Galectin-3 directs mitophagy in response to Parkin-/proteasome-dependent rupture of mitochondrial outer membrane.

PINK1/Parkin-dependent mitophagy is an intracellular process that selectively removes damaged, depolarized mitochondria. In this form of mitophagy, the mitochondrial outer membrane (OMM) undergoes focal rupture through a Parkin- and proteasome-dependent mechanism, which consequently results in the exposure of mitochondrial inner membrane (IMM) mitophagy receptors. The OMM rupture marks the damaged mitochondria and ensures their proper disposal. However, our understanding of the molecular events triggered by OMM rupture remains limited. Here, our proteomic study revealed that Galectin-3, a member of the β-galactoside-binding protein family, is significantly enriched in the ruptured OMM of damaged mitochondria. Galectin-3 is necessary for mitophagy, and it relocalizes from the cytosol to enclose the damaged mitochondria in response to mitophagy induction. The mitochondrial recruitment of Galectin-3 is Parkin- and proteasome-dependent, suggesting that the enclosure of mitochondria by Galectin-3 is a consequence of OMM rupture during PINK1/Parkin-mediated mitophagy. Functionally, Galectin-3 interacts with IMM protein PHB2 and recruits autophagy initiation factors ULK1 on the damaged mitochondria. Importantly, mutations in key residues that confer the liquid-liquid phase separation (LLPS) properties of Galectin-3 abrogates its mitochondrial relocalization, ULK1 recruitment, and mitophagy, suggesting that the capacity to form biomolecular condensates around the damaged mitochondria is crucial for the mitophagy function of Galectin-3. While much of the prior research on Galectin-3 focused on its extracellular functions, our findings shed light on its previously underexplored intracellular functions on the mitochondria and illuminated a novel mechanism by which Galectin-3 senses the damaged mitochondria and maintains organellar quality control. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13062-025-00692-1.