The Role of N-terminal Phosphorylation of the Photosystem IIProteins CP-43 and D2 as Probed by Site-Directed Mutagenesis

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9407187 Putnam-Evans This is a Research Planning Grant. The project focuses on the production and preliminary characterization of Synechocystis mutants lacking the N-terminal threonine residues in the Photosystem II proteins CP43 and D2. These threonine residues are phosphorylated in CP43 and D2 in higher plants. It is not yet clear what role(s) this phosphorylation may play in PSII function, although a number of hypotheses have been put forward. The goal of the project is to determine whether reversible phosphorylation/dephosphorylation of these two proteins is important to PS II function in Synechocystis. This cyanobacterium is a well defined genetic model system whose photosystems bear strong homology to those of higher plants. Replacement of endogenous genes with ones that have been altered by various methods is easily accomplished and provides a means for studying the functional role(s) of individual amino acids or small stretches of amino acids within proteins. The N-terminal threonine residue in both CP-43 and D2 of these proteins is conserved between higher plants and cyanobacteria. Oligonucleotide-directed mutagenesis will be employed in vitro to produce mutations in the N-terminal threonine codon in the genes encoding these proteins. Mutations will be re-introduced into Synechocystis along with a closely linked antibiotic resistance marker. Mutants will be screened for antibiotic resistance and possible loss of photoautotrophic growth. Mutants with altered PSII function will be selected and studied in greater depth. %%% Phosphorylation, or the addition of phosphate groups to certain serine or threonine residues of proteins, is a post- translational event. Phosphorylation-dephosphorylation reactions, in general, are means whereby cells control the activity of certain enzymes or structural proteins. One way to determine the role of phosphorylation on the function of a specific protein is to create a variant of the protein which lacks th e appropriate site (i.e., the serine or threonine residue), by molecular genetic manipulation, and look for alterations in function. This approach will be used to look at the role of phosphorylation in two proteins of Photosystem II, a multi-protein complex in chloroplasts which is responsible for the photosynthetic reactions that provide the chemical energy for most of life on earth. If successful, the results will be provide important information about how cells regulate the photosynthetic process. ***