Markwell 9303871 Protein Phosphorylation plays a major role in eukaryotic cellular regulation. Much recent attention has been focused on the protein phosphatase enzymes which reverse this covalent modification, but there remains a paucity of information about these enzymes in plants. Chloroplastic thylakoid membrane protein phosphorylation in higher plants senses environmental light conditions and correspondingly regulates steady-state photosynthetic energy production. Whereas much attention has been devoted to the protein kinases which phosphorylate thylakoid proteins, relatively little is known about the protein phosphatase enzymes which reverse this covalent modification. The native phosphoprotein substrates are membrane bound and lack of a soluble substrate for the protein phosphatase has been the major limitation in studies of this enzyme. Recent work by the P.I.s has found that synthetic substrate-analog phosphopeptides are exogenous substrates capable of selectively assaying the thylakoid protein phosphatase. Using these substrates, this project will purify and characterize the protein phosphatase activity from pea thylakoid membranes. Characterization will include kinetic analysis of the enzyme's substrate specificity utilizing model phosphopeptides, biochemical analysis of structure and relationship to previously characterized eukaryotic protein phosphatases. These studies will provide an understanding of the mechanisms underlying reversible chloroplastic protein phosphorylation and the role of thylakoid protein phosphatase in plant bioenergetics and physiology. %%% During a normal day, a plant is exposed to continually changing light conditions. In order to maintain efficient photosynthetic energy production, plants have evolved the ability to respond to qualitatively and quantitatively different light environments. The signal transduction for this compensatory mechanism involves the phosphorylation of chloroplast proteins involved in harvesti ng light energy. While many studies have been conducted on the kinase enzyme responsible for phosphorylation of the light-harvesting pigment-protein complexes, very little is known about the protein phosphatase enzyme which reverses the phosphorylation. The major goal of this project is to purify and characterize the thylakoid protein phosphatase enzyme. These studies will provide an understanding of the role this enzyme plays in the ability of plants to optimize photosynthetic energy production under the diverse light conditions which naturally occur. ***
