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. 2018 Oct 16;115(42):E9792-E9801.
doi: 10.1073/pnas.1811874115. Epub 2018 Sep 5.

Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex

Saikat Chowdhury  1 , Chinatsu Otomo  1 , Alexander Leitner  2 , Kazuto Ohashi  1 , Ruedi Aebersold  2   3 , Gabriel C Lander  4 , Takanori Otomo  4
Affiliations

Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex

Saikat Chowdhury et al. Proc Natl Acad Sci U S A. .

Abstract

Autophagy is an enigmatic cellular process in which double-membrane compartments, called "autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the PI3P-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

Keywords: ATG2; autophagy; chemical cross-linking coupled with mass spectrometry; membrane tethering; single-particle analysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structural analyses of the human ATG2A-WIPI4 complex and the ScATG2-ATG18 complex by negative-stain EM. (A) Diagram of the primary structure of ATG2. The lengths of human ATG2A/B and ScATG2 are indicated. The regions conserved among all species are indicated by ovals with solid outlines. The similarities suggested in ScATG2 to FMP27 and APT1 proteins are indicated as ovals with dashed outlines. (B) Affinity capture experiment with ATG2A immobilized on the beads and WIPI4 in solution. (C) Superose 6 size-exclusion chromatography profile of the mixture of ATG2A and an excess amount of WIPI4. (D and E) 2D class averages of the ATG2A-WIPI4 complex (D) and ATG2A alone (E). (F and G) Reconstructed 3D structures of the ATG2A-WIPI4 complex (F) and ATG2A alone (G). (H) 2D class averages of the ScATG2-ATG18 complex. Green asterisks in 2D class averages in D and H indicate the locations of WIPI4 and ATG18, respectively.
Fig. 2.
Fig. 2.
Structural characterizations of the ATG2A-WIPI4 complex. (A) CXL-MS analysis of the ATG2A-WIPI4 complex. Cross-links within ATG2A and between ATG2A and WIPI4 are indicated by dashed and solid lines, respectively. The cross-linked residues are labeled. The diagram of the primary structure of ATG2A is colored per the conservation score of each residue as indicated. The conservation score was calculated on the ConSeq server (56). Pfam conserved domains (Chorein_N, ATG2_CAD, and ATG_C) and the CLR are indicated. (B) 2D class averages of MBP-fused or inserted ATG2A in complex with WIPI4. For each construct, a representative 2D class in which the MBP is consistently located adjacent to a tip of ATG2A is shown; the number of particles (in percentage) that constitute all the similar 2D classes in each dataset is indicated below. The remaining particles for each dataset in which the MBP density is missing in the 2D classes are not shown. The green and blue asterisks indicate the locations of WIPI4 and MBP, respectively. (C) Superdex 200 size-exclusion chromatography profiles of 6xHis-GB1-ATG2A (residues 1358–1404), WIPI4, 6xHis-GB1, and their mixtures as indicated. SDS/PAGE shows the size-exclusion fraction contents of the sample mixtures containing stoichiometric amounts of WIPI4 and 6xHis-GB1-ATG2A (1358–1404) (Left) and of WIPI4 and 6xHis-GB1 (Right), respectively. (D) WIPI4-docked electron density map of the ATG2A-WIPI4 complex.
Fig. 3.
Fig. 3.
Interaction between ATG2A and liposomes and its visualization by EM. (A) Liposome flotation assay with 50 nM ATG2A. The liposomes composed of 99% DOPC and 1% 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) (indicated as “−PS”) or 74% DOPC, 25% DOPS, and 1% DiD (+PS) were prepared by sonication (SUVs) or extrusion (LUVs) using 100- or 400-nm filters. The inputs (4%) and the top layers after centrifugation (24%) were loaded onto SDS/PAGE. The percentage of ATG2A recovered in each of the top fractions was quantified and is shown below the gel image. (B) Micrographs of the negative-stained ATG2A-WIPI4-SUV complex. Colored arrowheads and arrows denote an elongated object that emanates perpendicularly (blue arrowheads) or tangentially (yellow arrowheads) from an SUV or is tethering two SUVs (green arrows). (C) 2D class averages of the ATG2A-WIPI4-SUV complex shown without (Upper) and with (Lower) a manually placed 3D model of ATG2A (shown in yellow). The green dot marks the WIPI4 density.
Fig. 4.
Fig. 4.
Membrane tethering by ATG2A. (A–C) The DLS profiles of SUVs (A), LUVs (100 nm) (B), and LUVs (400 nm) (C) in the absence (cyan) or presence (magenta) of 200 nM ATG2A. All liposomes consisted of 75% DOPC and 25% DOPS. The samples were incubated for 1 h before the measurements. (D) The final sample of A was mixed with proteinase K and remeasured after 1 (yellow) and 2 h (purple) incubation. (E) Auto-scaled autocorrelation functions of the four DLS measurements with SUVs (B and D) are plotted. (F) Fluorescence liposome-tethering assay. Liposomes consisting of 73.3% DOPC, 25% DOPS 0.2% biotinylated lipids, and 1.5% rhodamine-PE were mixed with liposomes of the same size consisting of 73% DOPC, 25% DOPS, and 2% 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) in the presence of 200 nM ATG2A. The fluorescence reports the number of DiD-containing liposomes associated with the biotinylated liposomes. For each experiment, the average of three repeats is shown; whiskers indicate the SD.
