PURIFICATION, CHARACTERIZATION AND SEQUENCING OF A FAMILY OF PETUNIA PETAL LIPID TRANSFER PROTEINS PHOSPHORYLATED BY PLANT CALCIUM-DEPENDENT PROTEIN-KINASE

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 Ca2+-dependent protein kinase (CDPK) whereas Pet3 is a very poor substrate for this enzyme. After tryptic digestion of [P-32]phosphoPet1 and [P-32]phosphoPet2 phosphorylated by CDPK and reversed phase HPLC-based purification of [P-32]phosphopeptides, Edman sequencing of the purified [P-32]phosphopeptides revealed major Ser phosphorylation sites of S-40 and S-70 in the sequences SQAS(40)TTP and GLPS(70)TCG 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 oil 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 [P-32]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.