Analysis of a number of PSII complexes detectable in the wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803 showed that the PsbH protein is present in the complexes containing CP47, including unassembled CP47. In a mutant lacking CP47, in which the PSII assembly is stopped at the level of the D1-D2-cytochrome b-559 reaction centre complex, a negligible amount of the PsbH protein was not bound to this complex but was detected in the free form. The results indicate that the PsbH protein has a high affinity for CP47 and during PSII assembly most probably first associates with CP47 and this pair is subsequently attached to the reaction centre complex. Similarly to CP47, the PsbH protein exhibits a slow light-induced degradation in the presence of protein synthesis inhibitor. The absence of the PsbH protein leads to a greatly increased D1 pool that is not associated with other PSII proteins or it is present as a part of the reaction centre complex. We conclude that PsbH is important for the prompt incorporation of the newly synthesized D1 protein into PSH complexes and for the fast D1 maturation.

Anabaena is a model to analyze the evolutionary development of plastids, cell differentiation, and the regulation of nitrogen fixation. Thereby, the outer membrane proteome is the place of sensing environmental differences and during plastid development, systems for intracellular communication had to be added to the proteome of this membrane. We present a protocol for the isolation of the outer membrane from Anabaena and the analysis of the proteome using different tools. 55 proteins were identified.

Vipp1 (vesicle-inducing protein in plastids 1) is found in Cyanobacteria and chloroplasts of photosynthetic eukaryotes where it is essential for the formation of the thylakoid membrane. Vipp1 is closely related to the phage shock protein A (PspA), a bacterial protein induced under diverse stress conditions. Vipp1 proteins differ from PspA by an additional C-terminal domain that is required for Vipp1 function in thylakoid biogenesis. We show here that in Cyanobacteria, green algae, and vascular plants, Vipp1 is part of a high molecular mass complex. The complex is formed by multiple copies of Vipp1, and complex formation involves interaction of the central alpha-helical domain that is common to Vipp1 as well as to PspA proteins. In chloroplasts of vascular plants, the Vipp1 complex can be visualized by green fluorescent protein fusion in discrete locations at the inner envelope. Green fluorescent protein fusion analysis furthermore revealed that complex formation is important for proper positioning of Vipp1 at the inner envelope of chloroplasts.

Accumulation of monomer and dimer photosystem (PS) II reaction center core complexes has been analyzed by two-dimensional Blue-native/SDS-PAGE in Synechocystis PCC 6803 wild type and in mutant strains lacking genes psbA, psbB, psbC, psbDIC/DII, or the psbEFLJ operon. In vivo pulse-chase radiolabeling experiments revealed that mutant cells assembled PSII precomplexes only. In ΔpsbC and ΔpsbB, assembly of reaction center cores lacking CP43 and reaction center complexes was detected, respectively. In ΔpsbA, protein subunits CP43, CP47, D2, and cytochrome b559 were synthesized, but proteins did not assemble. Similarly, in ΔpsbD/C lacking D2, and CP43, the de novo synthesized proteins D1, CP47, and cytochrome b559 did not form any mutual complexes, indicating that assembly of the reaction center complex is a prerequisite for assembly with core subunits CP47 and CP43. Finally, although CP43 and CP47 accumulated in ΔpsbEFLJ, D2 was neither expressed nor accumulated. We, furthermore, show that the amount of D2 is high in the strain lacking D1, whereas the amount of D1 is low in the strain lacking D2. We conclude that expression of the psbEFLJ operon is a prerequisite for D2 accumulation that is the key regulatory step for D1 accumulation and consecutive assembly of the PSII reaction center complex.

Alb3 homologs Oxa1 and YidC have been shown to be required for the integration of newly synthesized proteins into membranes. Here, we show that although Alb3.1p is not required for integration of the plastid-encoded photosystem II core subunit D1 into the thylakoid membrane of Chlamydomonas reinhardtii, the insertion of D1 into functional photosystem II complexes is retarded in the Alb3.1 deletion mutant ac29. Alb3.1p is associated with D1 upon its insertion into the membrane, indicating that Alb3.1p is essential for the efficient assembly of photosystem II. Furthermore, levels of nucleus-encoded light-harvesting proteins are vastly reduced in ac29; however, the remaining antenna systems are still connected to photosystem II reaction centers. Thus, Alb3.1p has a dual function and is required for the accumulation of both nucleus- and plastid-encoded protein subunits in photosynthetic complexes of C. reinhardtii.

