Vacuoles provide the source membrane for TORC1-containing signaling endosomes.

Organelle biogenesis is fundamental to eukaryotic cell biology. Yeast signaling endosomes were recently identified as a signaling platform for the evolutionarily conserved Target of Rapamycin Complex 1 (TORC1) kinase complex. Despite the importance of signaling endosomes for TORC1-mediated control of cellular metabolism, how this organelle is generated has been a mystery. Here, we developed a system to induce synchronized de novo formation of signaling endosomes, enabling real-time monitoring of their biogenesis. Using this system, we identify vacuoles as a membrane source for newly formed signaling endosomes. Membrane supply from vacuoles is mediated by the CROP membrane-cutting complex, consisting of Atg18 PROPPIN and retromer subunits. The formation of signaling endosomes requires TORC1 activity, suggestive of a tightly regulated process. This study unveiled the first mechanistic principles and molecular participants of signaling endosome biogenesis.