Radiolabeling and Degradation of Ubiquitin Substrates

Ub5-DHFR (a gift from Millennium Pharmaceuticals) was radiolabeled using PKA (Sigma) and [γ-³²P]ATP^{40 (link)}. Sic1PY was radiolabeled using casein kinase II and [γ-³²P]ATP (NEB), followed by ubiquitination using Rsp5 as described previously (Ubn-Sic1)^{46 (link)}. Degradation of these substrates (50 nM) by 26S proteasomes (1 nM) was assayed in the presence of 50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 2 mM ATP, 1 mM DTT, 0.01 mg/ml BSA (Sigma) as reported^{47 (link),48 (link)} and measured by after the conversion of the substrate to trichloroacetic acid (TCA)-soluble ³²P-labeled peptides. The reaction of 26S was carried out at 37 °C for 0, 10, 20 or 30 min with Ub5-DHFR and at 0, 3 or 9 min with Ubn-Sic1.