Fig. 5. Effects of E2 asparagine mutations on E3-dependent isopeptide bond synthesis. (A) Mdm2-dependent ubiquitylation of p53 was assayed with UbcH5B (5B, positive control), no E2 (–, negative control), or two different concentrations (1×, 2×) of wild-type UbcH5A (N) or UbcH5A-N77Q (Q). A western blot was developed with p53 antibodies. (B) Mdm2 autoubiquitylation was monitored with radiolabeled Ub [autoradiograph; annotation is the same as in (A)]. The blot shown in (A) was also stripped and re-probed with an Mdm2 antibody to confirm that autoubiquitylation was impaired by the N77Q mutation (data not shown). (C) AO7-catalyzed autoubiquitylation was assayed [autoradiograph, annotation is the same as in (A)]. (D) Ubc13-N79A cannot support DNA damage tolerance in vivo. A yeast mms2Δubc13Δ strain (Hofmann and Pickart, 1999) was transformed with empty vectors (diamonds), plasmids expressing Mms2 and HA-tagged wild-type Ubc13 (squares) or plasmids expressing Mms2 and HA-tagged Ubc13-N79A (circles). Inset, Ubc13-N79A is highly expressed (western blot developed with HA antibodies). Asterisk, cross-reacting band (loading control; the wild-type lane is underloaded). (E) KIAA10 (HECT) E3-dependent synthesis of K48-linked polyUb chains (Coomassie-stained gel). E3 was omitted from lanes 1 and 5 (negative controls). Numbers denote chains consisting of the indicated number of Ub units. Asterisk denotes bovine serum albumin (carrier protein).