Figure 1.

Nedd4-2 exhibits broad specificity for the Ubc4/5 E2 clade in assembling free lysine 63–linked polyubiquitin chains. A, kinetic assays of ¹²⁵I-polyubiquitin chain assembly were carried out for 20 min at 37 °C containing 2 nm GST-Nedd4-2 in the absence (lane 1) or presence (lanes 2–10) of a 200 nm concentration of the indicated E2 carrier proteins. Reactions were quenched by the addition of SDS sample buffer containing 2% (v/v) β-mercaptoethanol. Reaction products were resolved by SDS-PAGE at 4 °C, and the resulting gel was dried and visualized by autoradiography as described under “Materials and methods.” B, ¹²⁵I-polyubiquitin conjugation reactions containing 100 nm Uba1, 200 nm Ubc5B, 1 nm GST-Nedd4-2, and 5 μm ¹²⁵I-ubiquitin (lane 1); 1 μm ¹²⁵I-ubiquitin and 4 μm unlabeled wild-type ubiquitin (lane 2); or unlabeled single lysine-to-arginine ubiquitin point mutants (lanes 3–9) were carried out for 15 min at 37 °C. Reaction products were resolved by SDS-PAGE and visualized by autoradiography as described in A. The positions of mobility standards are shown to the left; the position for free ¹²⁵I-ubiquitin is shown to the right.