Extended Data Figure 5. Determining the specificity of ATG14 to autophagic SNAREs-mediated membrane fusion and characterizing recombinant ATG14 homo-oligomerization.

a, Scheme of the ensemble content-mixing assay using autophagic SNARE-reconstituted proteoliposomes (v- and t-proteoliposomes). b, Purified recombinant Flag–STX17, His-VAMP2, His-SNAP25 and His-Syntaxin1 were incubated with IgG Sepharose associated with recombinant ZZ–Flag–ATG14, and the ATG14 binding proteins were cleaved by TEV protease and detected by Coomassie blue staining (upper panel). ZZ–Flag–ATG14 bound to IgG Sepahrose was detected by western blotting in the bottom panel. ATG14 binds to STX17 but not to neuronal SNAREs. c, ATG14 had no detectable effect on promoting ensemble lipid-mixing of proteoliposomes reconstituted with neuronal SNAREs (n =3). d, The membrane-tethering activity of ATG14 CCD deletion mutant is largely intact. Shown is the mean number of tethered vesicles (±s.d.) (n =15) in the same sample channel. e, The ATG14 CCD deletion mutant fails to enhance ensemble lipid mixing of proteoliposomes reconstituted with autophagic SNAREs (n =3). f, The oligomeric states of recombinant ATG14 were determined by SEC–MALS. g, Membrane-tethering activities of ATG14 monomer and dimer measured by the single protein-free vesicle/liposome membrane-tethering assay. Shown are the mean numbers of tethered vesicles (±s.d.) (n =15) in the same sample channel. Representative images are shown in the bottom panels (n =15). h, Mapping ATG14 oligomerization sites to its N terminus. Flag-tagged full-length ATG14 or an ATG14 truncating mutant (lacking the first 70 residues) were transfected into HEK293T cells and treated with cross-linking agent DSS (0, 0.1, 0.2, 0.4 mM) for 30 min, then subjected to SDS–PAGE analysis. ATG14 was probed by anti-Flag antibody. i, ATG14 CCD deletion mutant still forms oligomer in the DSS cross-linking assay. j, Purified recombinant ATG14 (36 nM) was boiled in non-reducing SDS sample buffer with 12.5 mM TCEP (lane 2), 25 mM DTT (lane 3) or 2.5% β-mercaptoethanol (lane 4), or mock-treated (lane 1), and the samples were loaded on non-reducing SDS–PAGE and probed for anti-Flag antibody for ATG14. k, Flag–ATG14 was transfected into HEK293T cells and treated with rapamycin (500 nM for 12 h) and/or cross-linking agent DSS (0.2 μM) for 30 min, then subjected to SDS–PAGE analysis. ATG14 was probed by anti-Flag antibody.