PtdIns(4,5)P ₂ signaling regulates ATG14 and autophagy

Abstract

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 Iγ PIP kinase isoform 5 (PIPKIγi5), an enzyme that generates PtdIns(4,5)P 2 in mammalian cells. Autophagosomes have associated PIPKIγi5 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 PIPKIγi5. Loss of PIPKIγi5 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.M acroautophagy (referred to as autophagy from here on) is an intracellular self-digestion process conserved in all eukaryotes that maintains cell homeostasis and protects cells from various stresses. Autophagy involves the formation of doublemembrane autophagosomes that contain target cytoplasmic contents for lysosomal degradation (1). Autophagosome formation is driven by coordinated functions of a number of autophagy-related (ATG) proteins (2). The ATG14 (also called Barkor or ATG14L)-Beclin 1 (atg6 in yeast)-VPS34 (an enzyme involved in the cellular process of autophagy; class III PI3K) complex plays essential roles in the initiation steps of autophagy by generating phosphatidylinositol 3-phosphate [PtdIns(3)P] and recruiting PtdIns(3)P effectors (3-6). ATG14 functions by recruiting VPS34 to punctate structures associated with the endoplasmic reticulum (ER) (7), and then PtdIns(3)P, the product of VPS34, in turn binds the Barkor/Atg14 autophagosome targeting sequence (BATS) domain of ATG14 and regulates its membrane curvature sensing (8). The ATG14-BATS domain also binds PtdIns(4,5)P 2 without unknown functional relevance (8).Phosphoinositides are important lipid messengers in various membrane trafficking events. PtdIns(4,5)P 2 plays multiple roles at the plasma membrane, such as in endocytosis, exocytosis, focal adhesion assembly, and so on (9). However, recently studies have argued for roles for PtdIns(4,5)P 2 at intracellular compartments (10). The majority of PtdIns(4,5)P 2 in mammalian cells are generated by the type I phosphatidylinositol 4-phosphate (PIP) 5-kinase (PIPKI), of which there are three genes (α, β, and γ) each wit...

Phosphatidylinositol 4,5‐bisphosphate controls Rab7 and PLEKHM 1 membrane cycling during autophagosome–lysosome fusion

“…Although these studies suggested a direct role of PI4P or the PI4K2A protein itself in the control of these trafficking steps, several studies have shown that PI(4,5)P 2 also controls lysosomal degradation of integral membrane proteins (Sun et al , ; Schill et al , ; Tan et al , ). PI(4,5)P 2 was found to regulate autophagy initiation (Tan et al , ) and lysosome reformation from autophagosomes (Rong et al , ). The Tan study has established a physical connection between the PIP5Kγ i5 isoform and the ATG14 protein and showed that PI(4,5)P 2 binding to the C‐terminal Barkor/ATG14(L) autophagosome‐targeting sequence of ATG14 promotes its assembly with Vps34 and Beclin1 and hence is critical for autophagosome initiation (Tan et al , ).…”

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“…This provides a means to localize membrane binding proteins to particular compartments on the basis of electrostatics, lipid packing, and local geometry. Phosphoinositides (PI) in particular, seem to have an emerging role in regulating cellular processes where micron-scale curvature is present including forespore membrane extension in S. pombe [57], autophagy [100], and micropinocytosis [101] in mammalian cells. Septins may be locally recruited to membranes based on the local lipid composition.…”