Fig. 5.
Fig. 5.
Tethering of PI3P-containing LUVs by the ATG2-WIPI4 complex. (A–C) DLS profiles of LUVs (100 nm) consisting of 75% DOPC, 15% DOPS, and 10% PI3P in the absence (A–C: cyan) or the presence of 200 nM WIPI4 (A), 200 nM ATG2A (B), or both proteins (C). (D) Auto-scaled autocorrelation functions of the four DLS measurements. (E) Fluorescence-based liposome tethering assay. The higher the fluorescence, more associations there are between the liposomes with and without PI3P. LUVs consisting of 73.3% DOPC, 15% DOPS, 10% PI3P, 0.2% biotinylated lipids, and 1.5% rhodamine-PE were mixed with LUVs consisting of 73% DOPC, 25% DOPS, and 2% 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate in the presence of the indicated proteins. For each experiment, the average of three repeats is shown; whiskers indicate the SD.
Fig. 6.
Fig. 6.
Characterization of the CLR. (A) Liposome flotation assay with 100 nM MBP-fused ATG2A CLR (residues1723–1819) or MBP alone (control). The liposomes used are indicated as in Fig. 3A. The inputs (4%) and the top layers after the centrifugation (25%) were loaded onto SDS/PAGE. The percentage of MBP-CLR recovered in each of the top fractions was quantified and is shown below the gel image. (B) Preparation of the CLR-SUV complex by liposome flotation. The input containing MBP-CLR and SUVs (75% DOPC and 25% DOPS) was mixed with tobacco etch virus (TEV) to cleave off MBP, and the resulting CLR-SUV complex was isolated in the top fraction after centrifugation. (C) The CD spectrum of the CLR-SUV complex. (D) Predicated α-helical regions in the CLR. A multiple sequence alignment (MSA) of the CLR of Homo sapiens (Hs) ATG2A, HsATG2B, Drosophila melanogaster (Dm) ATG2, Schizosaccharomyces pombe (Sp) ATG2, and ScATG2 was generated by ClustalW. Secondary structure predictions were obtained using JPred (57) and PSIPred (58) servers. The three fragments generated for flotation assays (H1: residues 1721–1739; H2: residues 1751–1774; H3: residues 1777–1819) are shown as a cartwheel drawing generated by the HeliQuest server (59). The asterisks shown above the MSA and in the cartwheel drawings indicate the residues mutated to aspartic acid. (E) Liposome flotation assay with 1 µM MBP-fused CLR fragments and LUVs prepared by extruding a lipid mixture consisting of 74% POPC, 25% POPS, and 1% 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate through a 100-nm membrane.
Fig. 7.
Fig. 7.
The CLR is not involved in membrane tethering. (A) Flotation assay with the ATG2A12xD mutant and liposomes composed of 74% POPC, 25% POPS, and 1% 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate. The liposomes were prepared by sonication or extrusion using 30- or 100-nm filters. The result of SDS/PAGE is shown as in Figs. 3A and 6A. (B and C) DLS homotypic membrane-tethering assay with ATG2A12xD. The DLS profiles (B) and the autocorrelation functions (C) of the 75%DOPC/25%DOPS SUVs in the absence and presence of ATG2A12xD are shown. (D) Fluorescence homotypic membrane assay with ATG2A wild-type and ATG2A12xD performed and presented as in Fig. 4F. (E and F) DLS homotypic membrane-tethering assay with PI3P-incorporated LUVs (100 nm). The DLS profiles (E) and the autocorrelation functions (F) are shown. (G) Fluorescence heterotypic tethering assay with ATG2A12xD performed as shown in Fig. 5E. The experiments with the wild-type ATG2A repeat the data shown in Fig. 5E but were performed at the same time as the experiments with mutants. Although the fluorescence values are different from those in Fig. 5E, the results from both experiments with the wild type are consistent with each other.
Fig. 8.
Fig. 8.
Proposed models of the ER-phagophore/isolation membrane association mediated by the ATG2-WIPI/ATG18 complex. (A) Illustration of the ER–phagophore junction based on current knowledge from cell biological studies. Each gray line represents a lipid bilayer. (B) Structural model of the ATG2-WIPI/ATG18 complex tethering the omegasome to its neighboring membranes (ER, phagophore edge, ATG9 vesicle, or COPII vesicle). The dark red color of ATG2 represents conserved regions as in Fig. 2A. The WIPI/ATG18-binding region of ATG2 is represented as a black line emanating from the middle region of ATG2 to indicate the flexible attachment of WIPI/ATG18.
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