Turning Autophagy On and Off through the Class III PI-3 Kinase

Autophagy (self-eating) is an essential process for cellular self-renewal. The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. The V-shaped architecture of the four-subunit version of PI3KC3-C1 consists of VPS34, VPS15, BECN1, and ATG14. Chapter 2 shows that a putative fifth subunit, 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. We used hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) 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 and activates VPS34 through its N-terminal MIT domain.The ability of NRBF2 to activate the lipid kinase of VPS34 via an allosteric mechanism had not been determined. Chapter 3 shows precisely where the MIT domain of NRBF2 binds to PI3KC3-C1, that one copy of the MIT domain is insufficient for activation, and that full activation requires a second copy of the MIT domain. Dimeric NRBF2 cinches around the conformational flexible catalytic arm of the complex and stabilizes the lipid kinase domain of VPS34 and the putative kinase domain of VPS15.Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. Chapter 4 describes how a cryptic membrane binding site within BECN1 was identified. First, the interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first β sheet of the BECN1 BARA domain by hydrogen-deuterium exchange and cryo-EM. The interaction determinants were confirmed in cell-based assays of PI3KC3 activity and autophagy. These data suggested that 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 membrane