The Structure of the Arabidopsis PEX4-PEX22 Peroxin Complex-Insights Into Ubiquitination at the Peroxisomal Membrane

• Published in: Frontiers in cell and developmental biology 2022-02, Vol.10, p.838923
• Main Authors: Traver, Melissa S, Bradford, Sarah E, Olmos, Jr, Jose Luis, Wright, Zachary J, Miller, Mitchell D, Xu, Weijun, Phillips, Jr, George N, Bartel, Bonnie
• Format: Article
• Language: English
• Subjects: Arabidopis thaliana; Cell and Developmental Biology; organelle tether; peroxin; peroxisome; ubiquitin conjugating enzyme (E2); X-ray crystal analysis
• ISSN: 2296-634X; 2296-634X
• DOI: 10.3389/fcell.2022.838923

Peroxisomes are eukaryotic organelles that sequester critical oxidative reactions and process the resulting reactive oxygen species into less toxic byproducts. Peroxisome function and formation are coordinated by peroxins (PEX proteins) that guide peroxisome biogenesis and division and shuttle proteins into the lumen and membrane of the organelle. Despite the importance of peroxins in plant metabolism and development, no plant peroxin structures have been reported. Here we report the X-ray crystal structure of the PEX4-PEX22 peroxin complex from the reference plant . PEX4 is a ubiquitin-conjugating enzyme (UBC) that ubiquitinates proteins associated with the peroxisomal membrane, and PEX22 is a peroxisomal membrane protein that anchors PEX4 to the peroxisome and facilitates PEX4 activity. We co-expressed PEX4 as a translational fusion with the soluble PEX4-interacting domain of PEX22 in . The fusion was linked a protease recognition site, allowing us to separate PEX4 and PEX22 following purification and solve the structure of the complex. We compared the structure of the PEX4-PEX22 complex to the previously published structures of yeast orthologs. PEX4 displays the typical UBC structure expected from its sequence. Although PEX22 lacks notable sequence identity to yeast PEX22, it maintains a similar Rossmann fold-like structure. Several salt bridges are positioned to contribute to the specificity of PEX22 for PEX4 versus other UBCs, and the long unstructured PEX22 tether would allow PEX4-mediated ubiquitination of distant peroxisomal membrane targets without dissociation from PEX22. The PEX4-PEX22 structure also revealed that the residue altered in (P123L), a mutant previously isolated a forward-genetic screen for peroxisomal dysfunction, is near the active site cysteine of PEX4. We demonstrated UBC activity for the PEX4-PEX22 complex and found that the pex4-1 enzyme has reduced ubiquitin-conjugating activity and altered specificity compared to PEX4. Our findings illuminate the role of PEX4 and PEX22 in peroxisome structure and function and provide tools for future exploration of ubiquitination at the peroxisome surface.