The mechanism of isolation membrane formation in autophagy is receiving intensive study. We recently found that Atg9 translocates phospholipids across liposomal membranes and proposed that this functionality plays an essential role in the expansion of isolation 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. To test this idea, we analyzed the distributions of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol 4-phosphate by freeze-fracture electron microscopy. We found that all 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 symmetrical distribution in autophagosomes within 30 min after synthesis, whereas this symmetrical distribution is compromised in yeast expressing an Atg9 mutant. These results indicate that transbilayer phospholipid movement that is mediated by Atg9 is involved in the biogenesis of autophagosomes.

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