FIG. 6.
Autoubiquitination. Cdc34 autoubiquitination was assayed for Cdc34 and its various derivatives by using an in vitro ubiquitination reaction. Reaction mixtures contained Cdc34 or one of its derivatives (100 nM), Uba1 (10 nM), 35S-Ub (200 nM), and an ATP cocktail and were incubated for 8 h at 30°C, representing an end point assay for autoubiquitination. DTT (100 mM) was added to stop the reactions, and the reaction products were analyzed by SDS-PAGE followed by autoradiography. The positions of free Ub (Ub), di-Ub (Ub2), and multi-Ub chains covalently linked to Cdc34 (Cdc34-Ubn) are indicated. SS, S73K/S97D derivative; SSΔ12, S73K/S97D/Δ12 derivative; Δ12, catalytic domain insert deletion (residues 103 to 114 deleted); Δ209 and Δ185, carboxy-terminal truncation derivatives (residues 1 to 209 and 1 to 185, respectively). A downward shift in molecular mass in the Ub chains on the Δ12 derivatives is observed, consistent with the catalytic domain insert deletion. Bands not indicated are degradation products of Cdc34-Ub.