FIG. 5.
Effects of mutations in VHL exon II on the binding of pVHL to CCT and the assembly of VCB/VCB-Cul2. mRNAs encoding either wild-type or various mutant forms of pVHL were translated in the RRL in the presence of [35S]methionine for 45 min, and aliquots of the reactions were removed and analyzed by native-PAGE. The migration positions of the pVHL-containing complexes are indicated on the right, and the positions of native molecular mass standards are shown on the left. A control translation reaction (−mRNA) was analyzed in lane 1. An exon III mutant, C162F, which does not form VCB and VCB-Cul2 was analyzed as a negative control in lane 9. AS is the naturally occurring alternatively spliced mutant in which all of exon II is deleted. The particular pVHL polypeptide synthesized in each reaction is indicated at the top of the panel.