The ESCRT-0 protein HRS regulates hepatocellular lipid droplet catabolism

Lipid droplets (LDs) are dynamic organelles that regulate lipid storage and metabolism pathways central to metabolic liver disease. LD turnover occurs in part through lysosomal catabolism (lipophagy), whereby LDs are delivered to lysosomes via two distinct trafficking pathways: autophagosome-dependent macrolipophagy and autophagosome-independent microlipophagy. However, the molecular machinery that regulates these two pathways, especially that of microlipophagy in mammalian cells, is poorly understood. In yeast, microlipophagy has been shown to rely on the endosomal sorting complex required for transport (ESCRT) protein family. Here, we used an ESCRT-specific RNAi library in hepatocytes, which identified the ESCRT-0 protein hepatocyte growth factor receptor substrate (HRS) as a critical regulator of LD homeostasis. HRS depletion leads to significant LD accumulation, driven by impaired LD catabolism rather than increased LD biogenesis. While HRS-deficient cells retain lipolytic activity, LD targeting via RAB5-mediated microlipophagy is reduced, and LD targeting by autophagosomes is increased. Consistent with these findings, HRS knockdown suppressed mTORC1 signaling, enhanced autophagosome formation, and reduced autophagic cargo degradation. Notably, despite unchanged lysosomal abundance, HRS knockdown elevated lysosomal pH, potentially impairing autophagic degradation and promoting LD accumulation. Overall, these findings identify HRS as a key regulator of LD turnover in mammalian cells, modulating lipophagy through lysosomal function.