4.7 Article

The Extraction Mechanism of Monoubiquitinated PEX5 from the Peroxisomal Membrane

Journal

JOURNAL OF MOLECULAR BIOLOGY
Volume 435, Issue 2, Pages -

Publisher

ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2022.167896

Keywords

PEX1; PEX6; ubiquitin; peroxisome; protein import

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The study investigated the extraction mechanism of PEX5 using a cell-free in vitro system and various engineered molecules. It was found that ubiquitinated PEX5 can be extracted, and the N- and C-terminus of PEX5 are not crucial for the extraction process. The study also revealed that the PEX5-linked monoubiquitin is unfolded prior to extraction, and intramolecularly cross-linked ubiquitin at residue 11 of PEX5 inhibits extraction.
The AAA ATPases PEX1.PEX6 extract PEX5, the peroxisomal protein shuttling receptor, from the perox-isomal membrane so that a new protein transport cycle can start. Extraction requires ubiquitination of PEX5 at residue 11 and involves a threading mechanism, but how exactly this occurs is unclear. We used a cell-free in vitro system and a variety of engineered PEX5 and ubiquitin molecules to challenge the extraction machinery. We show that PEX5 modified with a single ubiquitin is a substrate for extraction and extend previous findings proposing that neither the N-nor the C-terminus of PEX5 are required for extraction. Chimeric PEX5 molecules possessing a branched polypeptide structure at their C-terminal domains can still be extracted from the peroxisomal membrane thus suggesting that the extraction machinery can thread more than one polypeptide chain simultaneously. Importantly, we found that the PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and, accordingly, an intra-molecularly cross-linked ubiquitin blocked extraction when conjugated to residue 11 of PEX5. Collectively, our data suggest that the PEX5-linked monoubiquitin is the extraction initiator and that the complete ubiquitin-PEX5 conjugate is threaded by PEX1.PEX6. (c) 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecom-mons.org/licenses/by/4.0/).

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