The Cytosolic Domain of Pex22p Stimulates the Pex4p-Dependent Ubiquitination of the PTS1-Receptor
Figure 3
Pex22(C)p enhances autoubiquitination of Pex4p and Pex4p/Pex12p-catalyzed ubiquitination of the PTS1-receptor in vitro.
(a) Autoubiquitination of GST-Pex4p. The effect of Pex22(C)p on the autoubiquitination of GST-Pex4p was examined in vitro using heterologously expressed and purified proteins as indicated. Autoubiquitination of GST-Pex4p depends on the presence of E1 and ubiquitin (Ub). As shown by immunoblot analysis with antibodies against GST and Ub, the autoubiquitination of GST-Pex4p is significantly enhanced in the presence of His-Pex22(C)p. (b) Ubiquitination of the PTS1-receptor Pex5p. Recombinant proteins were used for in vitro ubiquitination of Pex5p, which depends on the presence of E1, GST-Pex4p, Ub as well as the RING-domain of the E3 enzyme Pex12p and was significantly enhanced in presence of His-Pex22(C)p.