Peroxisome targeting signal type-1 (PTS1) receptor, Pex5p, is a key player in peroxisomal matrix protein import. Pex5p recognizes PTS1 cargoes in the cytosol, targets peroxisomes, translocates across the membrane, unloads the cargoes, and shuttles back to the cytosol. Ubiquitination of Pex5p at a conserved cysteine is required for the exit from peroxisomes. However, any potential ubiquitin ligase (E3) remains unidentified in mammals. Here, we establish an in vitro ubiquitination assay system and demonstrate that RING finger Pex10p functions as an E3 with an E2, UbcH5C. The E3 activity of Pex10p is essential for its peroxisome-restoring activity, being enhanced by another RING peroxin, Pex12p. The Pex10p·Pex12p complex catalyzes monoubiquitination of Pex5p at one of multiple lysine residues in vitro, following the dissociation of Pex5p from Pex14p and the PTS1 cargo. Several lines of evidence with lysine-to-arginine mutants of Pex5p demonstrate that Pex10p RING E3-mediated ubiquitination of Pex5p is required for its efficient export from peroxisomes to the cytosol and peroxisomal matrix protein import. RING peroxins are required for both modes of Pex5p ubiquitination, thus playing a pivotal role in Pex5p shuttling.
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