The membrane proteome of stroma thylakoids from Arabidopsis thaliana studied by successive in-solution and in-gel digestion.

From individual localization and large-scale proteomic studies, we know that stroma-exposed thylakoid membranes harbor part of the machinery performing the light-dependent photosynthetic reactions. The minor components of the stroma thylakoid proteome, regulating and maintaining the photosynthetic machinery, are in the process of being unraveled. In this study, we developed in-solution and in-gel proteolytic digestion methods, and used them to identify minor membrane proteins, e.

Global differences in specific histone H3 methylation are associated with overweight and type 2 diabetes.

Background: Epidemiological evidence indicates yet unknown epigenetic mechanisms underlying a propensity for overweight and type 2 diabetes. We analyzed the extent of methylation at lysine 4 and lysine 9 of histone H3 in primary human adipocytes from 43 subjects using modification-specific antibodies.

Results: The level of lysine 9 dimethylation was stable, while adipocytes from type 2 diabetic and non-diabetic overweight subjects exhibited about 40% lower levels of lysine 4 dimethylation compared with cells from normal-weight subjects.

Photosystem II function and dynamics in three widely used Arabidopsis thaliana accessions.

Columbia-0 (Col-0), Wassilewskija-4 (Ws-4), and Landsberg erecta-0 (Ler-0) are used as background lines for many public Arabidopsis mutant collections, and for investigation in laboratory conditions of plant processes, including photosynthesis and response to high-intensity light (HL). The photosystem II (PSII) complex is sensitive to HL and requires repair to sustain its function. PSII repair is a multistep process controlled by numerous factors, including protein phosphorylation and thylakoid membrane stacking.

Differential phosphorylation of thylakoid proteins in mesophyll and bundle sheath chloroplasts from maize plants grown under low or high light.

In C4 plants, such as maize, the photosynthetic apparatus is partitioned over two cell types called mesophyll (M) and bundle sheath (BS), which have different structure and specialization of the photosynthetic thylakoid membranes. We characterized protein phosphorylation in thylakoids of the two cell types from maize grown under either low or high light. Western blotting with phosphothreonine antibodies and ProQ phosphostaining detected light-dependent changes in the protein phosphorylation patterns.

Identification of a photosystem II phosphatase involved in light acclimation in Arabidopsis.

Reversible protein phosphorylation plays a major role in the acclimation of the photosynthetic apparatus to changes in light. Two paralogous kinases phosphorylate subsets of thylakoid membrane proteins. STATE TRANSITION7 (STN7) phosphorylates LHCII, the light-harvesting antenna of photosystem II (PSII), to balance the activity of the two photosystems through state transitions.

Phosphoproteomics of Arabidopsis chloroplasts reveals involvement of the STN7 kinase in phosphorylation of nucleoid protein pTAC16.

Light-regulated protein kinases STN7 and STN8 phosphorylate thylakoid membrane proteins and also affect expression of several chloroplast proteins via yet unknown mechanisms. Comparative phosphoproteomics of acetic acid protein extracts of chloroplasts from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutants yielded two previously unknown findings: (i) neither STN7 nor STN8 kinase was required for phosphorylation of Ser-48 in Lhcb1.1-1.

Differential phosphorylation of ribosomal proteins in Arabidopsis thaliana plants during day and night.

Protein synthesis in plants is characterized by increase in the translation rates for numerous proteins and central metabolic enzymes during the day phase of the photoperiod. The detailed molecular mechanisms of this diurnal regulation are unknown, while eukaryotic protein translation is mainly controlled at the level of ribosomal initiation complexes, which also involves multiple events of protein phosphorylation. We characterized the extent of protein phosphorylation in cytosolic ribosomes isolated from leaves of the model plant Arabidopsis thaliana harvested during day or night.

Phosphorylation of PSII proteins in maize thylakoids in the presence of Pb ions.

