Nucleotide sequence of cDNA clones encoding the complete precursor for subunit delta of thylakoid-located ATP synthase from spinach

The nucleotide sequence of the entire nuclear-encoded precursor for subunit delta of the ATP synthase from spinach thylakoid membranes was determined by cDNA sequencing. Appropriate recombinant DNAs were selected from pBR322 and lambda gt11 libraries made from polyadenylated RNA of greening spinach seedlings. The mature protein consists of 187 amino acid residues corresponding to a molecular weight of 20468. The precursor protein (257 amino acid residues; M _r=27676) is probably processed between a Met-Val bond. The predicted secondary structure of the transit sequence (70 residues; 7.2 kDa) resembles that of the Rieske Fe/S polypeptide, but shows little similarity with those of stromal or luminal proteins. The comparison of the chloroplast delta amino acid sequence with the published delta sequences from respiratory ATP synthases of bacterial and mitochondrial sources and from the thylakoid ATP synthase of the cyanobacterium Synechococcus suggests substantial divergence at the genic level although structural elements appear to be remarkably conserved.     

Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide

Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic ribosomes: the small subunit of ribulose 1,5-bisphosphate carboxylase and the apoprotein components of the chlorophyll a/b light harvesting complex. We have recently reported the isolation of two cDNA clones from pea which encode both the small subunit of ribulose 1,5-bisphosphate carboxylase (pSS15) and the polypeptide 15 (pAB96), the major chlorophyll a/b binding protein (Broglie, R., Bellemare, G., Bartlett, S., Chua, N.-H., and Cashmore, A. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7304-7308). To further characterize these clones, we determined their nucleotide sequence. Clone pSS15 contains a 691-base pair cDNA insert which encodes the entire 123 amino acids of the mature small subunit protein. In addition, this clone also encodes 33 amino acids of the NH2-terminal transit peptide extension and 148 nucleotides of the 3' noncoding region preceding the poly(A)tail. A second cDNA clone (pAB96) contains an 833-nucleotide insert which encodes most of polypeptide 15. The DNA sequence of this cloned cDNA was used to deduce the previously undetermined amino acid sequence of this integral thylakoid membrane protein. The nucleotide sequence of the cDNA clone, pSS15, should provide information concerning the role of the transit sequence in the transport of cytoplasmically synthesized chloroplast proteins. Similarly, the deduced amino acid sequence of polypeptide 15 will provide information for predicting its orientation in thylakoid membranes as well as its role in binding chlorophyll.     

The amino acid sequence of the polypeptide segment which regulates membrane adhesion (grana stacking) in chloroplasts

A positively charged amino acid sequence, located on the NH2 terminus of the polypeptides of the chlorophyll a/b light harvesting complex, stabilizes thylakoid membrane adhesion. Threonine residues in this segment are the site of light-induced, reversible phosphorylation; this covalent modification results in changes in excitation-energy distribution in chloroplast membranes. Removal of the positively charged peptide by treatment with trypsin or chemical modification of amino acids in the sequence disrupts thylakoid adhesion and inhibits regulation of excitation-energy distribution. Purified preparations of the chlorophyll a/b light harvesting complex consist of 2 major polypeptides of 27 and 26 kDa and 2 minor polypeptides of 29 and 25 kDa (based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Trypsin treatment of the isolated chlorophyll proteins decreases the apparent molecular mass of the 27- and 26-kDa polypeptides by 1-1.5 kDa and releases 3 peptides; [Lys, Arg], Ser-Ala-Thr-Thr-Lys-Lys, and Ser-Ala-Thr-Thr-Lys. These peptides probably form the overlap sequence, [Lys, Arg]-Ser-Ala-Thr-Thr-Lys-Lys. The polypeptides of the chlorophyll a/b light-harvesting complex were separated by isoelectric focusing into 5 chlorophyll protein fractions which had isoelectric points between 4.0 and 4.55. The 27-kDa polypeptides had an isoelectric point of 4.3, and bound 11 chlorophyll molecules/polypeptide.     

Structure and Function of Chloroplast Membrane XI. The Effects of Linolenic Acid on the Structure and the Absorption and Fluorescence Spectra of Wheat Chloroplast Membranes as well as Regulations by MgCl2

The effects of various concentrations of linolenic acid on the structure and the absorption and fluorescence spectra of wheat chloroplast membranes were studied. Linolenic acid increases the absorption peaks in both red and blue regions of the chloroplast membranes. The degree of increase in the absorption peaks is proportional to the concentration within a range of concentration. Linolenic acid increased the fluorescence yield of F685 and F738 of chloroplast membranes. Electron microscopical studies revealed that the increases were mainly due to the disappearance of grana stacks and the unfolding of thylakoid membranes. The causes of effects of linolenic acid on the absorption and fluorescence spectra and the structure of chloroplast membranes as well as the reversion and regulation by MgCl2 were discussed.     

