title: Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex.
abstract: The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.
authors.affiliation: a MRC Laboratory of Molecular Biology , Cambridge , United Kingdom.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Ohashi
authors.affiliation: a MRC Laboratory of Molecular Biology , Cambridge , United Kingdom.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Soler
authors.affiliation: a MRC Laboratory of Molecular Biology , Cambridge , United Kingdom.
authors.forename: Miguel
authors.initials: M
authors.lastname: García Ortegón
authors.affiliation: a MRC Laboratory of Molecular Biology , Cambridge , United Kingdom.
authors.forename: Lufei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: b European Molecular Biology Laboratory , Structural and Computational Biology Unit , Heidelberg , Germany.
authors.forename: Marie L
authors.initials: ML
authors.lastname: Kirsten
authors.affiliation: a MRC Laboratory of Molecular Biology , Cambridge , United Kingdom.
authors.forename: Olga
authors.initials: O
authors.lastname: Perisic
authors.affiliation: a MRC Laboratory of Molecular Biology , Cambridge , United Kingdom.
authors.forename: Glenn R
authors.initials: GR
authors.lastname: Masson
authors.affiliation: b European Molecular Biology Laboratory , Structural and Computational Biology Unit , Heidelberg , Germany.
authors.forename: John E
authors.initials: JE
authors.lastname: Burke
authors.affiliation: b European Molecular Biology Laboratory , Structural and Computational Biology Unit , Heidelberg , Germany.|c European Molecular Biology Laboratory, Hamburg Unit , Hamburg , Germany.
authors.forename: Arjen J
authors.initials: AJ
authors.lastname: Jakobi
authors.affiliation: a MRC Laboratory of Molecular Biology , Cambridge , United Kingdom.
authors.forename: Apostolos A
authors.initials: AA
authors.lastname: Apostolakis
authors.affiliation: a MRC Laboratory of Molecular Biology , Cambridge , United Kingdom.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Johnson
authors.affiliation: a MRC Laboratory of Molecular Biology , Cambridge , United Kingdom.
authors.forename: Maki
authors.initials: M
authors.lastname: Ohashi
authors.affiliation: d The Babraham Institute , Cambridge , UK.
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Ktistakis
authors.affiliation: b European Molecular Biology Laboratory , Structural and Computational Biology Unit , Heidelberg , Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Sachse
authors.affiliation: a MRC Laboratory of Molecular Biology , Cambridge , United Kingdom.
authors.forename: Roger L
authors.initials: RL
authors.lastname: Williams
mesh_terms: D001343:Autophagy; D000071183:Autophagy-Related Proteins / Q000737:chemistry / Q000378:metabolism*; D058546:Class III Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018360:Crystallography, X-Ray; D041961:Deuterium Exchange Measurement; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D021122:Protein Subunits / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: C584791:Atg38 protein, S cerevisiae; D000071183:Autophagy-Related Proteins; D046912:Multiprotein Complexes; C494986:NRBF2 protein, human; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D058546:Class III Phosphatidylinositol 3-Kinases
keywords: Atg14; Atg38; Beclin 1; EM structure; NRBF2; Vps15; Vps30; Vps34; complex I; crystal structure