Functional analysis of the PsbP-like protein (sll1418) in Synechocystis sp. PCC 6803

A recent proteomic analysis of the thylakoid lumen of Arabidopsis thaliana revealed the presence of several PsbP-like proteins, and a homologue to this gene family was detected in the genome of the cyanobacterium Synechocystis sp. PCC 6803 (Schubert M, Petersson UA, Haas BJ, Funk C, Schröder WP, Kieselbach T (2002) J Biol Chem 277, 8354–8365). Using a peptide-directed antibody against this cyanobacterial PsbP-like protein (sll1418) we could show that it was localized in the thylakoid membrane and associated with Photosystem II. While salt washes did not remove the PsbP-like protein from the thylakoid membrane, it was partially lost during the detergent-based isolation of PSII membrane fractions. In total cell extracts this protein is present in the same amount as the extrinsic PsbO protein. We did not see any significant functional difference between the wild-type and a PsbP-like insertion mutant.     

Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen

The oxygen-evolving complex (OEC) of photosystem II (PS II) consists of at least three extrinsic membrane-associated protein subunits, OE33, OE23, and OE17, with associated Mn2+, Ca2+, and Cl- ions. These subunits are bound to the lumen side of PS II core proteins embedded in the thylakoid membrane. Our experiments reveal that a significant fraction of each subunit is normally present in unassembled pools within the thylakoid lumen. This conclusion was supported by immunological detection of free subunits after freshly isolated pea thylakoids were fractionated with low levels of Triton X-100. Plastocyanin, a soluble lumen protein, was completely released from the lumen by 0.04% Triton X-100. This gentle detergent treatment also caused the release from the thylakoids of between 10 and 20%, 40 and 60%, and 15 and 50% of OE33, OE23, and OE17, respectively. Measurements of the rates of oxygen evolution from Triton-treated thylakoids, both in the presence and absence of Ca2+, and before and after incubation with hydroquinone, demonstrated that the OEC was not dissociated by the detergent treatment. Thylakoids isolated from spinach released similar amounts of extrinsic proteins after Triton treatment. These data demonstrate that physiologically active chloroplasts contain significant pools of unassembled extrinsic OEC polypeptide subunits free in the lumen of the thylakoids.     

The use of polyclonal antibodies to identify peptides exposed on the stroma side of the spinach thylakoid

We have raised polyclonal antibodies against an oxygen-evolving photosystem II preparation. Western Blot analysis of the whole serum revaals antibodies specific for at least 15 Coomassie visible bands ranging from 59 to 11 kDa. These antibodies are specific for proteins located on both sides of the membrane. Included are antibodies specific for Tris-removable peptides (33, 25 and 18 kda), which are thought to be exposed on the lumen surface of the PS II complex. Since the whole serum agglutinates thylakoids, antibodies specific for the stroma side of the PS II complex are also present. A sub-population of antibodies can be isolated by allowing the antibodies in whole serum to bind to EDTA-treated thylakoid membranes. The antibodies which specifically bind are cross-reactive with peptides with Mr of 59, 57, 34, 28, 27, 26, and 23 kDa. Our data indicate that these peptides have antigenic determinants exposed on the stroma side of the thylakoid membrane.     

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.     

Characterization of chloroplast thylakoid polypeptides in the 32-kDa region: polypeptide extraction and protein phosphorylation affect binding of photosystem II-directed herbicides

In order to distinguish between two photosystem II proteins with apparent molecular weights of about 32 kDa, mild extraction procedures were used to remove several thylakoid membrane components. A 32-kDa protein that stained intensely with Coomassie brilliant blue could be extracted from the thylakoid membranes without removing the 32-kDa herbicide receptor protein, which stained poorly with Coomassie brilliant blue. The nonextracted protein was readily detectable after in vivo polypeptide labeling with [35S]methionine or after in vitro covalent tagging with [14C]azidoatrazine. The procedures used to extract the intensely stained, 32-kDa polypeptide resulted in changes in herbicide-binding characteristics, presumably due to conformational changes in the herbicide-binding environment. Alterations of membrane surface charge by protein phosphorylation also influenced herbicide binding.     

Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins.

A reverse staining procedure is described for the detection of proteins in acrylamide and agarose gels with and without SDS. Protein detection occurs a few minutes after electrophoresis. The sensitivity on acrylamide gels is higher than that of Coomassie blue staining either on acrylamide gels or on electrotransferred membranes. Sequencing of protein bands only detected by reverse staining on the gel and not by Coomassie blue is demonstrated.     

