Autophagy is a regulated self-digestion pathway with fundamental roles in cell homeostasis and diseases. Autophagy is regulated by coordinated actions of a series of autophagy-related (ATG) proteins. The Barkor/ATG14(L)-VPS34 (a class III phosphatidylinositol 3-kinase) complex and its product phosphatidylinositol 3-phosphate PtdIns(3)P play key roles in autophagy initiation. ATG14 contains a C-terminal Barkor/ATG14(L) autophagosome-targeting sequence (BATS) domain that senses the curvature of PtdIns(3) P-containing membrane. The BATS domain also strongly binds PtdIns(4,5)P-2, but the functional significance has been unclear. Here we show that ATG14 specifically interacts with type I. PIP kinase isoform 5 (PIPKI gamma i5), an enzyme that generates PtdIns(4,5)P-2 in mammalian cells. Autophagosomes have associated PIPKI gamma i5 and PtdIns(4,5)P-2 that are colocalized with late endosomes and the endoplasmic reticulum. PtdIns(4,5)P-2 generation at these sites requires PIPKI gamma i5. Loss of PIPKI gamma i5 results in a loss of ATG14, UV irradiation resistance-associated gene, and Beclin 1 and a block of autophagy. PtdIns(4,5)P-2 binding to the ATG14-BATS domain regulates ATG14 interaction with VPS34 and Beclin 1, and thus plays a key role in ATG14 complex assembly and autophagy initiation. This study identifies an unexpected role for PtdIns(4,5)P-2 signaling in the regulation of ATG14 complex and autophagy.
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