Noncanonical and reversible cysteine ubiquitination prevents the overubiquitination of PEX5 at the peroxisomal membrane

Tânia Francisco; Ana G Pedrosa; Tony A Rodrigues; Tarad Abalkhail; Hongli Li; Maria J Ferreira; Gerbrand J van der Heden van Noort; Marc Fransen; Ewald H Hettema; Jorge E Azevedo

doi:10.1371/journal.pbio.3002567

Noncanonical and reversible cysteine ubiquitination prevents the overubiquitination of PEX5 at the peroxisomal membrane

PLoS Biol. 2024 Mar 12;22(3):e3002567. doi: 10.1371/journal.pbio.3002567. eCollection 2024 Mar.

Authors

Tânia Francisco^{1

2

3}, Ana G Pedrosa^{1

2

3}, Tony A Rodrigues^{1

2

3}, Tarad Abalkhail⁴, Hongli Li⁵, Maria J Ferreira^{1

2

3}, Gerbrand J van der Heden van Noort⁶, Marc Fransen⁵, Ewald H Hettema⁴, Jorge E Azevedo^{1

2

3}

Affiliations

¹ Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal.
² Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
³ Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal.
⁴ School of Biosciences, University of Sheffield, Sheffield, United Kingdom.
⁵ Laboratory of Peroxisome Biology and Intracellular Communication, Department of Cellular and Molecular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.
⁶ Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands.

Abstract

PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown. Using an established rat liver-based cell-free in vitro system, we found that, in contrast to wild-type PEX5, a PEX5 protein possessing a lysine at position 11 is polyubiquitinated at the peroxisomal membrane, a modification that negatively interferes with the extraction process. Wild-type PEX5 cannot retain a polyubiquitin chain because ubiquitination at cysteine 11 is a reversible reaction, with the E2-mediated deubiquitination step presenting faster kinetics than PEX5 polyubiquitination. We propose that the reversible nonconventional ubiquitination of PEX5 ensures that neither the peroxisomal protein translocon becomes obstructed with polyubiquitinated PEX5 nor is PEX5 targeted for proteasomal degradation.

Copyright: © 2024 Francisco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

MeSH terms

Animals
Carrier Proteins / metabolism
Cysteine* / metabolism
Lysine* / metabolism
Peroxisome-Targeting Signal 1 Receptor / chemistry
Peroxisome-Targeting Signal 1 Receptor / metabolism
Protein Transport
Rats
Ubiquitination

Substances

Carrier Proteins
Cysteine
Lysine
Peroxisome-Targeting Signal 1 Receptor
Pex5 protein, rat

Grants and funding

This work was funded by National Funds through FCT — Fundação para a Ciência e a Tecnologia, I.P., under the projects UIDB/04293/2020 (to JEA), EXPL/BIA-MOL/1664/2021 “Investigating the molecular mechanisms of mammalian pexophagy” (to TAR), 2022.08378.PTDC “A new component of the mammalian peroxisomal protein import machinery” (to JEA) and 2023.02428.RESTART "Unveiling peroxisomal homeostasis mechanisms – the role of LonP2” (to TF), funded by FEDER (Fundo Europeu de Desenvolvimento Regional), through COMPETE 2020-Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the projects: “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), and through Norte 2020 - Programa Operacional Regional do Norte, under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S” (NORTE-01-0145-FEDER-000008). TF, AGP, TAR and MJF are supported by Fundação para a Ciência e Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu. GJvdHvN acknowledges funding by NWO (VIDI grant VI.192.011; to GJvdHvN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.