EPG5 orchestrates iron compartmentalization through dual regulation in macroferritinophagy.

Intracellular iron is distributed across functionally distinct pools though the mechanisms regulating its compartmentalization remain unclear. Here we show that EPG5, a tethering factor required for autophagosome-lysosome fusion, governs iron compartmentalization through dual mechanisms: direct involvement in autophagy and indirect regulation of ferritinophagy initiation. A systematic comparison of autophagy genes indicates that EPG5 deficiency uniquely decreases hemosiderin and increases the labile iron pool, a profile distinct from ATG5, ATG14, FIP200, or WDR45 deficiencies. Mechanistically, EPG5 loss disrupts autophagosome maturation and endocytic trafficking, causing ferritin condensates and iron transporters to accumulate in aberrant autophagosomes and endosomes. This blockade triggers compensatory TBK1 activation. TBK1 subsequently phosphorylates TAX1BP1 and interacts with the ferritinophagy receptors NCOA4, inhibiting receptor homo-oligomerization and modulating ferritinophagic flux. These findings position EPG5 as a novel orchestrator of iron compartmentalization and highlight the role of TBK1-NCOA4/TAX1BP1 axis in iron homeostasis and related stress response.