lumen, a screen to identify the reducing equivalent acceptor proteins of HCF164 was carried out by using a resin-immobilized HCF164 single cysteine mutant, leading to the isolation of putative target thylakoid proteins. Among the newly identified target proteins, the reduction of the PSI-N subunit of photosystem I by HCF164 was confirmed both in vitro and in isolated thylakoids. Two components of the cytochrome b6 f complex, the cytochrome f and Rieske FeS proteins, were also identified as novel potential target proteins. The data presented here suggest that HCF164 serves as an important transducer of reducing equivalents to proteins in the thylakoid lumen.

Arabidopsis↗

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↗

PubMed1982

A M(r) 95,000 polypeptide in Porphyridium cruentum phycobilisomes and thylakoids: Possible function in linkage of phycobilisomes to thylakoids and in energy transfer.

Two pigmented polypeptides with the same molecular weight (M(r) 95,000) were isolated from the photosynthetic apparatus of Porphyridium cruentum by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A blue polypeptide from phycobilisomes had absorption and fluorescence emission spectra similar to those of allophycocyanin. A green-pigmented polypeptide from photosynthetic membranes (free of phycobilisomes) contained chlorophyll a. Several properties were common to the M(r) 95,000 polypeptides from both sources: (i) identical molecular weights, (ii) identical gel electrophoresis patterns after limited protease digestion, and (iii) immunological crossreactivity with an IgG fraction directed against the M(r) 95,000 polypeptide from phycobilisomes. On the basis of this evidence, a common polypeptide exists in phycobilisomes and thylakoids, and it probably anchors the phycobilisome to the thylakoid membrane. The fluorescence emission overlap of the blue and green polypeptides suggests that they are involved in the transfer of energy from phycobilisomes to thylakoids.

Journal Article↗

PubMed2004

On why thylakoids energize ATP formation using either delocalized or localized proton gradients - a ca(2+) mediated role in thylakoid stress responses.

By the early 1970s, the chemiosmotic hypothesis of Peter Mitchell was widely accepted by bioenergetics researchers as the best conceptual scheme to explain how ATP is formed in oxidative and photosynthetic phosphorylation. At about the same time, however, work from a few laboratories suggested that some aspects of that elegant, relatively simple hypothesis required revision - not abandonment, but refinement to accommodate more complex movements of protons in the ATP formation mechanism than originally envisioned by Peter Mitchell. In some situations it appeared that protons were constrained to localized domains rather than always delocalized within an enclosed vesicle as envisioned by chemiosmosis. This minireview tells that story from my perspective, as one of the researchers involved in the experimental approaches that revealed more complex energy coupling proton flux patterns. Ionic conditions during isolated thylakoid storage were found to reversibly switch the [Formula: see text] gradient driving ATP formation between delocalized and localized energy coupling modes. Thylakoid accessible Ca(2+) ions proved to be the switching factor that was responding to the ionic conditions in the storage treatment. The mechanism of Ca(2+) was at least partially demystified when it was shown that the reversible switching between [Formula: see text] energy coupling modes involved Ca(2+) interactions with the 8 kDa CF(0) (the H(+) channel) subunit in a type of H(+) flux gating action. Other experiments showed that the Ca(2+) gating of H(+) flux into the lumen may be a critical regulatory factor in controlling the lumen pH and thereby help regulate the activity of the violaxanthin de-epoxidase enzyme, a key part of the chloroplast photoprotective response to over-energization (excess light) stress.

Journal Article↗

PubMed1989

Galactolipids of Thylakoid Pigment Protein Complexes Separated Electrophoretically from Thylakoids of Dunaliella salina Labeled with Radioactive Fatty Acids.

Experiments have been conducted to determine whether mono- and digalactosyldiacylglycerols (MGDG and DGDG) labeled by short term exposure of the green alga Dunaliella salina to exogenous radioactive lauric, myristic, or palmitic acids are preferentially recovered in one or more thylakoid protein complexes separated by electrophoresis on nondenaturing gels. A selective retention of certain lipid classes and molecular species by the major electrophoretically separable fractions, oligomeric form (LHCP-1) and monomeric form (LHCP-3) of hight-harvesting chlorophyll a/b protein complex, and major photosystem I protein complex (CPla), and nonprotein-bound lipids, was indicated. After 5 min of labeling with (3)H-palmitate the specific radioactivities of MGDG and DGDG molecular species recovered from LHCP-3 and CPla were slightly higher and lower, respectively, than specific radioactivities of equivalent molecular species from LHCP-1 and nonprotein-bound lipid. However, the differences were smaller than those which have been observed between whole thylakoids and the more highly labeled chloroplast envelopes from briefly labeled cells.

Journal Article↗