Chemically monoubiquitinated PEX5 binds to the components of the peroxisomal docking and export machinery

Vera Hagmann,Henning D Mootz,Jan Bierlmeier,Gabriele Dodt,Patrick Fabian,Nadine Van Treel,Jorge E Azevedo,Stefanie Sommer,Dirk Schwarzer

doi:10.1038/s41598-018-34200-5

Vera Hagmann, Henning D Mootz + Show 7 more

Open Access

https://doi.org/10.1038/s41598-018-34200-5

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Abstract

Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. “Click” chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.

Highlights

Mammalian peroxisomes are single membrane-bound organelles that do not contain DNA or RNA
In vitro analysis revealed that H6-PEX5-Ub-Strep binds PEX7/PTS2 proteins (Fig. 2a and Supplementary Fig. 3) and interacts with the docking translocation module (DTM) components PEX14 and PEX13 (Fig. 4)
We have no evidence that the binding of H6-PEX5-Ub-Strep to PEX7/PTS2 proteins or to DTM proteins is affected by the ubiquitination

Summary

Introduction

Mammalian peroxisomes are single membrane-bound organelles that do not contain DNA or RNA. PEX5L contains an insert of 37 amino acids that is positioned after amino acids 214 of PEX5S This insert contains part of the binding-site for the PEX7/PTS2 cargo complex and only PEX5L (hereafter referred to as PEX5) can transport PTS2 proteins to the peroxisome[13,14]. After binding their cargos in the cytosol, PEX5 or PEX5/PEX7 interact with the peroxisomal membrane docking translocation module (DTM), which in mammals comprises the peroxins 14 and 1315 and the three RING (really interesting new gene) finger proteins PEX2, PEX10, and PEX1216. Using a copper(I)-catalysed alkyne-azide cycloaddition (CuAAC, known as “click” chemistry), a strategy frequently employed to produce proteins modified with ubiquitin and ubiquitin-like proteins[42,43,44], we have generated large amounts of recombinant human PEX5 possessing a single ubiquitin molecule covalently attached to its residue 11

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