Autophagy is a key cellular quality control mechanism that enables cells to selectively degrade a wide range of macromolecules. While nutrient stress triggers bulk autophagy through non-selective degradation of cytoplasmic material, selective autophagy specifically eliminates protein aggregates, damaged organelles, and other targeted cargos via autophagy receptors. Although phase separation has been observed for certain autophagy components in bulk autophagy, as the autophagy-related gene 1 (Atg1) complex, its regulation and functional role in selective autophagy remain poorly understood.
Here, we demonstrate that key autophagy biogenesis factors undergo phase separation into initiation hubs at cargo surfaces in yeast. These hubs subsequently mature into the phagophore assembly site (PAS), in which the phagophore nucleates. We show that this phase separation depends on multivalent, low-affinity interactions between autophagy receptors and cargo, creating a dynamic cargo surface. Importantly, high-affinity receptor-cargo interactions block initiation hub formation and prevent autophagy progression.
To validate these principles, we engineered the mammalian reovirus nonstructural protein μNS, which normally accumulates as undegraded particles in yeast cytoplasm, into a neo-cargo that is efficiently degraded by selective autophagy.
We further demonstrate that initiation hubs form on diverse cargos, both membrane-bound and membrane-less, in mammalian cells and are essential for establishing connections to the endoplasmic reticulum, where the phagophore assembly site forms to initiate phagophore biogenesis. Our findings reveal that regulated phase separation is a fundamental mechanism underlying the initiation of selective autophagy across evolutionarily diverse organisms.