--Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy.

During macroautophagy/autophagy, precursor cisterna known as phagophores expand and sequester portions of the cytoplasm and/or organelles, and subsequently close resulting in double-membrane transport vesicles called autophagosomes. Autophagosomes fuse with lysosomes/vacuoles to allow the degradation and recycling of their cargoes. We previously showed that sequential binding of yeast Atg2 and Atg18 to Atg9, the only conserved transmembrane protein in autophagy, at the extremities of the phagophore mediates the establishment of membrane contact sites between the phagophore and the endoplasmic reticulum. As the Atg2-Atg18 complex transfers lipids between adjacent membranes , it has been postulated that this activity and the scramblase activity of the trimers formed by Atg9 are required for the phagophore expansion. Here, we present evidence that Atg9 indeed promotes Atg2-Atg18 complex-mediated lipid transfer , although this is not the only requirement for its function . In particular, we show that Atg9 function is dramatically compromised by a F627A mutation within the conserved interface between the transmembrane domains of the Atg9 monomers. Although Atg9 self-interacts and binds to the Atg2-Atg18 complex, the F627A mutation blocks the phagophore expansion and thus autophagy progression. This phenotype is conserved because the corresponding human ATG9A mutant severely impairs autophagy as well. Importantly, Atg9 has identical scramblase activity like Atg9, and as with the wild-type protein enhances Atg2-Atg18-mediated lipid transfer. Collectively, our data reveal that interactions of Atg9 trimers via their transmembrane segments play a key role in phagophore expansion beyond Atg9's role as a lipid scramblase. BafA1: bafilomycin A; Cvt: cytoplasm-to-vacuole targeting; Cryo-EM: cryo-electron microscopy; ER: endoplasmic reticulum; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCS: membrane contact site; NBD-PE: -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl--glycero-3-phosphoethanolamine; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor Ape1; PtdIns3P: phosphatidylinositol-3-phosphate; SLB: supported lipid bilayer; SUV: small unilamellar vesicle; TMD: transmembrane domain; WT: wild type.