title: Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy.
abstract: The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen-deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720;
authors.forename: Lindsey N
authors.initials: LN
authors.lastname: Young
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720;
authors.forename: Kelvin
authors.initials: K
authors.lastname: Cho
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720;
authors.forename: Rosalie
authors.initials: R
authors.lastname: Lawrence
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720;
authors.forename: Roberto
authors.initials: R
authors.lastname: Zoncu
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 jimhurley@berkeley.edu.
authors.forename: James H
authors.initials: JH
authors.lastname: Hurley
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics; D001343:Autophagy; D000071183:Autophagy-Related Proteins / Q000235:genetics; D000071186:Beclin-1 / Q000235:genetics; D058546:Class III Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
chemical_list: C539000:ATG14 protein, human; D033942:Adaptor Proteins, Vesicular Transport; D000071183:Autophagy-Related Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; C494986:NRBF2 protein, human; D015534:Trans-Activators; D058546:Class III Phosphatidylinositol 3-Kinases
keywords: allostery; electron microscopy; hydrogen–deuterium exchange