FIG. 6.

Similar phosphorylation patterns obtained for the VPgs of PVA strains B11 and B11-M by using kinase activity from tobacco. (A) Schematic map of the recombinant VPg molecules. The amino acid differences between VPgs B11^wt, M^wt, B11^Ser185Leu, and M^Leu185Ser and the positions of the changed amino acids in VPg are indicated. (His)₆, six-His tag. (B) Recombinant VPgs B11^wt, M^wt, B11^Ser185Leu, and M^Leu185Ser were phosphorylated in the presence of 5 mM Mn ²⁺ in vitro with a total protein extract of tobacco as the kinase source. Phosphorylated proteins were purified by SDS-PAGE, blotted onto nylon membrane, and visualized with Ponceau S. VPg was digested on the membrane with trypsin, and the released peptides were lyophilized and separated on TLE/TLC plates. Radioactive ³³P-labeled peptides were autoradiographically visualized.