Light-dependent phosphorylation of thylakoid membrane polypeptides.

The phosphorylation of thylakoid membrane proteins was studied using isolated chloroplasts from $\text{Euglena gracilis}$. We have found, using [³²P] labelling, that this phenomenon was light-driven, reversible in the dark, and completely inhibited by Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP). Polyacrylamide gel electrophoresis containing SDS has revealed five main bands which have been found to be proteins. Amino acid analysis of the bands has shown that [³²P] is incorporated into phosphothreonine.     

The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii

The sites of synthesis of the major thylakoid membrane polypeptides have been studied in the green alga Chlamydomonas reinhardtii by pulse labeling of cells with [14C]acetate in the presence of inhibitors specific for chloroplast and cytoplasmic protein synthesis. The labeled membrane polypeptides were separated by an improved method of sodium dodecyl sulfate (SDS) gradient gel electrophoresis, and autoradiographs were made of the dried gels. The results demonstrate that of the 33 polypeptides resolved in the gels, at least nine are made on chloroplast ribosomes. Two of these (polypeptides 2 and 6) are associated with the reaction centers of photosystems I and II. Another polypeptide (polypeptide 5) appears from genetic data to be coded by chloroplast DNA. Experiments with a mutant whose chloroplast ribosomes are resistant to spectinomycyn (spr-u-1-6-2) show that polypeptides whose synthesis takes place on chloroplast ribosomes are made in the presence of spectinomycin in the mutant although their synthesis is blocked by this antibiotic in wild type cells.     

Two dimensional analysis of thylakoid membrane polypeptides of a barley mutant

Summary One dimensional SDS-PAGE and two dimensional electrophoresis of the thylakoid membrane proteins of a barley mutant and its original variety were carried out. It was found that (1) approximately 20 bands could be identified. The mutant lacked one band at 23 kd, and two bands at 17.5 nd 18.3 kd were fainter when compared with the original variety which served as the standard. (2) In two dimensional patterns the 23 kd band was resolved into two faint bands which were composed of three peptide spots having a pH of 6.35, 6.46 and 6.50, respectively. The band in the 17–18 kd region was resolved into five heavy peptide spots with varying pH values of 6.35–6.62.     

Preparative isolation of Band 3, the predominant polypeptides of the human erythrocyte membrane

Band 3 proteins are the predominant polypeptide components of the human erythrocyte membrane and have been implicated in various transport activities. Following extraction of membrane ghosts with dimethylmaleic anhydride to remove two polypeptides (Bands 4.2 and 6) associated with Band 3, Band 3 proteins were solubilized along with the other major membrane glycoproteins (PAS 1–3) with Triton X-100 under nondenaturing conditions. Band 3 proteins were then purified (>95%) on a large scale by chromatography via thioldisulfide interchange on activated thiol-Sepharose 4B [agarose-(glutathione-2-pyridyl disulfide) conjugate]. This procedure allows the preparation of 20 to 25 mg of purified Band 3 proteins in high yield (>80%) from ghosts in a soluble form suitable for physical, chemical, and functional characterization.     

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.     

Mutants of Chlamydomonas: tools to study thylakoid membrane structure, function and biogenesis.

The unicellular green alga Chlamydomonas reinhardtii is a model system for the study of photosynthesis and chloroplast biogenesis. C. reinhardtii has a photosynthesis apparatus similar to that of higher plants and it grows at rapid rate (generation time about 8 h). It is a facultative phototroph, which allows the isolation of mutants unable to perform photosynthesis and its sexual cycle allows a variety of genetic studies. Transformation of the nucleus and chloroplast genomes is easily performed. Gene transformation occurs mainly by homologous recombination in the chloroplast and heterologous recombination in the nucleus. Mutants are precious tools for studies of thylakoid membrane structure, photosynthetic function and assembly. Photosynthesis mutants affected in the biogenesis of a subunit of a protein complex usually lack the entire complex; this pleiotropic effect has been used in the identification of the other subunits, in the attribution of spectroscopic signals and also as a 'genetic cleaning' process which facilitates both protein complex purification, absorption spectroscopy studies or freeze-fracture analysis. The cytochrome b6f complex is not required for the growth of C. reinhardtii, unlike the case of photosynthetic prokaryotes in which the cytochrome complex is also part of the respiratory chain, and can be uniquely studied in Chlamydomonas by genetic approaches. We describe in greater detail the use of Chlamydomonas mutants in the study of this complex.     

