bioRxiv : the preprint server for biologyNov 24, 2025

Step-by-step process of how ULK1 attaches to membranes in autophagy

Longevity & Aging Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 7, 2026

Abstract

The ATG13:ATG101 dimer forms a tight complex with the PI3P-binding protein WIPI3, essential for autophagy induction.

PI3P generated by the PI3KC3-C1 complex is crucial for recruiting ULK1C to membranes.
The engagement of WIPI3 with ATG13:ATG101 is necessary for the phosphorylation of ATG16L1 by ULK1.
Molecular dynamics simulations indicate that WIPI3 and the ATG101 WF finger stabilize the complex on membranes.
The PVP motif within the ULK1 IDR is essential for ULK1's phosphorylation of ATG16L1 and supports mitophagy.
A stepwise pathway for ULK1 recruitment to the membrane surface is established downstream of PI3KC3-C1.

Simplified

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 stabilizes the complex on membranes, explaining the essential role of the WF residues in autophagy. Biochemical reconstitution and a cell-based assay show that WIPI3:ATG13 engagement is required for ATG16L1 phosphorylation by ULK1, ATG13 puncta formation, and bulk autophagic flux. We further showed that a kinase domain (KD)-proximal PVP motif within the ULK1 IDR docks onto the surface of the ATG13:ATG101 HORMA dimer and used molecular modeling to show how the ULK1 KD is brought close to the membrane surface. Biochemical reconstitution and cell-based assays show that the PVP motif is essential forULK1 phosphorylation of ATG16L1 and important for BNIP3/NIX-dependent mitophagy. These data establish a stepwise pathway for recruitment of the ULK1 KD to the vicinity of the membrane surface downstream of PI3KC3-C1. in vitro

Funding

Competing interests

J.H.H. is a co-founder and shareholder of Casma Therapeutics, and receives research funding from Hoffmann-La Roche. S.M. is a shareholder of Casma Therapeutics. The other authors declare no competing interests.

PubMed

Step-by-step process of how ULK1 attaches to membranes in autophagy

Abstract

Funding

Competing interests

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Abstract

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