Fig. 4.

Hrd1 RING is incapable of activating Ubc7_E118. (A) Hrd1-mediated activation of diUb formation. A single-round Ub-turnover assay was performed as described in Fig. 2C using Ubc7, Ubc7_R118, or Ubc7_E118 as Ub donors in the presence of Hrd1 RING. The reaction was incubated for 30 min at 30 °C; then diUb formation was determined. Arrow indicates the migration distance of diUb. (B) Hrd1 activation of polyUb chain formation by the indicated Ubc7 variants. The ubiquitylation reaction was carried out as described in Fig. 2D and was terminated after 30 min. (C) Kinetics of Ub∼Ubc7 thiol ester formation. The Ubc7:Cue1 dimer was incubated at 30 °C with E1 and FlagUb_R48 for the indicated time periods. Proteins were resolved on SDS/PAGE and were subjected to immunoblot with anti-Flag Abs. (D) Degradation of KHN in cells expressing the indicated Ubc7 variants was determined by CHX-chase as described in Fig. 1A.