Characteristics of D1 photosystem II reaction center protein phosphorylation: Identification of a calcium dependent protein kinase

Abstract

Several photosystem II (PSII) proteins - LHCII, D1, D2, CP43 - are known to undergo light dependent reversible phosphorylation. However, little is known about the kinases responsible for their phosphorylation. Using as substrate a synthetic peptide mimicking the N-terminus of D1, we have isolated, identified and characterized a thylakoidal protein kinase. The putative D1 kinase is an extrinsic membrane protein of 50 kD with an optimum pH of 7.4, isoelectric point of 5.5, and preference for manganese cations. The N-terminus of the purified protein was blocked. Therefore, several tryptic fragments of the protein were sequenced, based on which we synthesized degenerate oligonucleotide primers to clone the gene from rice, Arabidopsis and Spirodela. The cDNA sequence predicts a protein kinase with a mass of 59 kD based on the entire open reading frame. This size is compatible with processing of a primary translation product to produce a mature protein like that recovered from purification of the rice kinase. The protein kinase gene identified shows sequence similarities to members of the family of calcium dependent protein kinases (CDPK¿s; Breviario et al. Plant Mol. Biol. 27: 953-967, 1995). One of the CDPK isoforms from Spirodela but not the rice or Arabidopsis CDPK is imported into intact pea chloroplasts, although at a significantly slower rate than pLHCII. Spirodela kinase expressed in E. coli as an N-terminal 6-His fusion protein was purified. The purified kinase phosphorylated the N-terminal synthetic D1 peptide, but not LHCII or Rubisco peptides. Work is in progress on the role of CDPK in chloroplast function.