Pcu3p is modified by Ned8p on lysine 729. (a) Cells containing Pcu3p tagged with 13 Myc epitope tags at the C-terminus (pcu3-13myc cells) were transformed with a pRep3-based plasmid driving the overexpression of a fusion of GST with S. pombe Ned8p. Pcu3p-13Myc was immunoprecipitated and precipitates were analyzed for the presence of Pcu3p and GST-Ned8p with the antibodies indicated. The lower panel shows GST-Ned8p expression in the toal cell lysates. (b) N-terminally Myc epitope-tagged wild-type Pcu3p or point mutants where lysines 729 and 760 were replaced by arginine were expressed from pRep81. Cell lysates were blotted with Myc antibodies to reveal the neddylated and unmodified species of Pcu3p. The comparatively low level of Pcu3p neddylation in this experiment may be attributable to overexpression.