biostudies-literatureMuneshige KUniversity of AberdeenBiotechnology and Biological Sciences Research CouncilJapan Student Services Organizatione202407021https://www.ebi.ac.uk/biostudies/studies/S-EPMC11893502biostudies-literatureUnknown224(5)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.The Journal of cell biologyVacuoles provide the source membrane for TORC1-containing signaling endosomes.PMC11893502BB/X018229/1BB/V016334/1Hatakeyama RMuneshige KfalseVacuoles 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.2025-01-01T00:00:00Z2025 May2026-05-29T14:33:47.436Z2025-04-05T23:53:10.921ZS-EPMC118935024005292310.1083/jcb.202407021