Cryo-EM structure and biochemical analysis reveal the basis of the functional difference between human PI3KC3-C1 and -C2

Abstract

Phosphatidylinositol 3-phosphate (PI3P) plays essential roles in vesicular trafficking, organelle biogenesis and autophagy. Two class III phosphatidylinositol 3-kinase (PI3KC3) complexes have been identified in mammals, the ATG14L complex (PI3KC3-C1) and the UVRAG complex (PI3KC3-C2). PI3KC3-C1 is crucial for autophagosome biogenesis, and PI3KC3-C2 is involved in various membrane trafficking events. Here we report the cryo-EM structures of human PI3KC3-C1 and PI3KC3-C2 at sub-nanometer resolution. The two structures share a common L-shaped overall architecture with distinct features. EM examination revealed that PI3KC3-C1 "stands up" on lipid monolayers, with the ATG14L BATs domain and the VPS34 C-terminal domain (CTD) directly contacting the membrane. Biochemical dissection indicated that the ATG14L BATs domain is responsible for membrane anchoring, whereas the CTD of VPS34 determines the orientation. Furthermore, PI3KC3-C2 binds much more weakly than PI3KC3-C1 to both PI-containing liposomes and purified endoplasmic reticulum (ER) vesicles, a property that is specifically determined by the ATG14L BATs domain. The in vivo ER localization analysis indicated that the BATs domain was required for ER localization of PI3KC3. We propose that the different lipid binding capacity is the key factor that differentiates the functions of PI3KC3-C1 and PI3KC3-C2 in autophagy.

Figure 1

Purification and reconstruction of C1 and C2. (A) Domain organization of ATG14L and UVRAG. (B, C) Chromatography of C1 and C2 on Superose 6 10/300 GL columns. The dark curves show the UV absorbance at 280 nm, and the peaks containing C1 and C2 are marked by red lines along with the elution volume. SDS-PAGE analyses of the complexes in the peaks are shown at the right of the top panels in B and C. The corresponding proteins are labeled on the left of the PAGE images. The molecular weights of the standard markers are labeled on the right. Classical 2D class averages of cryo-EM images of C1 and C2 are shown in the bottom panels. (D) In vitro lipid kinase assay of C1 and C2. Data are represented as mean ± SD (n = 3). (E) 3D reconstruction volume of C1 (grey) with a threshold of 3.9 σ. The model in the bottom panel is rotated 90 degrees along the main axis compared to the model in the top panel. (F) 3D reconstruction volume of C2 (yellow) with a threshold of 3.8 σ. The model in the bottom panel is rotated 90 degrees along the main axis compared to the model in the top panel.

Figure 2

Atomic model building and analysis of C1 and C2. (A, C) The chimeric EM volume of C1 docked with the built atomic model (VPS34-CTD excluded). Within the atomic model, the Beclin1, ATG14L, VPS34 and P150 protein subunits are colored orange, light green, hot pink and cyan, respectively. The chimeric EM volume is presented as a translucent grey surface. A shows the top view and C represents the side view. (B, D) The refined EM volume of C2 docked with the hypothetical atomic model of C2 (VPS34-CTD excluded), which was directly extracted from the model of yeast PI3K complex 2 (PDB code: 5DFZ). Within the atomic model, the Beclin1, UVRAG, VPS34 and P150 subunits are colored orange, light green, hot pink and cyan, respectively. The chimeric EM volume is presented as a translucent grey surface. B shows the top view and D represents the side view. The dashed circle in D indicates the electron density from the β-barrel insertion within the NTD of UVRAG. (E, F) The built atomic model of C1 and the docked model of C2. The dashed rectangles indicate Beclin1, ATG14L and the P150 WD domain in C1 and Beclin1, UVRAG and the P150 WD domain in C2, which were extracted from the overall model and are presented in the bottom panels with the same orientation. The relative orientation of these two extracted parts is marked with the rotation axis and angle. The top right panels in E and F show cartoon models of the side and top views of C1 and C2. The green shapes indicate the Beclin1/ATG14L subunits in C1 and the Beclin1/UVRAG subunits in C2. The blue shapes indicate the P150/VPS34-NTD subunits in both complexes.

