PerCap isolates native peroxisomes to reveal peroxisomal proteins and regulators

Peroxisomes are highly heterogeneous organelles with remarkable functional diversity across species and tissues. Isolating peroxisomes is essential for proteomic investigating their biological roles and regulatory mechanisms. Here, we discover that the N-terminal region of ATF6α interacts directly with peroxisomal membrane protein ABCD1 and ABCD3 in vitro and develop an affinity-based strategy PerCap, leveraging these interactions to rapidly purify native peroxisomes. Using this approach, we isolated peroxisomes from human cells, validated their intactness and identified the potential peroxisomal proteins by proteomic analysis. We show that AKAP1, CPT1A and CYB5R1 also localize to peroxisomes and loss of AKAP1 affects peroxisome morphology. Peroxisomes were also isolated from mouse tissues to compare tissue-specific peroxisomal proteins. By monitoring peroxisomal proteome dynamics during Torin1-induced pexophagy, we identified UBE2V1 as a contributor to this process. In summary, PerCap enables isolation of native peroxisomes from human cells and mouse tissues for proteomic and functional analyses.