The use of polyvinylidenedifluoride membranes as a general blotting matrix

A fast chemical staining of protein by Coomassie blue on a stable nonshrinking membrane of polyvinylidenedifluoride is described. The procedure is especially useful for detection of nonlabeled marker proteins in comparison to specifically detected proteins. The membrane is highly suitable for other chemical stains like silver, gold, or india ink for immunostaining, lectin binding, and binding of radioactive ligands like 45Ca. Blocking of the background against nonspecific binding to proteins was studied using different reagents. A multiple slit plate is described for volume-saving simultaneous application of multiple reactants on one protein-loaded membrane.     

Proteome map of the chloroplast lumen of Arabidopsis thaliana.

The thylakoid membrane of the chloroplast is the center of oxygenic photosynthesis. To better understand the function of the luminal compartment within the thylakoid network, we have carried out a systematic characterization of the luminal thylakoid proteins from the model organism Arabidopsis thaliana. Our data show that the thylakoid lumen has its own specific proteome, of which 36 proteins were identified. Besides a large group of peptidyl-prolyl cis-trans isomerases and proteases, a family of novel PsbP domain proteins was found. An analysis of the luminal signal peptides showed that 19 of 36 luminal precursors were marked by a twin-arginine motif for import via the Tat pathway. To compare the model organism Arabidopsis with another typical higher plant, we investigated the proteome from the thylakoid lumen of spinach and found that the luminal proteins from both plants corresponded well. As a complement to our experimental investigation, we made a theoretical prediction of the luminal proteins from the whole Arabidopsis genome and estimated that the thylakoid lumen of the chloroplast contains approximately 80 proteins.     

A rapid method for staining proteins in acrylamide gels

A negative staining procedure for the rapid visualization of proteins in acrylamide gels is described. In the absence of proteins, staining of the gel occurs through the reduction of nitroblue tetrazolium by reduced glutathione. No staining occurs in the presence of proteins. The procedure can be completed in 20 min and is at least as sensitive as Coomassie brilliant blue staining.     

Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel

A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels. The transfer is carried out essentially as has been used for protein blotting on nitrocellulose membranes [Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350-4354], but is now used to determine the amino acid composition and partial sequence of the immobilized proteins. Protein transfer could be carried out after staining the proteins in the gels with Coomassie blue, by which immobilized proteins are visible as blue spots, or without previous staining, after which transferred proteins are detected as fluorescent spots following reaction with fluorescamine. The latter procedure was found to be more efficient and yielded binding capacities of +/- 20 micrograms/cm2. Fluorescamine detection was of equal or higher sensitivity than the classical Coomassie staining of proteins in the gel. Immobilized proteins could be hydrolyzed when still present on the glass fiber and reliable amino acid compositions were obtained for various reference proteins immobilized in less than 100 pmol quantities. In addition, and more importantly, glass-fiber-bound proteins could be subjected to the Edman degradation procedure by simply cutting out the area of the sheet carrying the immobilized protein and mounting the disc in the reaction chamber of the gas-phase sequenator. Results of this immobilization-sequencing technique are shown for immobilized myoglobin (1 nmol) and two proteolytic fragments of actin (+/- 80 pmol each) previously separated on a sodium-dodecylsulfate-containing gel.     

Dicyclohexylcarbodiimide-binding proteins related to the short circuit of the proton-pumping activity of photosystem II. Identified as light-harvesting chlorophyll-a/b-binding proteins

In photosynthesis of higher plants, photosystem II drives electron transfer from the water-oxidizing manganese centre at the lumenal side to bound plastoquinone at the stromal side of the thylakoid membrane. Proton release into the lumen and proton uptake from the stroma, i.e. net proton pumping, follows as consequence of vectoral electron transport. The proton pumping activity can be short circuited by covalent modification with N,N'-dicyclohexylcarbodiimide (cHxN)2C of certain proteins in the 20-28-kDa range. After modification, protons from water oxidation are no longer released into the thylakoid lumen, but instead transferred through the photosystem complex to protonate the photoreduced bound quinone at the other side of the membrane [Jahns, P., Polle, A. & Junge, W. (1988) EMBO J. 7, 589-594]. Here we identify the pertinent (cHxN)2C-binding proteins by amino acid sequence analysis and localize (cHxN)2C-binding sites within their primary structure. The proteins that are associated with the proton short circuit are light-harvesting chlorophyll-a/b-binding proteins. Our results imply that in addition to acting as antennae they may serve another function: the funneling into the thylakoid lumen of protons, which are liberated in the water-oxidizing Mn centre.     

