Autophagy depends on the repopulation of lysosomes to degrade intracellular components and recycle nutrients. How cells coordinate lysosome repopulation during basal autophagy, which occurs constitutively under nutrient-rich conditions, is unknown. Here, we identify an endosome-dependent phosphoinositide pathway that links PI3Ka signaling to lysosome repopulation during basal autophagy. We show that PI3Ka-derived PI(3)P generated by INPP4B on late endosomes was required for basal but not starvationinduced autophagic degradation. PI(3)P signals were maintained as late endosomes matured into endolysosomes, and served as the substrate for the 5-kinase, PIKfyve, to generate PI(3,5)P 2 . The SNX-BAR protein, SNX2, was recruited to endolysosomes by PI(3,5)P 2 and promoted lysosome reformation. Inhibition of INPP4B/PIKfyvedependent lysosome reformation reduced autophagic clearance of protein aggregates during proteotoxic stress leading to increased cytotoxicity. Therefore under nutrient-rich conditions, PI3Ka, INPP4B, and PIKfyve sequentially contribute to basal autophagic degradation and protection from proteotoxic stress via PI(3,5)P 2 -dependent lysosome reformation from endolysosomes. These findings reveal that endosome maturation couples PI3Ka signaling to lysosome reformation during basal autophagy.

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