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 ...