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.     

Rapid staining of proteins on polyacrylamide gels and nitrocellulose membranes using a mixture of fluorescent dyes

The present work describes a novel, fluorescence-based method for staining proteins on SDS-PAGE and membrane(s). In this method, proteins are stained using a mixed-dye (sulfo-rhodamine B and 1-anilino-8-naphthalene sulfonic acid (NH(4)(+))) solution. The mixed-dye staining protocol can detect proteins up to a concentration of 15 ng. This method is generally applicable to all proteins and is more sensitive than the conventional Coomassie blue method. The staining method is rapid and efficient. Staining-destaining of proteins using the mixed-dye protocol takes less than half an hour. Another interesting feature of the staining protocol described here is the applicability to the staining of proteins on nitrocellulose membranes.     

Immunodetection after complete destaining of coomassie blue-stained proteins on immobilon-PVDF.

A technique that simplifies the localization of an immunodetectable protein in relation to the other electrophoresed proteins is described. Proteins are transblotted onto a polyvinylidene difluoride (PVDF) membrane and visualized by staining with Coomassie brilliant blue R-250, and a photograph of the protein pattern is taken. The Coomassie blue-stained PVDF membrane is then completely destained using a 25% acetic acid/50% methanol solution that allows subsequent immunostaining on the same membrane. The technique uses common laboratory reagents, is rapid, and has been shown to be applicable for a variety of proteins using both monoclonal and polyclonal antibodies and a variety of transblots.     

叶绿体类囊体腔蛋白功能研究进展

【目的】类囊体是叶绿体光合作用中光反应进行的重要场所。类囊体腔是由类囊体膜包围形成的一个狭小空间。在类囊体腔中存在多种不同的蛋白家族,包括高叶绿素荧光(high chlorophyll fluorescence, HCF)蛋白、亲免蛋白、放氧复合物(oxygen-evolving complex, OEC)蛋白、PsbP类蛋白等,它们对植物的光合作用、核酸代谢以及氧化还原反应等都起着重要作用。【评论】文章分类综述了参与光合作用调控的类囊体腔蛋白在光系统组装、植物生长发育调节和高光逆境响应等生理活动中发挥的重要作用。【展望】文章可为未来研究类囊体腔蛋白的生理功能提供理论参考。     

Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function

The light‐dependent regulation of stromal enzymes by thioredoxin (Trx)‐catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx‐mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol‐dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx‐linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de‐epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox‐controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.     

Amino acid analysis on polyvinylidene difluoride membranes

A procedure for the amino acid analysis of proteins electrotransferred to polyvinylidene difluoride (PVDF) membranes is described. The proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a PVDF membrane. After staining with Coomassie brilliant blue, the visualized protein bands are excised from the membrane. Each band is placed in a vial and subjected to gas-phase hydrolysis in 6 N HCl in a vacuum desiccator at 110 degrees C. The amino acids are extracted from the membrane into 0.1 N HCl/30% CH3OH and analyzed by reverse-phase HPLC using postcolumn o-phthalaldehyde-derivatizing reagent. The method was shown to give reproducible and reasonably accurate compositions for several proteins, as well as to provide an estimate of protein content. As little as 10 pmol of a 67-kDa protein can be determined.     

Comparison of protein components of the plasma membrane fraction from rat liver and Morris hepatomas 5123D and 7777.

Isolated plasma membrane fractions from rat liver and Morris hepatoma 5123D and 7777 were labelled with radioiodine 125I by a chemical or enzymatic procedure and then were solubilized in 2 per cent solution of sodium dodecyl sulphate containing 1 per cent 2-mercaptoethanol. Solubilized proteins were separated into 20--22 zones staining with Coomassie Brilliant Blue R-250 after disc gel electrophoresis (7.5 per cent polyacrylamide gel). The high similarity of electrophoretic patterns of polypeptide components of all three preparations of cellular membranes was found in distinction to apparent differnces in the amount and disposition of substances stained with Schiff's reagent. Some tentative conclusions were drawn on the disposition of proteins within membrane structure studied by the method of labelling by chemical and enzymatic procedures (distinguishing between extrinsic and integral proteins).