Purification and sequencing of a family of wheat lipid transfer protein homologues phosphorylated by plant calcium-dependent protein kinase

Abstract

Four low molecular weight, basic proteins (WBP1A, WBP1B, WBP2 and WBP3) that are substrates for wheat germ Ca2+-dependent protein kinase (CDPK) were purified from wheat germ by a procedure involving batchwise cation exchange on carboxymethylcellulose (CM52), acid precipitation, cation exchange HPLC on an SP5PW column and reverse-phase HPLC on a C18 column. While WBP1A, WBP1B and WBP3 are phosphorylated by wheat germ CDPK exclusively on Ser residues, WBP2 is phosphorylated on both Ser and Thr residues. CDPK-catalysed phosphorylation sites on WBP1A and WBP1B were determined. With all four proteins the phosphorylated form comigrates with non-phosphorylated protein (Mrabout 9 kDa) on SDS-PAGE. Average molecular masses of reduced WBP1A, WBP1B, WBP2 and WBP3 measured using electrospray ionisation mass spectrometry (ESMS) are 9389 Da, 9274 Da, 9479 Da and 9467 Da, respectively. The complete amino-acid sequences of WBP1A and WBP1B (determined by Edman sequencing and ESMS of proteolytically derived fragments) and N-terminal sequences of WBP2 and WBP3 are highly homologous to each other and to sequences of low molecular weight, basic plant lipid transfer proteins (LTPs).