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 higherorder organization of the preautophagosomal structure.hydrogen-deuterium exchange | electron microscopy | allostery A utophagy is a pathway of subcellular engulfment and lysosomal transport that is conserved throughout eukaryotes (1). Survival during starvation is probably the primordial function of autophagy (2). Most current research focuses on a growing array of selective autophagy pathways in human cells (3). These include autophagy of mitochondria (mitophagy), endoplasmic reticulum (ER-phagy), and intracellular microbes (xenophagy). Autophagy is generally considered protective against a range of diseases, including several neurodegenerative diseases (4), microbial infections, and cancer (5). Autophagy can also promote tumor growth in late stages of cancer, and therefore agonists and antagonists of autophagy are of interest as potential therapeutics (5). The centrality of autophagy to both basic cell processes and human health has leant urgency to understanding its underlying molecular mechanisms.Bulk and selective autophagy share common machinery for the initiation and growth of the double-membrane sheet known as the phagophore. The phagophore matures and closes to form the autophagosome. The proteins dedicated to autophagy number ∼41 in yeast and more in humans. They include the ULK1 (unc-51 like autophagy activating kinase 1) protein kinase complex [Atg1 (autophagy-related 1) complex in yeast], the phosphatidylinositol 3-kinase complexes I and II (PI3KC3-C1 and -C2), the integral membrane protein ATG9, the WIPIs as PI(3P) receptors, and the ubiquitin-like LC3 protein family and their conjugation pathway. PI3KC3-C1 functions in autophagy initiation (6, 7), whereas PI3KC3-C2 is important for autolysosome formation at a later step in autophagy (8). The two PI3KC3 complexes share three subunits in common: the lipid kinase subunit VPS34 (vacuolar protein sorting 34) and the regulatory subunits BECN1 (Beclin 1) and VPS15. PI3KC3-C2 contains the unique subunit UVRAG (UV radiation resistance associated), which is involved in regulating the fusion of autophagosomes to lysosomes (9). PI3KC3-C1 is targeted to ...

Mechanisms of Autophagy Initiation

“…NRBF2/Atg38 contain an N-terminal three-helix bundle MIT (Microtubule-Interacting and Transport) domain and a coiled-coil-containing domain at the C-terminus (105) that induces dimerization. NRBF2 binds to the base of complex I through interactions with the N-terminus of ATG14L and BECN1, enhances kinase activity in vitro and leads to the dimerization of human complex I (106) (Fig 3B). Although yeast Atg38 is dimeric as well, it does not facilitate yeast PI3KC3-C1 dimerization (105).…”

Sensing Membrane Curvature in Macroautophagy

“…How these surfaces are oriented with respect to one another could impart a second level of curvature dependence, possibly making the autophagosome-directed Beclin-containing complex significantly more dependent on local curvature 58 , as seen when comparing activity on small vesicles or GUVs 59 . In fact, the complex ordinarily exists as at least a heteropentamer (including the subunit NRBF2) 60, 61 , and the heteropentamer organizes into a dimer around a motif in NRBF2 62 , potentially allowing membrane tethering to be sensed by or driven by the VPS34 complex during autophagosome construction.…”

Vps34 Kinase Domain Dynamics Regulate the Autophagic PI 3-Kinase Complex

“…The N-terminal regulatory sequences of ATG14 and BECN1 have not been visualized in either the EM or crystal structures, but are presumably attached to the base. These regions contain the binding sites for important regulators of PI3KC3 such as Bcl-2 (Oberstein et al, 2007; Pattingre et al, 2005) and NRBF2 (Ohashi et al, 2016; Young et al, 2016) and important sites of regulatory phosphorylation by ULK1, AMPK, DAPK, MAPKAP2, and PGK1 (Kim et al, 2013; Qian et al, 2017; Russell et al, 2013; Wei et al, 2015). The VPS34 catalytic subunit makes almost no contacts between the regulatory BECN1 and ATG14 subunits.…”