FIG. 1.
Order of events in the assembly pathway of the VCB and VCB-Cul2 complexes. (Top) VHL mRNA was translated in the RRL in the presence of [35S]methionine for 4 min, and then inhibitors of initiation of translation were added, followed by continued incubation. Equal aliquots were removed at the times indicated after the beginning of the reaction and analyzed by native-PAGE. A fluorogram of the gel is shown. The migration positions of different pVHL-containing complexes are indicated on the right as I, II, and III (based on their apparent order of appearance). Native molecular mass standards are shown on the left. A control translation reaction in which no mRNA was added (−mRNA) was analyzed on the left. The position of migration of species not dependent upon VHL mRNA (i.e., which was present in the no-added mRNA column) is indicated by an asterisk. (Bottom) Aliquots of the same samples examined in the top panel were analyzed by SDS-PAGE. A fluorogram of the gel is shown. The migration positions of molecular mass standards are shown on the left.