Cdc34 forms ubiquitin thiolester and monoubiquitinates substrates, but is defective in substrate polyubiquitination. All assays were performed at 25°C in 10-20 μL and included 1 pmol of Uba1 E1 and 1.3 nmol of ubiquitin or its derivative. (A). Formation of ubiquitin-thiolester. The indicated Cdc34244 proteins with wt, tm or Δ12 E2 core (1-4 pmol) were incubated for 5 minutes with Uba1 and ubiquitin followed by SDS-PAGE under reducing (+ βME) or non-reducing (- βME) conditions and α-Cdc34 WB. (B). Autoubiquitination of full length Cdc34 and tmCdc34. Full length Cdc34 or tmCdc34 (5 pmol) was incubated for the times indicated with Uba1 and ubiquitin or methylated ubiquitin followed by α-Cdc34 WB. (C). FSCFCdc4-dependent polyubiquitination of Sic1. The indicated Cdc34244 proteins (5 pmol) were incubated for the times indicated with Uba1, the Sic1/Clb5/GstCdc28 substrate complex (2 pmol) and FSCFCdc4 (2 pmol) followed by 10% SDS-PAGE and α-Sic1 WB. Reactions shown in lanes 4, 8 and 12 do not have Sic1. (D). GstSCFMet30-dependent polyubiquitination of Met4. Tests were performed as described in C except that with the GstSCFMet30 E3 and FMet4 substrate. (E). GstSCFMet30-dependent monoubiquitination of FMet4. Tests as in D, but analyzed with shorter (1 instead of 5 minutes) western blot exposure time, which is necessary to visualize unubiquitinated and monoubiquitinated FMet4 as separate species due to similarities in their molecular weights.