The Extraction Mechanism of Monoubiquitinated PEX5 from the Peroxisomal Membrane

• Published in: Journal of molecular biology 2023-01, Vol.435 (2), p.167896-167896, Article 167896
• Main Authors: Pedrosa, Ana G., Francisco, Tânia, Rodrigues, Tony A., Ferreira, Maria J., van der Heden van Noort, Gerbrand J., Azevedo, Jorge E.
• Format: Article
• Language: English
• Subjects: adenosinetriphosphatase; ATPases Associated with Diverse Cellular Activities - metabolism; Cell-Free System; crosslinking; Humans; Membrane Proteins - metabolism; molecular biology; peroxisome; Peroxisome-Targeting Signal 1 Receptor - metabolism; Peroxisomes - metabolism; PEX1; PEX6; polypeptides; protein import; Protein Transport; ubiquitin; Ubiquitin - metabolism; Ubiquitination
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2022.167896

[Display omitted] •Extraction of PEX5 from the peroxisome membrane requires its ubiquitination but the role of this modification is unclear.•We found that the ubiquitin moiety of the ubiquitin-PEX5 conjugate is unfolded at a pre-extraction stage.•Accordingly, modification of PEX5 with a difficult-to-unfold ubiquitin species blocks extraction at a pre-extraction stage.•This suggests that the PEX5-linked ubiquitin is the extraction initiator and that the complete ubiquitin-PEX5 conjugate is threaded by the receptor extraction machinery. The AAA ATPases PEX1•PEX6 extract PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal 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.