Figure 1.

Pex22p acts as a specific co-activator for Pex4p in vitro. (A–E) E2 self-ubiquitination assays containing some of the following: 0.2 μM E1 enzyme, 11 μM wild type (WT) or a lysine 48 to arginine mutant form (K48R) of Ub (Sigma), 5 mM ATP, 1.2 μM Pex4^15–183, 1.2 μM Ubc4p and 1.7 μM Pex22^S, probed with either anti-Pex4p (A, C) or anti-Ub antibodies (B, D and E). ^*Defines an anti-Ub crossreactive species, likely to be Ubc4p. The presence of Pex22^S stimulates the ubiquitin-conjugating activity of Pex4^15–183 and additionally allows Pex4^15–183 to produce Ub chains (A, B), linked through lysine 48 in Ub (C, D) but does not affect Ubc4p's activity (E).