9.A.79.  The Autophagosomal Phospholipid Transmembrane Translocase (Atg9) Family

Orii et al. 2021 found that Atg9, a lipid scramblase, translocates phospholipids across liposomal membranes and proposed that this function plays an essential role in the expansion of membranes. The distribution of phosphatidylinositol 3-phosphate in both leaflets of yeast autophagosomal membranes supports this proposal, but if Atg9-mediated lipid transport is crucial, symmetrical distribution in autophagosomes should be found broadly for other phospholipids. Therefore, Orii et al. 2021 analyzed the distributions of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol 4-phosphate by freeze-fracture electron microscopy and found that these phospholipids are distributed with comparable densities in the two leaflets of autophagosomes and autophagic bodies. Moreover, de novo-synthesized phosphatidylcholine is incorporated into autophagosomes preferentially and shows a symmetrical distribution in autophagosomes within 30 min after synthesis, whereas this symmetrical distribution is compromised in yeast expressing an Atg9 mutant. Thus, transbilayer phospholipid movement that is mediated by Atg9 is involved in the biogenesis of autophagosomes. Backbone NMR assignments of the nucleotide binding domain of BmrA in the post-hydrolysis state have been determined (Coudevylle et al. 2022). Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy (Chumpen Ramirez et al. 2022).

The dynamin, Vps1, (of 704 aas and 2 TMSs, see TC# 9.A.63.1.1) mediates Atg9 transport to the sites of autophagosome formation (Arlt et al. 2023). Autophagy is a key process in eukaryotes for maintaining cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. Atg9 is the only conserved transmembrane protein within this core Atg machinery. In the yeast, Saccharomyces cerevisiae, the retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and impair the autophagic flux by affecting two separate autophagy steps. Arlt et al. 2023 provided evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants, associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth, are unable to sustain autophagy and affect Atg9 trafficking.  ATG9 promotes autophagosome formation through interaction with LC3 (Xu et al. 2025).  Thus, LC3 is an essential membrane component of the autophagosome, thereby allowing ATG9 to incorporate into the autophagosome membrane.

Autophagy proteins coordinate the biogenesis of a phagophore, the formation and maturation of an autophagosome (Millard and Tooze 2025). Genetic mutations of these proteins can result in dysregulated autophagy, stalled autophagosome biogenesis, and cell death. ATG9, the sole transmembrane ATG (autophagy related) protein governs nucleation of the phagophore. ATG9 can redistribute lipids across a lipid bilayer. ATG9-positive vesicles can also deliver lipid-modifying enzymes to alter the lipid composition of membranes. Both functions are required for autophagy. However, ATG proteins, including ATG9, play key molecular roles in pathways unrelated to autophagy. ATG9 has been shown to function in multiple pathways at the Golgi, plasma membrane, and lysosomes. It can also play a role in immune signalling. The trafficking of ATG9 in ATG9-positive vesicles is essential to many of these pathways. Millard and Tooze 2025 highlight the functions of ATG9 in autophagy and autophagy-unrelated pathways, here referred to as 'non-canonical functions', and summarise the broader role of ATG9A in cell homeostasis.

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