Macroautophagy (hereafter autophagy) is a conserved intracellular degradation pathway that is essential for maintaining cellular homeostasis. Autophagosome (AP) formation involves pre-autophagosomal structure (PAS) organization and expansion of the isolation membrane (IM). Although phosphatidylinositol 4-phosphate (PtdIns4P) localizes to the IM and is required for autophagy, the specific functional role it plays in AP formation remains unclear. Pik1, a PtdIns 4-kinase localized to the Golgi, plays a critical role in this process. We used temperature-sensitive pik1 mutant cells and found that PAS localization of Atg9, Atg17, Atg1 and Atg13 remained normal at the restrictive temperature, indicating that PAS scaffold formation was unaffected. In contrast, the recruitment of downstream Atg proteins, the PtdIns 3-kinase complex I including Atg14, the Atg2-Atg18 complex, and Atg8, was impaired under the same condition. These findings demonstrate that Pik1-generated PtdIns4P is essential for PAS organization. Since Atg9 vesicles are derived from the Golgi, we hypothesized that PtdIns4P is transported to the PAS on Atg9 vesicles to mediate recruitment of downstream Atg proteins. To test this, we performed immunoprecipitation analysis using a PtdIns4P-binding protein and found that Atg9 was co-immunoprecipitated at the permissive temperature, but not at the restrictive temperature. This result indicates that PtdIns4P is enriched on Atg9 vesicles through Pik1 activity. Moreover, our assessment in pik1 mutant cells showed that IM expansion is impaired at the restrictive temperature. Collectively, these results identify PtdIns4P as a key component of PAS organization.
