Abstract
Combination of reversed genetics with analyses of in vivo protein phosphorylation in Arabidopsis thaliana revealed that STN8 protein kinase is specific in phosphorylation of N-terminal threonine residues in D1, D2, and CP43 proteins, and Thr-4 in the PsbH protein of photosystem II. Phosphorylation of D1, D2, and CP43 in the light-exposed leaves of two Arabidopsis lines with T-DNA insertions in the stn8 gene was found significantly reduced in the assays with anti-phosphothreonine antibodies. Protein phosphorylation in each of the mutants was quantified comparatively to the wild type by mass spectrometric analyses of phosphopeptides released from the photosynthetic membranes and differentially labeled with stable isotopes. The lack of STN8 caused 50-60% reduction in D1 and D2 phosphorylation, but did not change the phosphorylation level of two peptides that could correspond to light-harvesting proteins encoded by seven different genes in Arabidopsis. Phosphorylation of the PsbH protein at Thr-4 was completely abolished in the plants lacking STN8. Phosphorylation of Thr-4 in the wild type required both light and prior phosphorylation at Thr-2, indicating that STN8 is a light-activated kinase that phosphorylates Thr-4 only after another kinase phosphorylates Thr-2. Analysis of the STN8 catalytic domain suggests that selectivity of STN8 in phosphorylation of the very N-terminal residues in D1, D2, and CP43, and Thr-4 in PsbH pre-phosphorylated at Thr-2 may be explained by the long loops obstructing entrance into the kinase active site and seven additional basic residues in the vicinity of the catalytic site, as compared with the homologous STN7 kinase responsible for phosphorylation of light-harvesting proteins.
Highlights
These analyses revealed a specific decrease in photosystem II (PSII) core protein phosphorylation because of the loss of the stn8 gene in Arabidopsis
In this work we determined that in vivo substrate specificity of STN8 protein kinase in A. thaliana is directed toward the PSII core proteins
This information has been obtained by comparison of endogenous thylakoid protein phosphorylation levels in STN8 knock-out mutants with that in the wild type plants
Summary
Cleavage of the Surface-exposed Peptides from the Thylakoids—The isolated thylakoids were resuspended in 25 mM NH4HCO3, 10 mM NaF to a final concentration of 2.5 mg of chlorophyll/ml and incubated with sequencing grade-modified trypsin (Promega) (5 g of enzyme/mg of chlorophyll) for 3 h at 22 °C. The supernatant was collected while the membranes were resuspended in water and centrifuged again Both supernatants containing released thylakoid peptides were pooled and centrifuged at 100,000 ϫ g. Preparation of Peptide Methyl Esters—The peptides released by trypsin from the thylakoids isolated from the wild type or stn plants were lyophilized and methyl-esterified with 2 N methanolic HCl or DCl. The reagent was prepared by dropwise addition of 80 l of acetylchloride (Aldrich) to 500 l of anhydrous d0-methyl alcohol (Aldrich) or d3-methyl d-alcohol (Aldrich) with stirring. The difference in intensity of two signals in each doublet reflected the ratio in phosphorylation of a particular site between the wild type and stn plants
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