Pex2 and pex12 function as protein-ubiquitin ligases in peroxisomal protein import.

The PTS1-dependent peroxisomal matrix protein import is facilitated by the receptor protein Pex5 and can be divided into cargo recognition in the cytosol, membrane docking of the cargo-receptor complex, cargo release, and recycling of the receptor. The final step is controlled by the ubiquitination status of Pex5. While polyubiquitinated Pex5 is degraded by the proteasome, monoubiquitinated Pex5 is destined for a new round of the receptor cycle. Recently, the ubiquitin-conjugating enzymes involved in Pex5 ubiquitination were identified as Ubc4 and Pex4 (Ubc10), whereas the identity of the corresponding protein-ubiquitin ligases remained unknown. Here we report on the identification of the protein-ubiquitin ligases that are responsible for the ubiquitination of the peroxisomal protein import receptor Pex5. It is demonstrated that each of the three RING peroxins Pex2, Pex10, and Pex12 exhibits ubiquitin-protein isopeptide ligase activity. Our results show that Pex2 mediates the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.

Ubiquitination of the peroxisomal import receptor Pex5p is required for its recycling.

Pex5p, which is the import receptor for peroxisomal matrix proteins harboring a type I signal sequence (PTS1), is mono- and polyubiquitinated in Saccharomyces cerevisiae. We identified Pex5p as a molecular target for Pex4p-dependent monoubiquitination and demonstrated that either poly- or monoubiquitination of the receptor is required for the ATP-dependent release of the protein from the peroxisomal membrane to the cytosol as part of the receptor cycle. Therefore, the energy requirement of the peroxisomal import pathway has to be extended by a second ATP-dependent step, namely receptor monoubiquitination.