Role of <i>N</i> ‐glycosylation as a determinant of <scp>ATG9A</scp> conformations and activity

Here, we investigate the effects of glycosylation at position N99 on the structural dynamics and lipid scrambling activity of ATG9A, a key autophagy protein, using microsecond all-atom molecular dynamics simulations. ATG9A is an integral membrane protein involved in autophagosome biogenesis, and glycosylation at N99 has previously been implicated in intracellular trafficking, although its precise role remains unclear. The simulations reveal that the hydrophilic central cavity of ATG9A supports lipid reorientation and partial trans-bilayer movements, consistent with experiments on its lipid scrambling activity. We propose that N-glycosylation at N99 enhances cooperative interactions between protomers, facilitating lipid insertion and translocation within the central cavity. These findings suggest a mechanism by which glycosylation may influence lipid redistribution across the phagophore membrane during autophagy. To test this hypothesis, we generate N99 variants (ATG9A^N99A and ATG9A^N99D) lacking N-glycosylation. These mutants show no significant changes in autophagy flux, suggesting that N99 glycosylation may not be essential for bulk autophagic processing. However, the analysis of autophagosome size indicates that the variants fail to rescue the enlarged vesicle phenotype of ATG9A-KO cells, unlike wild-type ATG9A. Thus, glycosylation might fine-tune ATG9A function, influencing vesicle morphology through conformational dynamics and lipid transport. We also observe asymmetric protomer conformations in ATG9A, in contrast to the symmetric structures obtained from cryo-EM, suggesting that structural heterogeneity could be further explored with experimental methods. Overall, our study highlights the importance of including glycosylation in computational models of membrane proteins and provides mechanistic insight into lipid transport during autophagy, with potential implications for other lipid scramblases and flippases.