Unique requirements for mono- and polyubiquitination of the peroxisomal targeting signal co-receptor, Pex20

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.

FIGURE 1.

Pex20-HA complements the pex20Δ strain. A, growth of Pex20-HA strain compared with wild-type PPY12h and pex20Δ in oleate. The strains used were slxq63, PPY12h, and pex20Δ. B, subcellular fractionation of Pex20-HA. The strains used were slxq63 and PPY12h. C, assessment of peroxisomal protein import. Immunofluorescence microscopy of Pex20-HA and peroxisomal matrix (thiolase) and membrane (Pex12) markers in the slxq63 strain. DIC, differential interference contrast.

FIGURE 2.

Cys⁸ and Lys¹⁹ are required for Pex20 recycling degradation, respectively. A, growth of Pex20-HA and its mutants in oleate. The strains used were slxq63, slxq66, slxq71, slxq96, PPY12h, and pex20Δ. B, stabilities of Pex20-HA and its mutants. The strains used were slxq63, slxq66, slxq71 and slxq96.

FIGURE 3.

Pex20 is mono/diubiquitinated on Cys⁸ and polyubiquitinated on Lys¹⁹. A, ubiquitination of Pex20-HA and its mutants in His₆-Myc-Ub (K48R) overexpression strains. The strains used were slxq65, slxq68, slxq73, and slxq97. B, ubiquitination of Pex20-HA in His₆-Myc-Ub (K48R) and Ub (K48R) overexpression strains. The strains used were slxq65 and slxq174. C, the deletion of the first 6 amino acids and mutation of Pex20 (C8A/K19R) or other lysines do not affect the DTT-resistant monoubiquitination of Pex20 (C8S/K19R)-HA. The strains used were slxq160, slxq170, slxq152 to slxq159, and slxq97. D, the DTT-resistant monoubiquitination band of Pex20 (C8S/K19R)-HA (strain slxq97) was susceptible to hydroxylamine and NaOH. Cell extracts made for the ubiquitination assay were incubated with hydroxylamine or NaOH at 30 °C for 1 h before SDS-PAGE.

FIGURE 4.

The majority of peroxins (Pex20-HA, Pex2, Pex10, and Pex12), PTS2 cargo thiolase and G6PDH (cytosolic marker) are mostly correctly localized in RING domain point mutants. The strains used were slxq167, slxq168, slxq169, and slxq63.

FIGURE 5.

All three RING peroxins are required for both mono- and polyubiquitination of Pex20. A, growth curve in oleate for RING domain point mutants. The strains used were slxq167, slxq168, slxq169, and slxq63. B, stabilities of RING peroxins in WT and mutants. The strains used were slxq167, slxq168, slxq169, slxq63, pex2Δ, pex10Δ, and pex12Δ. C, RING domain point mutations affect the ubiquitination of Pex20-HA. The strains used were slxq126, slxq127, slxq128, and slxq65. D, RING domain point mutations affect the mono/diubiquitination of Pex20 (K19R)-HA. The strains used were slxq73, slxq103, slxq104, and slxq105. E, RING domain point mutations affect the polyubiquitination of Pex20 (C8S)-HA. The strains used were slxq99, slxq100, slxq101, and slxq68.

FIGURE 6.

Pex4 is required for Pex20 mono/diubiquitination on Cys⁸ and also its polyubiquitination on Lys¹⁹. A, the pex4Δ deletion affected Pex20 (K19R)-HA mono/diubiquitination. The strains used were slxq73 and slxq84. B, the pex4Δ deletion affected Pex20 (C8S)-HA polyubiquitination at shorter time points (1–3 h). The strains used were slxq83, slxq68, and slxq129. C, the pex4Δ deletion stabilized Pex20 (C8S)-HA. The strains used were slxq175 and slxq66.

FIGURE 7.

Pex7 is only required for Pex20 polyubiquitination. A, the pex7Δ deletion did not affect Pex20 (K19R)-HA mono/diubiquitination. The strains used were slxq73 and slxq140. B, the S280F mutation or pex7Δ deletion affected Pex20 (C8S)-HA polyubiquitination. The strains used were slxq68, slxq57, and slxq139. C, the S280F mutation or the pex7Δ deletion stabilized Pex20 (C8S)-HA. The strains used were slxq53, slxq59, slxq66, and slxq63. D, subcellular fractionation and protease protection assay of the P200 fraction isolated from a PNS of Pex20 (C8S)-HA + pex7Δ and Pex20-HA. The strains used were slxq59 and slxq63.

FIGURE 8.

All three RING peroxins are required for both mono- and polyubiquitination of Pex5. A, the ubiquitination of Pex5-HA and its mutants in His₆-Myc-Ub (K48R) overexpression strains. The strains used were slxq122, slxq123, slxq124, and slxq125. B, RING domain point mutations affect the ubiquitination of Pex5-HA and its mutants. The strains used were slxq122, slxq189, slxq190, slxq191, slxq124, slxq144, slxq145, slxq146, slxq123, slxq141, slxq142, and slxq143.

FIGURE 9.

Model for Pex20 ubiquitination during its recycling and RADAR. The numbers denote Pex proteins assigned these designated numbers. C8 and K19 represent Cys⁸ and Lys¹⁹ on Pex20, respectively. Single or multiple circles with Ub denote mono- or poly-Ub on Cys⁸ or Lys¹⁹ of Pex20, respectively. Pex20 interacts with Pex7 during PTS2 cargo import. After cargo release, Pex20 is recycled back to the cytosol for the next round of import. The mono- and diubiquitination of Pex20 occurs on Cys⁸, and its recycling does not require Pex7 or interaction between Pex20 and Pex7. When the recycling machinery is impaired by mutation, Pex20 interaction with Pex7 and its subsequent polyubiquitination on Lys¹⁹ are necessary for Pex20 degradation (Pex20 cloud) by the ubiquitin proteasome system (UPS). Pex2, Pex10, and Pex12 function as E3 ubiquitin ligases, which are required for both mono- and polyubiquitination of Pex20. Pex4 functions as the E2 ubiquitin-conjugating enzyme, which is not only required for Pex20 mono- and diubiquitination on Cys⁸ during its recycling but also as a nonessential kinetic component for a step in the polyubiquitination of Pex20 on Lys¹⁹.