Abstract
Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first β sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA β sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 β sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes.
Copyright © 2018 Elsevier Inc. All rights reserved.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Video-Audio Media
MeSH terms
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Autophagy*
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Autophagy-Related Proteins
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Beclin-1 / chemistry
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Beclin-1 / genetics
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Beclin-1 / metabolism*
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Binding Sites
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Class III Phosphatidylinositol 3-Kinases / chemistry
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Class III Phosphatidylinositol 3-Kinases / genetics
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Class III Phosphatidylinositol 3-Kinases / metabolism*
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Cryoelectron Microscopy
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Enzyme Activation
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Gene Expression Regulation
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HEK293 Cells
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HeLa Cells
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Humans
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism
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Molecular Dynamics Simulation
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Phosphatidylinositol Phosphates / metabolism
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Protein Binding
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Protein Conformation, beta-Strand
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Protein Interaction Domains and Motifs
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Signal Transduction
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Structure-Activity Relationship
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nef Gene Products, Human Immunodeficiency Virus / genetics
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nef Gene Products, Human Immunodeficiency Virus / metabolism
Substances
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Autophagy-Related Proteins
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BECN1 protein, human
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Beclin-1
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Intracellular Signaling Peptides and Proteins
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Phosphatidylinositol Phosphates
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RUBCN protein, human
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nef Gene Products, Human Immunodeficiency Virus
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nef protein, Human immunodeficiency virus 1
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phosphatidylinositol 3-phosphate
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Class III Phosphatidylinositol 3-Kinases