UC Berkeley

A fundamental question in the central cellular recycling pathway of macroautophagy (hereafter autophagy) is how the autophagosome membrane is shaped around its cargo. At its heart, autophagosome biogenesis is a membrane remodeling event that sequesters cytoplasmic inclusions targeted for lysosomal degradation. And, while the biochemical basis for the recruitment of core autophagy proteins and individual subreactions has been largely worked out, critically, the physical mechanisms for membrane engulfment of cargo have yet to be elucidated. Among the most important outstanding questions are how membrane shape participates in the recruitment of early autophagy proteins and how the highly curved phagophore membrane is stabilized during autophagy initiation.Here, we use in vitro reconstitution on membrane nanotubes investigate how core autophagy proteins in the LC3 lipidation cascade and PI3K kinase complexes interact with curved membranes, providing insight into the possible roles in regulating membrane shape during autophagosome biogenesis. I found that ATG12–ATG5-ATG16L1 was up to 100-fold enriched on highly curved nanotubes relative to flat membranes. At high surface density, ATG12–ATG5-ATG16L1 binding increased the curvature of the nanotubes. While WIPI2 binding directs membrane recruitment, the amphipathic helix α2 of ATG16L1 is responsible for curvature sensitivity. Molecular dynamics simulations revealed that helix α2 of ATG16L1 inserts shallowly into the membrane, explaining its curvature-sensitive binding to the membrane. These observations show how the binding of the ATG12–ATG5-ATG16L1 complex to the early phagophore rim could stabilize membrane curvature and facilitate autophagosome growth. Further, reconstitution of the Class III PI3K complexes on membrane nanotubes shows that both Complex I and Complex II are similarly curvature sensitive, opening questions about the role of membrane curvature in the differential regulation of PI3K complex localization and regulation in the cell. Finally, these insights were facilitated by improvements and simplifications to existing membrane tube and in vitro reconstitution methods, which accelerated the work of precise biophysical quantification. Together, this work provides mechanistic insight into the physical basis of membrane remodeling in autophagosome biogenesis.