Abstract
In Pichia pastoris, the peroxisomal targeting signal 2 (PTS2)-dependent peroxisomal matrix protein import pathway requires the receptor, Pex7, and its co-receptor Pex20. A conserved lysine (Lys(19)) near the N terminus of Pex20 is required for its polyubiquitination and proteasomal degradation, whereas a conserved cysteine (Cys(8)) is essential for its recycling. In this study, we found that Cys(8) is required for the DTT-sensitive mono- and diubiquitination of Pex20. We also show that the PTS2 cargo receptor, Pex7, is required for Pex20 polyubiquitination. Pex4, the E2 ubiquitin-conjugation enzyme, is required for monoubiquitination of Pex20. However, it is also necessary for polyubiquitination of Pex20, making its behavior distinct from the ubiquitination described for other PTS receptors. Unlike the roles of specific RING peroxins in Pex5 ubiquitination, we found that all the RING peroxins (Pex2, Pex10, and Pex12) are required as E3 ubiquitin ligases for Pex20 mono- and polyubiquitination. A model for Pex20 ubiquitination is proposed based on these observations. This is the first description of the complete ubiquitination pathway of Pex20, which provides a better understanding of the recycling and degradation of this PTS2 cargo co-receptor.
Highlights
Pex20 recycling and degradation depend on its ubiquitination
This is the first description of the complete ubiquitination pathway of Pex20, which provides a better understanding of the recycling and degradation of this peroxisomal targeting signal 2 (PTS2) cargo co-receptor
This is the first description of the complete ubiquitination pathway of Pex20, which provides new roles of Pex4, Pex7, and RING peroxins in Pex20 mono/polyubiquitination, thereby providing a better understanding of the recycling and degradation of this PTS2 cargo co-receptor
Summary
Pex recycling and degradation depend on its ubiquitination. Results: Ubiquitination of Pex and the roles of the E2 enzyme Pex, the RING peroxins (Pex2/Pex10/Pex12), and Pex in Pex ubiquitination were determined in vivo. Most of these insights on the sites and enzymes involved in these ubiquitination steps and the biological role of mono- or polyubiquitination of PTS receptors have come from studies on the PTS1 receptor, Pex, and to a far lesser extent from studies on the PTS2 pathway co-receptor Pex18/Pex20 Understanding these processes for the Pex family of proteins, the subject of this paper, is essential for a complete understanding of the PTS2 import pathway, as well as for an appreciation of the coevolution of the PTS1 and PTS2 import pathways. A model for Pex ubiquitination is proposed based on these observations This is the first description of the complete ubiquitination pathway of Pex, which provides new roles of Pex, Pex, and RING peroxins in Pex mono/polyubiquitination, thereby providing a better understanding of the recycling and degradation of this PTS2 cargo co-receptor
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