Proposals for the naming of chloroplast genes. II. Update to the nomenclature of genes for thylakoid membrane polypeptides

The nomenclature for genes for components of the photosynthetic membranes has been reviewed and updated. Newly discovered genes have been added to the existing convention for gene nomenclature. Genes designatedpetA throughpetI are described for components of the photosynthetic electron transport systems,psaA throughpsaK for photosystem I components, andpsbA throughpsbR for photosystem II, including the extrinsic polypeptides of the oxygen-evolving complex. References for representative examples of each gene are given.     

Structural similarities between the major polypeptides of thylakoid membranes from Chlamydomonas reinhardtii

The major polypeptides of thylakoid membranes from Chlamydomonas reinhardtii were purified by preparative gel electrophoresis and examined for structural similarities. The largest of these polypeptides has an apparent molecular mass of 29,500 ± 500 daltons, whereas the other two both have an apparent mass of 26,000 ± 500 daltons. The amino acid compositions and uv-absorption spectra of the 29K- and 26K-dalton polypeptides are very similar. The same pattern of release of amino acids was obtained from both fractions by digestion with carboxypeptidase Y. Endoproteolytic digestion with trypsin, chymotrypsin, staphylococcal protease, and mild acid yielded identical patterns of N-terminal amino acids from both the 29K- and 26K-dalton polypeptides. However, different patterns of peptides were found after electrophoresis of fragments generated by digestion with staphylococcal protease. Conditions of electrophoresis were defined that permitted separation of the 26K-dalton fraction into two components, designated as polypeptides 16 and 17 in the identification system of Chua and Bennoun (1975, Proc. Nat. Acad. Sci. USA72, 2175–2179). Amino acid compositions of these two polypeptides are nearly identical. Polypeptide 16 contained N-terminal isoleucine, but no free N-terminal amino group was detected in polypeptide 17. Electrophoretic analysis of staphylococcal protease digests of these two polypeptides revealed significant differences in the patterns of peptides. These data confirm that there are three distinct major polypeptides in these membranes, which are present at nearly equal amounts. However, the data also suggest that significant similarities in amino acid sequence exist between these polypeptides.     

New isolation procedure and further biochemical characterization of glycoproteins IIb and IIIa from human platelet plasma membrane.

Mucus glycoproteins (mucins) were obtained from human cervical and pig gastric mucus as well as from chronic-bronchitic sputum after low-shear extraction. The mucus gel was solubilized in guanidinium chloride supplemented with proteinase inhibitors, and the macromolecules were purified by using isopycnic density-gradient centrifugation. The macromolecules were spread in monolayers of benzyldimethylalkyl-ammonium chloride and studied with electron microscopy after staining with uranyl acetate and/or shadowing with platinum/carbon. The mucins appeared as flexible linear threads with lengths varying from approx. 200 nm to about 400 nm. No regularly branched or star-shaped structures were observed. The macromolecular architecture of cervical, respiratory and gastric mucins is thus similar.     

Composition and biosynthesis of thylakoid membrane polypeptides in the red alga Cyanidium caldarium: Comparison with the thylakoid polypeptide composition of higher plants and cyanobacteria

The polypeptide composition of thylakoid membranes of the red alga Cyanidium caldarium was studied by PAGE in the presence of lithium dodecyl sulfate. The thylakoid membranes were shown to contain 65 polypeptides with mol wt from 110 to 10 kDa. PS I isolated from C. caldarium cells is composed of at least 5 components, one of which is the chlorophyll-protein complex with mol wt of 110 kDa typical of higher plants. Cyt f, c ₅₅₂, b ₆ and b ₅₅₉ were identified. Inhibition of carotenoid biosynthesis with norflurazon caused no changes in the polypeptide composition of thylakoid membranes of the algae grown in dark. The suppression of the biosynthesis rate of some thylakoid polypeptides in the algae grown with norflurazon in light is a result of membrane photodestruction. Thylakoid membranes from C. caldarium cells are more similar in the number of protein components to thylakoid membranes from cells of the cyanobacterium Anacystis nidulans than to those of higher plants (Pisum sativum), which was proved by immune-blotting assays: Thylakoid membranes of the red alga and cyanobacteria contain 28 homologous polypeptides, while thylakoid membranes of the alga and pea, only 15.Abbreviations CD circular dichroism - CP chlorophyll-protein complex - LDS lithium dodecyl sulfate - NF norflurazon     

Organization of the thylakoid membrane from the heterotrophic cyanobacterium, Aphanocapsa 6714

