Reconstitution of multistep recruitment of ULK1 to membranes in autophagy

Duan, Y.; Lu, Y.; Paul, S.; Betz, J.; Wilhelm, L. P.; Cook, A. S. I.; Ren, X.; Adriaenssens, E.; Martens, S.; Ganley, I.; Hummer, G.; Hurley, J. H.

doi:10.1101/2025.11.07.687251

bioRxiv · 10.1101/2025.11.07.687251

Reconstitution of multistep recruitment of ULK1 to membranes in autophagy

Duan, Y.·Lu, Y.·Paul, S.·Betz, J.·Wilhelm, L. P.·Cook, A. S. I.·Ren, X.·Adriaenssens, E.·Martens, S.·Ganley, I.·Hummer, G.·Hurley, J. H.

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Abstract

The Human ULK1 autophagy-initiating complex consists of ULK1, FIP200, and the HORMA domain heterodimer ATG13:ATG101. PI3P is essential to recruit 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 WIPI3, as well as WIPI2. Bound to WIPI2-3, ATG13:ATG101 inserts its Trp-Phe (WF) finger into the membrane. MD simulations show that WIPIs and the WF finger cooperatively stabilizes 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 PVP motif in the ULK1 IDR 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.

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Duan, Y., Lu, Y., Paul, S., Betz, J., Wilhelm, L. P., Cook, A. S. I., Ren, X., Adriaenssens, E., Martens, S., Ganley, I., Hummer, G., Hurley, J. H.. 2025-11-09. Reconstitution of multistep recruitment of ULK1 to membranes in autophagy. https://doi.org/10.1101/2025.11.07.687251

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