Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation

Abstract

Autophagosome formation is a complex process that begins with the nucleation of a pre-autophagosomal structure (PAS) that expands into a phagophore or isolation membrane, the precursor of the autophagosome. A key event in the formation of the phagophore is the production of PtdIns3P by the phosphatidylinsitol kinase Vps34. In yeast the two closely related proteins, Atg18 and Atg21, are the only known effectors of PtdIns3P that act in the autophagy pathway. The recruitment of Atg18 or Atg21 to the PAS is an essential step in the formation of the phagophore. Our bioinformatic analysis of the Atg18 and Atg21 orthologues in all eukaryotes shows that WIPI1 and WIPI2 are both mammalian orthologues of Atg18. We show that WIPI2 is a mammalian effector of PtdIns3P and is ubiquitously expressed in a variety of cell lines. WIPI2 is recruited to early autophagosomal structures along with Atg16L and ULK1 and is required for the formation of LC3-positive autophagosomes. Furthermore, when WIPI2 is depleted, we observe a remarkable accumulation of omegasomes, ER-localized PtdIns3P-containing structures labeled by DFCP1 (double FYVE domain-containing protein 1), which are thought to act as platforms for autophagosome formation. In view of our data we propose a role for WIPI2 in the progression of omegasomes into autophagosomes.

SNX 18 regulates ATG 9A trafficking from recycling endosomes by recruiting Dynamin‐2

“…DyLight‐680‐ and DyLight‐800‐conjugated secondary antibodies were obtained from Invitrogen (SA5‐10170 and SA5‐10044, 1:5,000). Antibodies used for immunofluorescence are as follows: mouse anti‐WIPI2 (from Sharon Tooze 40, 1:2,000), rabbit anti‐ATG16L1 (MBL, PM040, 1:200), hamster anti‐ATG9A (from Sharon Tooze 41, 1:1,000), rabbit anti‐ATG9A (Abcam, ab108338, 1:400), mouse anti‐myc (IF and WB—DSHB, cl9E10, 1:20), rabbit anti‐TBC1D14 (from Sharon Tooze 8, 1:200), chicken anti‐SNX18 (from Sven Carlsson, 26, 30, 1:200), goat anti‐myc (Abcam, ab9132, 1:500), mouse anti‐TfR (Santa Cruz Biotechnology, sc‐65877, 1:200), mouse anti‐Dynamin (Upstate/Millipore, 05‐319, 1:100). Alexa 488‐conjugated secondary antibodies were obtained from Invitrogen (A‐21206 and A‐21202, 1:500), and Cy3 and Cy5‐conjugated secondary antibodies were obtained from Jackson ImmunoResearch (Cy3: 715‐165‐151, 127‐165‐099 and 711‐165‐152, Cy5: 711‐495‐152 and 715‐175‐150).…”

Autophagy and human diseases

“…The hit gene WDR45 (also known as WIPI4) is one of the four mammalian homologues of yeast Atg18, which plays an important role in autophagosome formation [16][17][18][19]. Atg18/WIPIs belong to the PROPPIN family of proteins.…”

“…The crystal structure of Hsv2, a yeast Atg18 paralogue, shows two phosphoinositide-binding sites at blades five and six, and an Atg2-binding region at blade 2 [23][24][25]. Atg18/ WIPIs are recruited to the autophagosome formation site through binding to phosphatidylinositol 3-phosphate, which is synthesized by the class III PtdIns 3-kinase complex [18,21]. Caenorhabditis elegans has two Atg18 homologues, ATG-18 and EPG-6 [19].…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…16 WIPI proteins (WIPI1 to WIPI4), mammalian homologs of yeast Atg18, may have the ability to sense and regulate PtdIns3P levels and participate in the initiation and maturation of the omegasome. 7,27,94,95 WDFY3 is implicated in cargo selection and delivery in selective autophagy. 17,27,96 It is further conjectured that the PtdIns3P synthesized in situ, together with its effector proteins, signals the de novo synthesis of other phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine), which constitute the autophagosomal membrane.…”