The polypeptide composition of thylakoid membrane fractions from the heterotrophic cyanobacterium Aphanocapsa 6714 was examined by electrophoretic and immunoblotting procedures. We have identified thylakoid cytochromes f, b6, c-550 and c-553 by tetramethylbenzidine staining of lithium dodecyl sulfate polyacrylamide gels; we also have identified the Rieske Fe-S center protein and subunit 4 of the cytochrome b6/f complex. We have characterized phycobilisomes and active core preparations of PS I and PS II. PS I is comprised of five polypeptides (62 kDa, 14.5 kDa, 10 kDa, and two proteins of less than 10 kDa), and our PS II preparation is highly enriched for three chlorophyll-binding proteins of 48, 45 and 36 kDa. Furthermore, we have resolved the chlorophyll-binding complexes on non-denaturing gels and have determined the polypeptide composition of each chlorophyll-containing band. Three bands are associated with PS I (I, IIa and IIb) and three bands are PS II components (III', IIIa and IIIb) as judged by low-temperature fluorescence emission spectra. Band III' contains a 64 kDa antenna polypeptide, IIIa contains the 48 kDa and 45 kDa polypeptides, and IIIb is comprised solely of a 36 kDa protein. The IIIb apoprotein represents a novel PS II component; its possible role in photochemistry is discussed.     

Azide-sensitive thylakoid membrane insertion of chimeric cytochrome f polypeptides imported by isolated pea chloroplasts

Post-translational integration of cytochrome f into thylakoid membranes was observed after import by isolated pea chloroplasts of a chimeric protein consisting of the presequence of the small subunit of ribulose 1,5-bisphosphate carboxylase fused to the cytochrome f precursor. Import of a similar chimeric protein lacking the C-terminal 33 amino acid residues resulted in a soluble cytochrome f protein in the thylakoid lumen, indicating that the C-terminal region contains a stop-transfer sequence for membrane integration. Azide inhibited the insertion of cytochrome f into the thylakoid membrane, whereas the ionophores nigericin and valinomycin had little effect on membrane insertion. The precursor of the 33 kDa protein, but not the 23 kDa protein, of the photosystem II oxygen-evolving complex inhibited the thylakoid insertion of cytochrome f , suggesting competition for a component of the transport pathway. These experiments suggest that the post-translational insertion of cytochrome f into the thylakoid membrane uses a SecA-dependent pathway.     

Functional flexibility and acclimation of the thylakoid membrane.

Light is an elusive substrate for the function of photosynthetic light reactions of photosynthesis in the thylakoid membrane. Therefore structural and functional dynamics, which occur in the timescale from seconds to several days, are required both at low and high light conditions. The best characterized short-time regulation mechanism at low light is a rapid state transition, resulting in higher absorption cross section of PSI at the expense of PSII. If the low light conditions continue, activation of the lhcb-genes and synthesis of the light-harvesting proteins will occur to optimize the functions of PSII and PSI. At high light, the transition to state 2 is completely inhibited, but the feedback de-excitation of absorbed energy as heat, known as the energy-dependent quenching (q(E)), is rapidly set up. It requires, at least, the DeltapH-dependent activation of violaxanthin de-epoxidase and involvement of the PsbS protein. Another crucial mechanism for protection against the high light stress is the PSII repair cycle. Furthermore, the water-water cycle, cyclic electron transfer around PSI and chlororespiration are important means induced under high irradiation, functioning mainly to avoid an excess production of reactive oxygen species.     

Penicillin-binding proteins of Rhodopseudomonas sphaeroides and their membrane localization.

Cytoplasmic membranes (CM) prepared from both chemotrophic and phototrophic cells of Rhodopseudomonas sphaeroides possess penicillin-binding proteins (PBPs), as demonstrated by binding of [125]furazlocillin to isolated membranes, the subsequent separation of the constituent PBPs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their detection by autoradiography. The major PBP present in CM from R. sphaeroides corresponds in molecular weight to PBP-5, the predominant PBP present in CM of Escherichia coli. In contrast, the outer membrane of R. sphaeroides shows only low-level furazlocillin-binding activity on a per milligram of protein basis compared with chemotrophic CM. The intracytoplasmic membrane (ICM) derived from phototrophic cells contains less than 5% of the furazlocillin-binding activity of the CM. Based on the specific localization of PBPs in the CM, it is possible to provide quantitative estimates of the extent of CM present in preparations of ICM. This method demonstrates that highly purified preparations of ICM contain less than 5% CM. Additionally, the assay for PBPs demonstrates that during ICM remodeling, which occurs upon a shift from phototrophic to chemotrophic growth, there is no significant insertion of PBPs into the ICM over the first two generations after a shift to chemotrophic growth.