Phosphopeptides as substrates for thylakoid protein phosphatase activity.
Lack of a suitable substrate has been a major obstacle in studying the chloroplastic thylakoid membrane protein phosphatase activity. In this study, the suitability of synthetic phosphopeptides for this purpose was investigated. Phosphothreonine-containing phosphopeptides mimicking the N-terminal phosphorylation site of the major thylakoid phosphoprotein, the light-harvesting chlorophyll a/b-binding protein (LHCP-II), were dephosphorylated by isolated peak thylakoid membranes. Phosphopeptides representing unrelated sequences or in which the target phosphothreonine had been changed to a phosphoserine were not dephosphorylated. The dephosphorylation of phosphopeptides by thylakoid membranes was similar to the dephosphorylation of endogenous LHCP-II in its pH-dependence profile, sensitivity to inhibitors, and bivalent cation requirement. The same phosphopeptide analogs of the LHCP-II phosphorylation site inhibited endogenous LHCP-II dephosphorylation in isolated thylakoids, whereas the dephospho-analogs and nonsubstrate phosphopeptides had no effect. Collectively, these results suggest that phosphopeptides mimicking a thylakoid phosphoprotein dephosphorylation site can be exploited for further study of the thylakoid protein phosphatase activity.
Sun
G
G
Department of Biochemistry, University of Nebraska-Lincoln 68583-0718.
Sarath
G
G
Markwell
J
J
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002451:Cell Compartmentation; D002736:Chloroplasts / Q000201:enzymology*; D007887:Fabaceae / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology*; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D010946:Plants, Medicinal; D013379:Substrate Specificity
D002413:Cations, Divalent; D045342:Light-Harvesting Protein Complexes; D010748:Phosphopeptides; D045322:Photosynthetic Reaction Center Complex Proteins; D010749:Phosphoprotein Phosphatases