Figure 3

Membrane binding orientation of C1. (A) Orientation of C1 on carbon films. The upper panel presents the five top 2D class averages of WT C1 on carbon. The lower panel presents the Euler angle distribution of all the particles generated by SPIDER. The sizes of the dark dots correlate with the number of particles in a certain orientation. A classical surface view coupled with the projection for the preferred orientation is shown on the right. The cartoon model indicates the general appearance of the particles on carbon film in their preferred orientation. (B) Orientation of C1 on lipid monolayers. The upper panel presents the five top 2D class averages of WT C1 bound to lipid monolayers. The lower panel presents the Euler angle distribution of all the particles generated by SPIDER. The sizes of the dark dots correlate with the number of particles in a certain orientation. Two classical surface views corresponding to the projections of the preferred orientations are shown on the left and right. The cartoon models indicate the general appearance of WT C1 particles on lipid monolayers in their preferred orientations. (C) Orientation of truncated C1 with VPS34 CTD deletion. The descriptions of the figure panels are the same as in A. (D) Flotation assay to examine the binding of C1 WT, C1 VPS34 ΔCTD and C1 ATG14L ΔBATs to liposomes containing 6% PI in a sucrose gradient (from top to bottom: 0%, 20%, 25%, 30%). All 14 fractions from top to bottom were immunoblotted using an antibody against ATG14L. (E) The flotation ratio from D for ATG14L and Supplementary information, Figure S4E for P150, VPS34 and Beclin1 was calculated by dividing the total intensity of the bands in all the fractions with the intensity of the bands in the top 8 fractions, then normalizing to the value for C1 WT. Data are represented as mean ± SD (n = 3). (F) Membrane binding model of C1. The structural areas responsible for membrane binding (ATG14L BATs domain) and the orientation of the complex (VPS34 CTD) are colored in hot pink.

Figure 4

The BATs domain determines the ER-binding capacity of PI3KC3 complexes in vitro. (A) Flotation assay to examine the interaction of C1 and C2 with liposomes containing 6% PI3P in a sucrose gradient (from top to bottom: 0%, 20%, 25%, 30%). All 14 fractions from top to bottom were immunoblotted using an antibody against P150. (B) The flotation ratio from C for P150 and Supplementary information, Figure S5C for VPS34, ATG14L/UVRAG and Beclin1 was calculated by dividing the band intensities from all the fractions with the band intensities of the top 8 fractions, then normalizing to the value for C1. Data are represented as mean ± SD (n = 2 or 3). (C) Flotation assay to examine the binding of C1, C2 and C2-BATs to liposomes containing 6% PI in a sucrose gradient (from top to bottom: 0%, 20%, 25%, 30%). All 14 fractions from top to bottom were analyzed by immunoblotting using an antibody against P150. (D) The flotation ratio from A for P150 and Supplementary information, Figure S5B for VPS34, ATG14L/UVRAG and Beclin1 was calculated by dividing the band intensities of all the fractions with the band intensities of the top 8 fractions, then normalizing to the value for C1. Data are represented as mean ± SD (n = 2 or 3). (E) Flotation assay to examine the interaction of C1, C1 ΔBATs, C2 and C2-BATs with the ER fraction in a sucrose gradient (from top to bottom: 0%, 20%, 25%, 30%). All 14 fractions from top to bottom were analyzed using an antibody against P150. (F) The flotation ratio from E for P150 and Supplementary information, Figure S5F for VPS34, ATG14L/UVRAG and Beclin1 was calculated by dividing the input with the top 8 fractions, then normalizing to the value for C1. Data are represented as mean ± SD (n = 2 or 3). (G) Confocal analyses of the subcellular co-localization of GFP-ATG14L WT or GFP-ATG14L ΔBATs with the ER marker calnexin in HEK293 cells. The insets in the top right corner show a high magnification of the selected areas. (H) Quantification of co-localization of GFP-ATG14L WT or GFP-ATG14L ΔBATs with calnexin (data are shown as mean ± SD, n = 30 cells obtained by gathering data from three independent experiments). ^***P < 0.001. (I) Confocal analyses of the subcellular co-localization of GFP-UVRAG WT or GFP-UVRAG-BATs with the ER marker calnexin in HEK293 cells. The insets in the top right corner show a high magnification of the selected areas. (J) Quantification of co-localization of GFP-UVRAG WT or GFP-UVRAG-BATs with calnexin (data are shown as mean ± SD, n = 30 cells obtained by gathering data from three independent experiments). ^***P < 0.001.