ER-endosome contacts generate a local environment promoting phagophore formation.

Autophagy starts with the formation of a double-membrane vacuole called the autophagosome, initiated by a transient structure known as the phagophore. Previous studies reported that phagophore biogenesis primarily occurs at endoplasmic reticulum (ER) omegasome subdomains, but other evidence suggests that the phagophore derives from recycling endosomes. Our study demonstrates the importance of ER-endosome interactions, revealing the dynamic mobilization of endosome-ER contact sites (EERCSs) in response to starvation. We characterize a sequential tethering of Rab5 and Rab11 endosomes to omegasomes, facilitating phagophore biogenesis. Detailed analyses reveal that EERCS-associated molecular machinery creates a confined environment that promotes local Ca2+ accumulation and liquid-liquid phase separation at ER exit sites. This environment primes de novo phagophore formation through a Rab3a-RAB3GAP1/2-mediated nano-vesicle fusion. We propose that EERCS mobilization generates transient cytoplasmic confinement, fostering localized accumulation of components for phagophore biogenesis. Our study reveals a novel role for the ER-endosome interface in the nutrient deprivation response, emphasizing organelle coordination during autophagy initiation.