Purification, characterization and sequencing of a family of Petunia petal lipid transfer proteins phosphorylated by plant calcium-dependent protein kinase

Abstract

A number of small basic proteins (Pet1, Pet2, Pet3, Pet4 and Pet5) were purified to homogeneity from petals of petunia ( Petunia hybrida var. Old Glory Blue) by a procedure involving batchwise cation exchange chromatography on carboxymethyl-cellulose (CM52) and cation exchange HPLC on a sulphopropyl-based SP5PW column. Pet1 to Pet5 were identified from extensive N-terminal sequencing as having a high degree of sequence homology to each other and to other plant phospholipid transfer proteins. Pet1, Pet2, Pet4 and Pet5 are phosphorylated by wheat embryo Ca 2+-dependent protein kinase (CDPK) whereas Pet3 is a very poor substrate for this enzyme. After tryptic digestion of [ 32P]phosphoPet1 and [ 32P]phosphoPet2 phosphorylated by CDPK and reversed phase HPLC-based purification of [ 32P]phosphopeptides, Edman sequencing of the purified [ 32P]phosphopeptides revealed major Ser phosphorylation sites of S 40 and S 70 in the sequences SQAS 40TTP and GLPS 70TCG in Pet1 and Pet2, respectively. These phosphorylation site sequences differ from the Basic-X-X-Ser(Thr) motif found with many synthetic peptide substrates of plant CDPK but are similar to each other and to phosphorylation sites on some other CDPK substrates involving A G , S T and P residues. The complete primary structure of Pet2 (90 residues) has been determined by Edman sequencing and electrospray ionization mass spectrometry of the native Pet2 and of proteolytically-generated fragments. While Pet2(19–90) and Pet2(32–90) retain activity as CDPK substrates, all smaller fragments tested were inactive. A CDPK is present in Petunia petals. After in vivo labelling of Petunia petals with [ 32P]phosphate the major labelled protein resolved by SDS-PAGE has a mass of 9.4 ± 0.6 kDa, similar to the M r of Pet1, Pet2 and Pet3.