In the postgenomic era, the transformation of genetic information into biochemical meaning is required. We have analyzed the proteome of the chloroplast outer envelope membrane by an in silico and a proteomic approach. Based on its evolutionary relation to the outer membrane of Gram-negative bacteria, the outer envelope membrane should contain a large number of beta-barrel proteins. We therefore calculated the probability for the existence of beta-sheet, beta-barrel, and hairpin structures among all proteins of the Arabidopsis thaliana genome. According to the existence of these structures, a number of candidates were selected. This protein pool was analyzed by TargetP to discard sequences with signals that would direct the protein to other organelles different from chloroplasts. In addition, the pool was manually controlled for the presence of proteins known to function outside of the chloroplast envelope. The approach developed here can be used to predict the topology of beta-barrel proteins. For the proteomic approach, proteins of highly purified outer envelope membranes of chloroplasts from Pisum sativum were analyzed by ESI-MS/MS mass spectrometry. In addition to the known components, four new proteins of the outer envelope membranes were identified in this study.

Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis. Using these methods, we were able to select single chloroplasts of Nicotiana tabacum. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube. Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast. The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.

Hcf136 encodes a hydrophilic protein localized in the lumen of stroma thylakoids. Its mutational inactivation in Arabidopsis thaliana results in a photosystem II (PHII)-less phenotype. Under standard illumination, PSII is not detectable and the amount of photosystem I (PSI) is reduced, which implies that HCF136p may be required for photosystem biogenesis in general. However, at low light, a comparison of mutants with defects in PSII, PSI, and the cytochrome b(6)f complex reveals that HCF136p regulates selectively biogenesis of PSII. We demonstrate by in vivo radiolabeling of hcf136 that biogenesis of the reaction center (RC) of PSII is blocked. Gel blot analysis and affinity chromatography of solubilized thylakoid membranes suggest that HCF136p associates with a PSII precomplex containing at least D2 and cytochrome b(559). We conclude that HCF136p is essential for assembly of the RC of PSII and discuss its function as a chaperone-like assembly factor.

Summary Root cell wall proteins involved in the incompatible interaction between soybean (Glycine max L.) and the phytopathogenic oomycete Phytophthora sojae were investigated by a proteomic approach using ESI-MS/MS analysis. Success of infection and subsequently induced defence reactions was proven by staining the cell wall localized defence-related 1,3-beta-glucan, callose, in semi-thin sections of the roots. Cell wall proteins of roots were extracted three weeks post-infection, separated by SDS-PAGE and eight selected protein bands were digested with trypsin. The resulting peptides were subjected to mass spectrometry-based sequencing. For four proteins, amino acid sequence information was obtained and used to identify the corresponding proteins by a homology search in databases. All four proteins (cationic peroxidase, peroxidase precursor, amine oxidase and lipoxygenase) were defence-related and their involvement in the oxidative burst is discussed. Three of these proteins have not been described for soybean before.

Protein export systems derived from prokaryotes are used to transport proteins into or across the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoid membrane. Signal recognition particle (SRP) and its receptor are essential components used exclusively for cotranslational export of endomembrane and secretory proteins to the endoplasmic reticulum in eukaryotes and export of polytopic membrane proteins to the cytoplasmic membrane in prokaryotes. An organellar SRP in chloroplasts (cpSRP) participates in cotranslational targeting of chloroplast synthesized integral thylakoid proteins. Remarkably, cpSRP is also used to posttranslationally localize a subset of nuclear encoded thylakoid proteins. Recent work has begun to reveal the basis for cpSRP's unique ability to function in co- and posttranslational protein localization, yet much is left to question. This review will attempt to highlight these advances and will also focus on the role of other soluble and membrane components that are part of this novel organellar SRP targeting pathway.

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit, PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ, Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b(559). Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in etioplasts. We therefore conclude that in etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a-triggered accumulation of reaction center monomers.

