Regulation of pexophagy by a novel TBK1-MARCHF7-PXMP4-NBR1 axis in PEX1-depleted HeLa cells.

Peroxisomes are essential for lipid metabolism and redox balance, with pexophagy playing a critical role in maintaining cellular homeostasis. However, the regulatory mechanisms of pexophagy remain unclear. Through functional screening, we identified MARCHF7 as a novel E3 ligase regulating pexophagy. MARCHF7 depletion impaired pexophagic flux under PEX1 knockdown conditions. MARCHF7 binds to PXMP4 and promotes its ubiquitination at lysine 20 in PEX1-deficient cells. Depletion of PXMP4 impairs pexophagy, and reconstitution with the PXMP4 lysine 20 ubiquitination-defective mutant failed to rescue pexophagy. PEX1 depletion also induces TBK1 phosphorylation at serine 172, activating TBK1, which subsequently phosphorylates MARCHF7. This activation is driven by ROS accumulation, which reduces PXMP4 ubiquitination and prevents peroxisome loss. Furthermore, downregulation of MARCHF7 or PXMP4 impairs NBR1 recruitment to peroxisomes, suggesting that ubiquitinated PXMP4 acts as a recognition signal for NBR1. Collectively, our findings establish the TBK1-MARCHF7-PXMP4-NBR1 axis as a key regulatory pathway for pexophagy in response to PEX1 depletion.