Figure 3

Purified ^tm 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). ^FSCF^Cdc4-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 ^FSCF^Cdc4 (2 pmol) followed by 10% SDS-PAGE and α-Sic1 WB. Reactions shown in lanes 4, 8 and 12 do not have Sic1. (D). ^GstSCF^Met30-dependent polyubiquitination of Met4. Tests were performed as described in C except that with the ^GstSCF^Met30 E3 and ^FMet4 substrate. (E). ^GstSCF^Met30-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.