Translation of the large subunit of ribulose-1,5-bisphosphate carboxylase (LSU) was investigated by labeling of isolated barley plastids with [35S]-methionine. In both chloroplasts and etioplasts, labeling of LSU was severely impaired if plastid membranes were removed from the reaction mixtures. Removal of membrane-bound polysomes with high salt or puromycin greatly decreased translation of LSU. Pulse-labeled chloroplast membranes were shown to release LSU if chased with unlabeled methionine in the presence of stroma. Immunoprecipitation detected higher amounts of labeled LSU translation intermediates associated with the membrane fraction than in the soluble fraction. We therefore conclude that, in plastids, membrane-bound polysomes are required not only for translation of membrane-intrinsic proteins but also for translation of a soluble protein.

Intact and lysed chloroplasts isolated from the day or night phase of seedling growth exhibit a higher rate of [35S]Met incorporation into the D1 protein in the light than in darkness. In the presence of the translation initiation inhibitor lincomycin, radiolabel incorporation remains unaffected for 7.5-10 min of the in vitro translation reaction, indicating that radiolabel incorporation is regulated by translation elongation. The rate of [35S]Met incorporation into D1-protein can be increased by addition of exogenous ATP to the in vitro translation reactions; however, ATP cannot replace light, and at physiological concentrations of stromal ATP (40 microM), the rate is at least 25-fold higher in the light than in darkness. This indicates that translation elongation is arrested in darkness. Separation of translation-elongation reactions into polysome-bound and membrane-integrated D1 proteins demonstrates that the rate of translation elongation is higher in the presence of light. In the light, less time is required to transiently radiolabel a D1 translation intermediate of about 17 kDa and to chase the translation intermediate into mature D1 protein. We propose that light regulates the enzymatic activity of the translation-elongation process in chloroplasts.

Upon transfer of lysed chloroplasts from darkness to light, the accumulation of membrane and stromal chloroplast proteins is strictly regulated at the level of translation elongation. In darkness, translation elongation is retarded even in the presence of exogenously added ATP and dithiothreitol. In the light, addition of the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea inhibits translation elongation even in the presence of ATP. This inhibition can be overcome by addition of artificial electron donors in the presence of light, but not in darkness. Electron flow between photo-system II and I induced by far red light of 730 nm is sufficient for the activation of translation elongation. This activation can also be obtained by electron donors to photosystem I, which transport protons into the thylakoid lumen, Release of the proton gradient by uncouplers prevents the Light-dependent activation of translation elongation. Also, the induction of translation activation is switched off rapidly upon transfer from light to darkness. Hence, we propose that the formation of a photosynthetic proton gradient across the thylakoid membrane activates translation elongation in chloroplasts.

Light-harvesting chlorophylla/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-pheophytin a/b, synthesizedin situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Zn-pheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Zn-pheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll- or Zn-pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll- or Zn-pheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.

Methods for the cryopreservation of protein import and integration in pea chloroplasts and of protein import or protein synthesis in tobacco mitochondria were modified to yield enzymatically active cryopreserved etioplasts from barley (Hordeum vulgare L.). The cryoprotectants ethylene glycol and dimethy sulfoxide were about 64 and 77% effective, respectively, for the cryopreservation of etioplast intactness. Phototransformation of protochlorophyllide a, esterification of chlorophyllide a or zinc-pheophorbide a, and stabilization of the de novo synthesized plastid-encoded chlorophyll-apoproteins P700, CP47, CP43, D2, and D1 were successfully preserved in liquid nitrogen. Cryopreservation of freshly prepared intact etioplasts completely retained enzymatic activities for accumulation of chlorophyll a or resulted in a slightly decreased yield of zinc-pheophytin a.

Chlorophyll a was compared with Zn-pheophytin a for stabilization of chlorophyll binding apoproteins, P700, CP47, CP43, D2, and D1, in intact etioplasts from barley (Hordeum vulgare L.). Intact etioplasts were shown to effectively translate the chlorophyll apoproteins, to take up and esterify the exogenously added substrates, chlorophyllide a and Zn-pheophorbide a, with geranylgeraniolpyrophosphate. For stabilization of P700, CP47, D2, and D1, the product, Zn-pheophytin a, was shown to substitute for chlorophyll a. Stabilization of CP43 was selectively increased in the presence of Zn-pheophytin a. The degree of stabilization was shown to depend on the amount of newly synthesized Zn-pheophytin a and on the central atom of the chlorophyll molecule.