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Updated: Jul 9, 2025
Monitoring Stub1-Mediated Pexophagy
Published on: May 12, 2023
The N1 domain of the peroxisomal AAA-ATPase Pex6 is required for Pex15 binding and proper assembly with Pex1
Bashir A Ali1, Ryan M Judy1, Saikat Chowdhury2
1Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California, USA.
Insights
The N1 domain of Pex6 is crucial for peroxisome formation and function, mediating interactions with Pex15 and Pex1 to coordinate ATPase activity.
Area of Science:
- Cell Biology
- Protein Biochemistry
- Molecular Motors
Background:
- Peroxisome biogenesis and maintenance rely on the AAA-ATPase Pex1/Pex6 complex.
- AAA-ATPases function as molecular motors, utilizing ATP hydrolysis to unfold and translocate substrate proteins.
- Substrate recruitment in AAA-ATPases often involves N-terminal domains or associated cofactors.
Purpose of the Study:
- To investigate the role of the N1 domain of Pex6 in the Pex1/Pex6 complex.
- To elucidate the structural and functional characteristics of the Pex6 N1 domain.
- To understand how the Pex6 N1 domain contributes to peroxisomal targeting and ATPase activity.
Main Methods:
- X-ray crystallography to determine the structure of the isolated Pex6 N1 domain.
- Cryo-electron microscopy (cryo-EM) reconstruction of the Pex1/Pex6 complex.
- AlphaFold2 predictions for structural modeling.
- Biochemical assays to assess ATPase activity and binding interactions.
Main Results:
- The Pex6 N1 domain shares a conserved fold with other AAA-ATPase N-terminal domains.
- While Pex1/ΔN1-Pex6 retains ATPase activity in vitro, it fails to support peroxisomal function in vivo.
- The Pex6 N1 domain directly binds to the peroxisomal membrane protein Pex15 and an extended loop of Pex1's D2 ATPase domain.
Conclusions:
- The Pex6 N1 domain is essential for the in vivo function of the Pex1/Pex6 complex.
- Structural and biochemical data reveal the Pex6 N1 domain acts as a critical hub for cofactor and substrate recruitment.
- The N1 domain's interactions stabilize the Pex1/Pex6 heterohexamer and coordinate its motor activity for peroxisome maintenance.
Abstract:
The heterohexameric ATPases associated with diverse cellular activities (AAA)-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motor's N-terminal domains or N terminally bound cofactors. Here, we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast, Saccharomyces cerevisiae. We found that although Pex1/ΔN1-Pex6 is an active ATPase in vitro, it does not support Pex1/Pex6 function at the peroxisome in vivo. An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N-terminal domains of PEX1, CDC48, and NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of cofactors and substrates with Pex1/Pex6 ATPase activity.
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