ATG2A engages Rab1a and ARFGAP1 positive membranes during autophagosome biogenesis

Fuller, D. M.; Wu, Y.; Schueder, F.; Rasool, B.; Nag, S.; Korfhage, J. L.; Garcia-Milian, R.; Melnyk, K. D.; Bewersdorf, J.; De Camilli, P.; Melia, T.

doi:10.1101/2025.03.24.645038

bioRxiv · 10.1101/2025.03.24.645038

ATG2A engages Rab1a and ARFGAP1 positive membranes during autophagosome biogenesis

Fuller, D. M.·Wu, Y.·Schueder, F.·Rasool, B.·Nag, S.·Korfhage, J. L.·Garcia-Milian, R.·Melnyk, K. D.·Bewersdorf, J.·De Camilli, P.·Melia, T.

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Abstract

Autophagosomes form from seed membranes that expand through bulk-lipid transport via the bridge-like lipid transporter ATG2. The origins of the seed membranes and their relationship to the lipid transport machinery are poorly understood. Using proximity labeling and a variety of fluorescence microscopy techniques, we show that ATG2A localizes to extra-Golgi ARFGAP1 puncta during autophagosome biogenesis. ARFGAP1 itself is dispensable during macroautophagy, but among other proteins associating to these membranes, we find that RAB1 is essential. ATG2A co-immunoprecipitates strongly, albeit indirectly, with RAB1A, and siRNA-mediated depletion of RAB1A/B blocks autophagy downstream of LC3B lipidation, similar to ATG2A depletion. Further, when either autophagosome formation or the early secretory pathway is perturbed, ARFGAP1 and RAB1A accumulate at ectopic locations with autophagic machinery. Our results indicate that ATG2A engages a RAB1A complex on select early secretory membranes in support of autophagosome biogenesis. Significance StatementThis study expounds upon the role of early secretory membranes in autophagosome biogenesis. The authors demonstrate that RAB1/ARFGAP1-positive membranes are essential to autophagy and are recruited to the phagophore assembly site at an early step of autophagosome biogenesis. These membranes interact with the bridge-like lipid transport protein ATG2A and are positive for LC3B and WIPI2, suggesting that RAB1 membranes are a direct source for autophagosome formation.

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Fuller, D. M., Wu, Y., Schueder, F., Rasool, B., Nag, S., Korfhage, J. L., Garcia-Milian, R., Melnyk, K. D., Bewersdorf, J., De Camilli, P., Melia, T.. 2025-03-25. ATG2A engages Rab1a and ARFGAP1 positive membranes during autophagosome biogenesis. https://doi.org/10.1101/2025.03.24.645038

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