MFP1 is a thylakoid-associated,nucleoid-binding protein with a coiled-coil structure

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.     

RT-PCR克隆籼稻叶绿素a/b结合蛋白基因全长cDNA及序列的in silico分析

根据日本晴cab4基因序列(GenBank:AK104499.1)设计引物,用RT-PCR的方法从籼稻9311中克隆了叶绿素a/b结合蛋白基因的全长cDNA,命名为cab-9311(cab gene from 9311)。insilico分析表明:cab-9311与cab4基因同源性为99%,编码的蛋白含有244个氨基酸,与cab4基因编码的蛋白同源性为98%。蛋白分子质量为26.9kD,理论等电点为6.52。第54位～第216位氨基酸是一个典型的叶绿素a/b结合蛋白功能域(chlorophyll a/bbinding domain)。跨膜分析和蛋白质三级预测显示,该蛋白在C端有一个典型的跨膜区。亚细胞定位分析表明该蛋白定位于叶绿体,是一个叶绿体内囊体膜上的锚定蛋白。     

Involvement of a chloroplast homologue of the signal recognition particle receptor protein, FtsY, in protein targeting to thylakoids

We isolated an Arabidopsis thaliana cDNA whose translated product shows sequence similarity to the FtsY, a bacterial homologue of SRP receptor protein. The Arabidopsis FtsY homologue contains a typical chloroplast transit peptide. The in vitro-synthesized 37 kDa FtsY homologue was imported into chloroplasts, and the processed 32 kDa polypeptide bound peripherally on the outer surface of thylakoids. Antibodies raised against the FtsY homologue also reacted with a thylakoid-bound 32 kDa protein. The antibodies inhibited the cpSRP-dependent insertion of the light-harvesting chlorophyll alb-binding protein into thylakoid membranes suggesting that the chloroplast FtsY homologue is involved in the cpSRP-dependent protein targeting to the thylakoid membranes.     

Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

Various chimeric precursors and deletions of the 33 kd oxygen-evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase, a stromal protein, resulted in redirection of mature OEE1 into the stromal compartment of the chloroplast. Utilizing chimeric transit peptides and block deletions we demonstrated that the 85 residue OEE1 transit peptide contains separate signal domains for importing and targeting the thylakoid lumen. The importing domain, which mediates translocation across the two membranes of the chloroplast envelope, is present in the N-terminal 58 amino acids. The thylakoid lumen targeting domain, which mediates translocation across the thylakoid membrane, is located within the C-terminal 27 residues of the OEE1 transit peptide. Chimeric precursors were constructed and used in in vitro import experiments to demonstrate that the OEE1 transit peptide is capable of importing and targeting foreign proteins to the thylakoid lumen.     

Changes in fatty acid composition of lipids in chloroplast membranes of tobacco plants during cold hardening

Changes in fatty acid composition of chloroplast membrane lipids were investigated using tobacco (Nicotiana tabacum L., cv. Samsun) plants subjected to cold hardening for 6 days at 8°C. Under optimal growing temperature (22°C), the lipids of thylakoid membranes were characterized by elevated content of 16:3n-3 and 18:3n-3 fatty acids (FA). Compared to the lipids of chloroplast envelope membranes, the thylakoid lipids were less rich in the content of saturated, mono- and diunsaturated FA. The relative content of unsaturated FA in chloroplast membranes increased substantially during cold hardening, which was mainly due to the accumulation of 18:3n-3 FA. It is concluded that the observed changes in FA composition of chloroplast lipids during cold hardening adjust the fluidity of these membranes to the level sufficient for functioning of tobacco photosynthetic apparatus, which is a prerequisite for accumulation of assimilates and allows the hardened tobacco plants to survive under conditions of hypothermia.     

