One of the major signaling events in autophagy is when the autophagy-specific class III PI 3-kinase complex synthesizes PI(3)P at the nascent phagophore. The phagophore is the precursor structure to the autophagosome. PI(3)P is recognized by various effector proteins, but the most important are Atg18 in yeast and WIPI 1-4 in mammals. All of these proteins are biochemically competent to bind both PI(3)P and PI(3,5)P2, however, the latter lipid is not known to have a direct role in autophagy. The ability of Atg18 to bind PI(3,5)P2 is important for a second biological function of this protein in vacuole homeostasis. The key autophagic lipid sensors are Atg18 in yeast and the WIPI proteins in mammals. Atg18 and the WIPIs belong to the PROPPIN family of proteins. PROPPINs are seven bladed β-propellers that bind to phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). In order to understand how PROPPINs bind phosphoinositides, we have determined the crystal structure of a representative, biochemically tractable PROPPIN, Hsv2 of Kluveromyces lactis. The structure revealed that PROPPINs contain two phosphoinositide binding sites which cooperate with a hydrophobic anchoring loop in membrane binding. These three binding elements cooperate in function, as demonstrated by the incremental loss of function in ATG18 alleles impaired in combinations of the two phosphoinositide binding site and the hydrophobic loop.
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