Reconstitution of multistep recruitment of ULK1 to membranes in autophagy

The human Unc51-like kinase 1 (ULK1) autophagy-initiating complex consists of ULK1, FIP200, and the Hop/Rev7/Mad2 (HORMA) domain heterodimer ATG13:ATG101. Phosphatidylinositol 3-phosphate (PI3P) is essential to recruit ULK1 complex (ULK1C) to membranes for ULK1, but ULK1C subunits do not contain PI3P-binding domains. Here, we show that the ATG13:ATG101 dimer forms a complex with the PI3P-binding protein WD40 interacting with phosphoinositide protein 3 (WIPI3), as well as WIPI2. Bound to WIPI2 and WIPI3, ATG13:ATG101 inserts its Trp-Phe (WF) finger into the membrane. Molecular dynamics simulations show that WIPIs and the WF finger cooperatively stabilize the complex on membranes. Biochemical reconstitution and cell-based assays show that WIPI3:ATG13 engagement promotes ATG16L1 phosphorylation, autophagy, and mitophagy. A kinase domain (KD)-proximal Pro-Val-Pro (PVP) motif in the ULK1 intrinsically disordered region docks onto the ATG13:ATG101 HORMA dimer brings the ULK1 KD close to the membrane. The PVP motif is essential for in vitro ULK1 phosphorylation of ATG16L1 and important for autophagy and mitophagy. These data establish a stepwise pathway for recruitment of the ULK1 KD to the vicinity of the membrane surface.