Chloroplast lipid synthesis and lipid trafficking through ER-plastid membrane contact sites

Plant chloroplasts contain an intricate photosynthetic membrane system, the thylakoids, and are surrounded by two envelope membranes at which thylakoid lipids are assembled. The glycoglycerolipids mono- and digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol as well as phosphatidylglycerol, are present in thylakoid membranes, giving them a unique composition. Fatty acids are synthesized in the chloroplast and are either directly assembled into thylakoid lipids at the envelope membranes or exported to the ER (endoplasmic reticulum) for extraplastidic lipid assembly. A fraction of lipid precursors is reimported into the chloroplast for the synthesis of thylakoid lipids. Thus polar lipid assembly in plants requires tight co-ordination between the chloroplast and the ER and necessitates inter-organelle lipid trafficking. In the present paper, we discuss the current knowledge of the export of fatty acids from the chloroplast and the import of chloroplast lipid precursors assembled at the ER. Direct membrane contact sites between the ER and the chloroplast outer envelopes are discussed as possible conduits for lipid transfer.     

Isolation and characterization of oxygen-evolving thylakoid membranes and photosystem II particles from a glaucocystophyte, Cyanophora paradoxa

Conditions for preparing oxygen-evolving thylakoid membranes and PSII complexes, and those for observing the PSII activity were investigated in a glaucocystophyte, Cyanophora paradoxa. The active thylakoid membranes were isolated either with a medium containing glycerol or with that containing high concentrations of sucrose, phosphate, and citrate. Active PSII particles were solubilized by octyl-beta-D-glucoside from thylakoid membranes and were separated by sucrose density gradient centrifugation. The thylakoid membranes and PSII particles showed an oxygen-evolving activity only in high-ionic-strength media. The extrinsic 33 kDa protein (PsbO) and the cytochrome c(550) (PsbV) were found to be present in the PSII particles as in cyanobacteria or red algae, but no 12 kDa protein (PsbU) was detected. The PsbO protein was classified as a land-plant type by its N-terminal amino acid sequence.     

Nucleus-encoded precursors to thylakoid lumen proteins of Euglena gracilis possess tripartite presequences.

The complete presequences of the nucleus-encoded precursors to two proteins, cytochrome c6 and the 30-kDa protein of the oxygen-evolving complex, that reside in the thylakoid lumen of the chloroplasts of Euglena gracilis are presented. Sorting of these proteins involves translocation across four membranes, the three-membraned chloroplast envelope and the thylakoid membrane. The tripartite presequences show the structure: signal sequence transit sequence signal sequence. Three hydrophobic domains become apparent: two of them correspond to signal sequences for translocation across the endoplasmic reticulum (ER) membrane and the thylakoid membrane, respectively, whereas the third constitutes the stop-transfer signal contained in the long stroma-targeting part of the tripartite presequence.     

Growth temperature effects on thylakoid membrane lipid and protein content of pea chloroplasts

The lipid composition and level of unsaturation of fatty acids has been determined for chloroplast thylakoid membranes isolated from Pisum sativum grown under cold (4°/7°C) or warm (14°/17°C) conditions. Both the relative amounts of lipid classes and degree of saturation were not greatly changed for the two growth conditions. In cold-grown plants, there was a slightly higher linolenic and lower linoleic acid content for the glycolipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol. In contrast to thylakoid membranes, a non-thylakoid leaf membrane fraction including the chloroplast envelope, had a higher overall level of fatty acid unsaturation in cold-grown plants due mainly to an increase in the linolenic acid content of MGDG, DGDG, phosphatidylglycerol, and phosphatidylcholine. The most clear cut change in the thylakoid membrane composition was the lipid to protein ratio which was higher in the cold-grown plants.     

Transit peptides of nuclear-encoded chloroplast proteins share a common amino acid framework.

We have identified three major blocks of amino acid homology shared by the transit peptides of two nuclear-encoded chloroplast proteins, the light-harvesting chlorophyll a/b-protein (LHCP) II of the thylakoid membrane and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of the stroma. These previously unrecognized homology blocks lie at the beginning, middle and end of both transit sequences, and are separated by differing lengths of unshared (interblock) sequence in the two proteins. These interblocks may be dispensible or they might confer a specific property on the individual proteins, such as facilitating proper compartmentalization within the chloroplast. We propose that these three shared sequence elements form a common framework in transit-bearing chloroplast precursors which mediates the common functions performed by each transit peptide. Ferredoxin, the only other such nuclear-encoded protein for which a published transit sequence exists, conforms to the predictions of this hypothesis. These findings stand in contrast to mitochondrial leader sequences and the well-studied signal peptides of secretory and certain integral membrane proteins in which no such framework has been observed.     