Lead is potentially toxic to all organisms including plants. Many physiological studies suggest that plants have developed various mechanisms to contend with heavy metals, however the molecular mechanisms remain unclear. We studied maize plants in which lead was introduced into detached leaves through the transpiration stream.

High light induced disassembly of photosystem II supercomplexes in Arabidopsis requires STN7-dependent phosphorylation of CP29.

Photosynthetic oxidation of water and production of oxygen by photosystem II (PSII) in thylakoid membranes of plant chloroplasts is highly affected by changes in light intensities. To minimize damage imposed by excessive sunlight and sustain the photosynthetic activity PSII, organized in supercomplexes with its light harvesting antenna, undergoes conformational changes, disassembly and repair via not clearly understood mechanisms. We characterized the phosphoproteome of the thylakoid membranes from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutant plants exposed to high light.

Histone variants and their post-translational modifications in primary human fat cells.

Epigenetic changes related to human disease cannot be fully addressed by studies of cells from cultures or from other mammals. We isolated human fat cells from subcutaneous abdominal fat tissue of female subjects and extracted histones from either purified nuclei or intact cells. Direct acid extraction of whole adipocytes was more efficient, yielding about 100 µg of protein with histone content of 60%-70% from 10 mL of fat cells.

Thylakoid phosphoproteins: identification of phosphorylation sites.

Redox-dependent thylakoid protein phosphorylation regulates both the short- and long-term acclimation of the photosynthetic apparatus to changes in environmental conditions. The major thylakoid phosphoproteins belong to photosystem II (D1, D2, CP43, PsbH) and its light-harvesting antenna (Lhcb1, Lhcb2, CP29), but a number of minor phosphoproteins have also been identified. The detection methods traditionally include the radiolabeling techniques, electrophoretic separation of the phosphorylated and unphosphorylated forms of the protein, and the use of phosphoamino acid antibodies or phosphoprotein-specific dyes.

A protein phosphorylation threshold for functional stacking of plant photosynthetic membranes.

Phosphorylation of photosystem II (PSII) proteins affects macroscopic structure of thylakoid photosynthetic membranes in chloroplasts of the model plant Arabidopsis. In this study, light-scattering spectroscopy revealed that stacking of thylakoids isolated from wild type Arabidopsis and the mutant lacking STN7 protein kinase was highly influenced by cation (Mg(++)) concentrations. The stacking of thylakoids from the stn8 and stn7stn8 mutants, deficient in STN8 kinase and consequently in light-dependent phosphorylation of PSII, was increased even in the absence of Mg(++).

Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism.

Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established.

The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis.

The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II.

Stt7-dependent phosphorylation during state transitions in the green alga Chlamydomonas reinhardtii.

Photosynthetic organisms are able to adapt to changes in light conditions by balancing the light excitation energy between the light-harvesting systems of photosystem (PS) II and photosystem I to optimize the photosynthetic yield. A key component in this process, called state transitions, is the chloroplast protein kinase Stt7/STN7, which senses the redox state of the plastoquinone pool. Upon preferential excitation of photosystem II, this kinase is activated through the cytochrome b(6)f complex and required for the phosphorylation of the light-harvesting system of photosystem II, a portion of which migrates to photosystem I (state 2).

Phosphorylation of photosystem II controls functional macroscopic folding of photosynthetic membranes in Arabidopsis.

Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein.

Peptidyl-prolyl isomerase activity in chloroplast thylakoid lumen is a dispensable function of immunophilins in Arabidopsis thaliana.

Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins and 11 FK506-binding proteins (FKBPs), which are considered protein folding catalysts, although only two of them, AtFKBP13 and AtCYP20-2, possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. To address the question of the physiological significance of this activity, we obtained and characterized Arabidopsis mutants deficient in the most active PPIase, AtFKBP13, and a double mutant deficient in both AtFKBP13 and AtCYP20-2. Two-dimensional gel electrophoresis of isolated thylakoid lumen, as well as immunoblotting analyses of major photosynthetic membrane protein complexes did not reveal differences in protein composition between the mutants and the wild type.

