Identification of Ubiquitin-specific Protease 9X (USP9X) as a Deubiquitinase Acting on Ubiquitin-Peroxin 5 (PEX5) Thioester Conjugate

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Title Identification of Ubiquitin-specific Protease 9X (USP9X) as a Deubiquitinase Acting on Ubiquitin-Peroxin 5 (PEX5) Thioester Conjugate
Author Grou, Cláudia P.; Francisco, Tânia; Rodrigues, Tony A.; Freitas, Marta O.; Pinto, Manuel P.; Carvalho, Andreia F.; Domingues, Pedro; Wood, Stephen Andrew; Rodriguez-Borges, José E.; Sa-Miranda, Clara; Fransen, Marc; Azevedo, Jorge E.
Journal Name Journal of Biological Chemistry
Year Published 2012
Place of publication United States
Publisher American Society for Biochemistry and Molecular Biology, Inc.
Abstract Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.
Peer Reviewed Yes
Published Yes
Alternative URI http://dx.doi.org/10.1074/jbc.M112.340158
Volume 287
Issue Number 16
Page from 12815
Page to 12827
ISSN 0021-9258
Date Accessioned 2012-05-25; 2012-09-21T04:17:29Z
Date Available 2012-09-21T04:17:29Z
Research Centre Eskitis Institute for Drug Discovery
Faculty Faculty of Science, Environment, Engineering and Technology
Subject Biochemistry and Cell Biology
URI http://hdl.handle.net/10072/46986
Publication Type Journal Articles (Refereed Article)
Publication Type Code c1

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