title: Structure-based analyses reveal distinct binding sites for Atg2 and phosphoinositides in Atg18.
abstract: Autophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by an autophagosome and delivered to a lysosome/vacuole. Atg18 plays a critical role in autophagosome formation as a complex with Atg2 and phosphatidylinositol 3-phosphate (PtdIns(3)P). However, little is known about the structure of Atg18 and its recognition mode of Atg2 or PtdIns(3)P. Here, we report the crystal structure of Kluyveromyces marxianus Hsv2, an Atg18 paralog, at 2.6 Å resolution. The structure reveals a seven-bladed β-propeller without circular permutation. Mutational analyses of Atg18 based on the K. marxianus Hsv2 structure suggested that Atg18 has two phosphoinositide-binding sites at blades 5 and 6, whereas the Atg2-binding region is located at blade 2. Point mutations in the loops of blade 2 specifically abrogated autophagy without affecting another Atg18 function, the regulation of vacuolar morphology at the vacuolar membrane. This architecture enables Atg18 to form a complex with Atg2 and PtdIns(3)P in parallel, thereby functioning in the formation of autophagosomes at autophagic membranes.
authors.affiliation: Institute of Microbial Chemistry, Tokyo, Tokyo 141-0021, Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Takafumi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Hayashi
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ohsumi
authors.forename: Nobuo N
authors.initials: NN
authors.lastname: Noda
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001343:Autophagy; D000071183:Autophagy-Related Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray / Q000379:methods; D004252:DNA Mutational Analysis; D006801:Humans; D007716:Kluyveromyces / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: C441008:ATG18 protein, S cerevisiae; C434064:ATG2 protein, S cerevisiae; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; C532949:HSV2 protein, S cerevisiae; D008055:Lipids; D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D029701:Saccharomyces cerevisiae Proteins; C055525:phosphatidylinositol 3-phosphate