Intrinsically unstructured phosphoprotein TSP9 regulates light harvesting in Arabidopsis thaliana.

Thylakoid-soluble phosphoprotein of 9 kDa, TSP9, is an intrinsically unstructured plant-specific protein [Song, J., et al. (2006) Biochemistry 45, 15633-15643] with unknown function but established associations with light-harvesting proteins and peripheries of both photosystems [Hansson, M.

Determination of in vivo protein phosphorylation in photosynthetic membranes.

Light- and redox-controlled reversible phosphorylation of thylakoid proteins regulates short- and long-term acclimation of plants to environmental cues. The major phosphoproteins in thylakoids belong to photosystem II and its light-harvesting antenna but phosphorylation of subunits of other thylakoid protein complexes has been detected as well. The detection methods include electrophoretic separation of proteins and detection of phosphoproteins with a phosphoaminoacid-specific antibody or phosphoprotein-specific dye.

AtCYP38 ensures early biogenesis, correct assembly and sustenance of photosystem II.

AtCYP38 is a thylakoid lumen protein comprising the immunophilin domain and the phosphatase inhibitor module. Here we show the association of AtCYP38 with the photosystem II (PSII) monomer complex and address its functional role using AtCYP38-deficient mutants. The dynamic greening process of etiolated leaves failed in the absence of AtCYP38, due to specific problems in the biogenesis of PSII complexes.

Light regulation of CaS, a novel phosphoprotein in the thylakoid membrane of Arabidopsis thaliana.

Exposure of Arabidopsis thaliana plants to high levels of light revealed specific phosphorylation of a 40 kDa protein in photosynthetic thylakoid membranes. The protein was identified by MS as extracellular calcium-sensing receptor (CaS), previously reported to be located in the plasma membrane. By confocal laser scanning microscopy and subcellular fractionation, it was demonstrated that CaS localizes to the chloroplasts and is enriched in stroma thylakoids.

Acute effects of insulin on the activity of mitochondrial GPAT1 in primary adipocytes.

The mitochondrial enzyme 1-acyl-sn-glycerol-3-phosphate acyltransferase (mtGPAT1) catalyzes a rate-limiting step in triacylglycerol and glycerophospholipid biosynthesis, which can be modulated by protein kinases in cell free analyses. We report that treatment of primary rat adipocytes with insulin acutely affects the activity of mtGPAT1 by increasing V(MAX) and K(M) for the substrates glycerol-3-phosphate and palmitoyl-CoA. Proteolytic cleavage of isolated mitochondrial membranes and mass spectrometric peptide sequencing identify in vivo phosphorylation of serine 632 and serine 639 in mtGPAT1.

Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-2.

The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses.

The mobile thylakoid phosphoprotein TSP9 interacts with the light-harvesting complex II and the peripheries of both photosystems.

The localization of the plant-specific thylakoid-soluble phosphoprotein of 9 kDa, TSP9, within the chloroplast thylakoid membrane of spinach has been established by the combined use of fractionation, immunoblotting, cross-linking, and mass spectrometry. TSP9 was found to be exclusively confined to the thylakoid membranes, where it is enriched in the stacked grana membrane domains. After mild solubilization of the membranes, TSP9 migrated together with the major light-harvesting antenna (LHCII) of photosystem II (PSII) and with PSII-LHCII supercomplexes upon separation of the protein complexes by either native gel electrophoresis or sucrose gradient centrifugation.

The Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction centre D1 protein.

The extrinsic photosystem II (PSII) protein of 33 kDa (PsbO), which stabilizes the water-oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isoforms. Two T-DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were retarded in growth in comparison with the wild type, while differing from each other phenotypically. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less efficient than PsbO1 under photoinhibitory conditions.