Fig. 3.

Activity in xUB transfer by the E6AP mutants identified from yeast cell selection. a Activity of the HECT mutants YW1–6 in auto-ubiquitination by xUB through the xUba1–xUbcH7 pair. b Activity of the full-length E6AP mutants fYW1, fYW4, and fYW6 in auto-ubiquitination by the xUba1–xUbcH7 pair (right panel). While wt E6AP can be auto-ubiquitinated by the wt Uba1–UbcH7 pair (left panel), it cannot be conjugated to xUB by the xUba1–xUbcH7 pair (right panel). c p53 ubiquitination by wt E6AP and fYW4 and fYW6 in the presence of E6. p53 can be efficiently ubiquitinated by wt UB through the wt Uba1–UbcH7–E6AP cascade in the presence of E6 (left panel). p53 can also be efficiently modified by xUB through the engineered cascade of xUba1–xUbcH7–fYW6 in the presence of E6 (right panels). In contrast, xUB has very low activity in ubiquitinating p53 through the crossover cascade of xUba1–xUbcH7–wt E6AP in the presence of E6. All blots are representative of at least three independent experiments