The Saccharomyces cerevisiae (Sc) PTS1 import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and a Pex4p (Ubc10p)-dependent manner. Both of these modifications require the RING domain-containing protein Pex10p in vivo, but the actual role this protein plays in the ubiquitination of Pex5p has so far, remained enigmatic. Here, we report that the RING domain of Pex10p exhibits E-3 ligase activity in vitro, in combination with the human E-2 enzyme UbcH5a, a homologue of ScUbc4p, but not when ScPex4p was used as an E-2 enzyme in the reaction. We have further characterised Pex10p's E-3 ligase activity using mutants designed to disturb this activity and show that Pex10p acts as the E-3 ligase for Ubc4p-dependent ubiquitination of Pex5p but not Pex4p-dependent ubiquitination in vivo. These data imply that the two distinct Pex5p modifications require different E-3 ligases, as well as different E-2 enzymes. (c) 2008 Elsevier Inc. All rights reserved.