Dessus, A. N.; Ohashi, Y.; Bourguet, M.; Morgan, T. E.; Nunez, A.; Manifava, M.; Ktistakis, N. T.; Williams, R. L.

Macroautophagy/autophagy is a cellular process enabling degradation of intracellular components during starvation. In mammalian cells, autophagosomes can reach diameters of over 1000 nm within 30 min after triggering starvation, but how such substantial amounts of membranes can be synthesized within a brief time remains elusive. A protein complex central to the phagophore initiation is the lipid kinase PIK3C3-Complex 1 (PtdIns3K-C1), which produces phosphatidylinositol-3-phosphate (PtdIns3P). PtdIns3P recruits a variety of downstream proteins, among which is PtdIns3P-binding WIPI2 that facilitates lipidation of mammalian ATG8 (mATG8) family proteins on phagophores. Here we show that upon inhibition of mATG8 lipidation in cells, there is a decreased accumulation of WIPI2, suggesting a feedback loop between mATG8s and PtdIns3P production. The role of PtdIns3K-C1 in this feedback was demonstrated by in vitro experiments where recombinant membrane-coupled mATG8s bind to and potently activate PtdIns3K-C1, with GABARAP being the most potent activator among all mATG8s. By a combination of cryo-electron microscopy, structural mass spectrometry, activity assays and mutagenesis, we show that GABARAP binds two sites in PtdIns3K-C1, with one site showing an atypical bipartite interaction with the mATG8. We also confirm both sites are essential for GABARAP to activate PtdIns3K-C1. We propose that once GABARAP is indirectly recruited by PtdIns3P generated by basal activity of PtdIns3K-C1, a positive feedback loop is formed where PtdIns3K-C1 interacts with GABARAP and becomes activated to produce more PtdIns3P, thereby further stimulating GABARAP lipidation. This mechanism would be central for autophagosome biogenesis, where enlarged membranes need to be synthesized within a brief period.