Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

Abstract

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.DOI: http://dx.doi.org/10.7554/eLife.05115.001

Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy

“…High-quality peptides were analyzed, clearly separated from other peptides, and inspected individually. HDX-MS analysis of PI3KC3-C1 is largely consistent with our previous analysis of VPS34 and VPS15 (12). All four subunits contained at least one peptide for which the percent hydrogen exchange changed by more than 10% in the presence of NRBF2 (Fig.…”

“…The complex undergoes at least two large-scale structural movements. The V is capable of opening and closing to the extent of about 45°, and the VPS34 lipid kinase undergoes dislodging from the rest of the complex (12).…”

“…The structure of the four-subunit human PI3KC3-C1 assembly was determined at low resolution by our group by negative-stain EM and revealed an overall V-shaped architecture (12). One arm of the V contains the VPS34 lipid kinase and VPS15 pseudokinase domains, whereas the other includes BECN1, ATG14, and the WD40 (WD repeat) domain of VPS15.…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…75,76,86 In the PIK3C3 complexes, PIK3R4 is required for the kinase activity of PIK3C3, [89][90][91][92] and it serves as a bridge linking PIK3C3 with ATG14-BECN1, or with UVRAG-BECN1. 87 For clarity, the compositions of Vps34/PIK3C3 complexes in yeast and mammals and their functional specificity are summarized in Table 2. In the following sections, we will discuss in more detail how each of the components in class III PtdIns3K complexes functions as a point in the autophagy-regulatory network under different circumstances and the composition of the corresponding complexes (Fig.…”

“…84,86 Atg14/ATG14 and Vps38/UVRAG are present exclusively in 2 Vps34/PIK3C3 complexes (PtdIns3K complex I and II in yeast, and class III PtdIns3K-C1 and PtdIns3K-C2 in mammalian cells, respectively), which are organized in similar V-shape conformations with Vps15/PIK3R4 at the base. 87 In yeast, PtdIns3K complex I and II play a role in autophagy and the vacuolar protein sorting pathway, respectively. 74 In mammalian cells, PtdIns3K-C1 and PtdIns3K-C2 mainly function in autophagy and endosomal trafficking, respectively; 75,88,89 however, both are implicated in autophagy.…”