biostudies-literatureUnknownDuan YNINDS NIH HHSThe ULK1 complex (ULK1C) and the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) act together to initiate autophagy. Human ULK1C consists of ULK1 itself, FIP200, and the HORMA domain heterodimer ATG13:ATG101. PI3P generated by PI3KC3-C1 is essential to recruit and stabilize ULK1C on membranes for ULK1 to phosphorylate its membrane-associated substrates in autophagy induction, even though ULK1C subunits do not contain any PI3P-binding domains. Here we show that the ATG13:ATG101 dimer forms a tight complex with the PI3P-binding protein WIPI3, as well as with WIPI2. Bound to WIPI2-3, ATG13:ATG101 aligns with the membrane to insert its Trp-Phe (WF) finger into the membrane. Molecular dynamics simulations show that alignment of WIPIs and the ATG101 WF finger cooperatively stabilizbioRxiv : the preprint server for biology2025.11.07.687251https://www.ebi.ac.uk/biostudies/studies/S-EPMC12637519biostudies-literatureReconstitution of multistep recruitment of ULK1 to membranes in autophagy.PMC12637519R01 NS134598Cook ASIRen XHurley JHMartens SLu YBetz JAdriaenssens EPaul SWilhelm LHummer GGanley IGDuan YfalseReconstitution of multistep recruitment of ULK1 to membranes in autophagy.The ULK1 complex (ULK1C) and the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) act together to initiate autophagy. Human ULK1C consists of ULK1 itself, FIP200, and the HORMA domain heterodimer ATG13:ATG101. PI3P generated by PI3KC3-C1 is essential to recruit and stabilize ULK1C on membranes for ULK1 to phosphorylate its membrane-associated substrates in autophagy induction, even though ULK1C subunits do not contain any PI3P-binding domains. Here we show that the ATG13:ATG101 dimer forms a tight complex with the PI3P-binding protein WIPI3, as well as with WIPI2. Bound to WIPI2-3, ATG13:ATG101 aligns with the membrane to insert its Trp-Phe (WF) finger into the membrane. Molecular dynamics simulations show that alignment of WIPIs and the ATG101 WF finger cooperatively stabiliz2025-01-01T00:00:00Z2025 Nov2026-06-11T03:12:23.048Z2026-06-11T03:08:10.463ZS-EPMC126375194127870810.1101/2025.11.07.687251