FIGURE 5.

Fusion of human Cdc34-Δ190 to Cul1 can partially rescue the defect of Cdc34 tail deletion in ubiquitylation reactions. A, di-ubiquitin synthesis assay comparing wild type and Cdc34-Δ190 in the presence of Rbx1+Cul1. B, comparison of wild type (WT) and Cdc34-Δ190 fusions to Cul1. Reactions containing either 300 nm Rbx1+Cul1 and 300 nm Cdc34 or containing 300 nm Rbx1+Cul1-Cdc34, 0.7 μm human E1, 6 μm ³²P-labeled K48R ubiquitin, and 50 μm D77 ubiquitin were incubated at 20–22 °C for the specified times and quenched with SDS-PAGE buffer. No products were observed when Cdc34-Δ190 was assayed as an unfused species. C and D, di-ubiquitin synthesis assay comparing Cul1 fusions to either wild type Cdc34 (C) or Δ190 Cdc34 (D). The reactions containing 300 nm Rbx1+Cul1-Cdc34, 0.7 μm human E1, 6 μm ³²P-labeled K48R ubiquitin, and 50 μm D77 ubiquitin were incubated at 20–22 °C for the specified times and quenched with SDS-PAGE buffer. E, Ub-β-catenin (25) ubiquitylation reactions comparing unfused wild type and Cdc34-Δ190 (300 nm) in the presence of Rbx1+Cul1 (300 nm) or of wild type and Cdc34-Δ190 fusions (1.2 μm). SCF^βTrCP complex formation was accomplished by briefly mixing equimolar amounts of either Rbx1+Cul1 or the Rbx1+Cul1-Cdc34 fusions with βTrCP prior to initiation of the reaction by the addition of substrate. The reactions also contained 0.7 μm E1, 75 μm Ub, and 3 μm ³²P-labeled Ub-β-catenin peptide. The reactions were incubated at 20–22 °C for the specified times. All of the experiments were done in duplicate.