Distinct ubiquitination cascades act on the peroxisomal targeting signal type 2 co-receptor Pex18p

Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by a peroxisomal targeting sequence (PTS) and ferry them to the peroxisomal membrane. Subsequently, the cargo is translocated into the peroxisomal lumen, whereas the receptor is released to the cytosol for further rounds of protein import. This cycle is controlled by the ubiquitination status of the receptor, which is best understood for the PTS1-receptor. While polyubiquitination of PTS-receptors results in their proteasomal degradation, the monoubiquitinated PTS-receptors are exported to the cytosol and recycled for further rounds of protein import. Here, we describe the identification of two ubiquitination cascades acting on the PTS2 co-receptor Pex18p. Using in vivo and in vitro approaches, we demonstrate that the polyubiquitination of Pex18p requires the ubiquitin-conjugating enzyme (E2) Ubc4p, which cooperates with the RING (really interesting new gene)-type ubiquitin-protein ligases (E3) Pex2p as well as Pex10p. Monoubiquitination of Pex18p depends on the E2 enzyme Pex4p (Ubc10p), which functions in concert with the E3 enzymes Pex12p and Pex10p. Our findings for the PTS2-pathway complement the data on PTS1-receptor ubiquitination and add up to a unified concept of the ubiquitin-based regulation of peroxisomal import.