The carboxyl terminus of the epsilon subunit of the chloroplast ATP synthase is exposed during illumination

The epsilon subunit of the chloroplast ATP synthase is an inhibitor of activity of the enzyme. Recombinant forms of the epsilon subunit from spinach chloroplasts lacking the last 10, 32, or 45 amino acids were immobilized onto activated Sepharose. A polyclonal antiserum raised against the epsilon subunit was passed over these immobilized protein columns, and the purified antibodies which were not bound recognized the portions of the epsilon subunit missing from the recombinant form present on the column. The full polyclonal antiserum can strip the epsilon subunit from the ATP synthase in illuminated thylakoid membranes [Richter, M. L., and McCarty, R. E. (1987) J. Biol. Chem. 262, 15037-15040]. Exposure of illuminated thylakoid membranes to antibodies recognizing the last 32 amino acids of the epsilon subunit collapses the proton gradient and hinders ATP synthesis with similar efficiency as the full polyclonal preparation. These results indicate that antibodies against the last 32 amino acids of the epsilon subunit are capable of stripping the subunit from the ATP synthase in illuminated membranes. Neither of these effects was seen when the membranes were exposed to the antibodies in the dark. This is direct evidence that the chloroplast ATP synthase undergoes a conformational shift during its activation by the electrochemical proton gradient which specifically alters the conformation of the carboxyl-terminal domain of the epsilon subunit from protected to solvent-exposed. The relation between this shift and activation of the enzyme by the electrochemical proton gradient is discussed.     

The nuclear-coded chloroplast 22-kDa heat-shock protein of Chlamydomonas. Evidence for translocation into the organelle without a processing step

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.     

Localization and targeting of the VP14 epoxy-carotenoid dioxygenase to chloroplast membranes

Abscisic acid (ABA) is a key regulator of seed dormancy and plant responses to environmental challenges. ABA is synthesized via an oxidative cleavage of 9-cis epoxy-carotenoids, the first committed and key regulatory step in the ABA biosynthetic pathway. Vp14 of maize encodes an epoxy-carotenoid dioxygenase that is soluble when expressed in E. coli. An important goal has been to determine how the soluble VP14 protein is targeted to epoxy-carotenoid substrates that are located in the thylakoid and envelope membranes of chloroplasts and other plastids. Using an in vitro chloroplast import assay, we have shown that VP14 is imported into chloroplasts with cleavage of a short stroma-targeting domain. The mature VP14 exists in two forms, one which is soluble in stroma and the other bound to thylakoid membranes. Analysis of a series of truncated VP14 mutants mapped the membrane targeting signal to the 160 amino acid N-terminal sequence. A putative amphipathic alpha-helix within this region is essential, but not sufficient, for the membrane targeting. Either deletion of or insertion of helix breaking residues into this region abolished the membrane binding, whereas a chimeric protein carrying just the amphipathic region fused with bacterial glutathione S-transferase failed to associate with the thylakoid membrane. The membrane-bound VP14 was partially resistant to chaotropic washes such as 0.1 M Na2CO3 (pH 11.5) and 6 M urea. Unlabelled recombinant VP14 inhibited the tight binding of imported VP14, suggesting that VP14 is associated with specific components of the thylakoid membrane.     

Photosynthetic oxygen evolution is stabilized by cytochrome c550 against heat inactivation in Synechococcus sp. PCC 7002.

We investigated the factors responsible for the heat stability of photosynthetic oxygen evolution by examining thylakoid membranes from the cyanobacterium Synechococcus sp. PCC 7002. We found that treatment of the thylakoid membranes with 0.1% Triton X-100 resulted in a remarkable decrease in the heat stability of oxygen evolution, and that the heat stability could be restored by reconstituting the membranes with the components that had been extracted by Triton X-100. The protein responsible for the restoration of heat stability was purified from the Triton X-100 extract by two successive steps of chromatography. The purified protein had a molecular mass of 16 kD and exhibited the spectrophotometric properties of a c-type Cyt with a low redox potential. The dithionite-minus-ascorbate difference spectrum revealed an alpha band maximum at 551 nm. We were able to clone and sequence the gene encoding this Cyt from Synechococcus sp. PCC 7002, based on the partial amino-terminal amino acid sequence. The deduced amino acid sequence revealed a gene product consisting of a 34-residue transit peptide and a mature protein of 136 residues. The mature protein is homologous to Cyt c550, a Cyt with a low redox potential. Thus, our results indicate that Cyt c550 greatly affects the heat stability of oxygen evolution.