hATG8 proteins are required for basal mitophagy. (A) Lysates from WT, hATG8 KO, and hATG8 KO HeLa cells reconstituted with individual Myc-tagged hATG8 proteins were immunoblotted with indicated antibodies. (B and C) hATG8 KO cells were reconstituted with individual Myc-tagged human ATG8 proteins, and cells were treated or not with BafA1 for 24h. Lysates were immunoblotted with indicated antibodies (B), and the ability of individual ATG8 proteins to restore basal mitophagy monitored as an increase in mitochondrial protein level upon treatment with BafA1 were quantified (C). Values are mean ± SD. ***, P < 0.001; **, P < 0.005; *, P < 0.01; †, NS; one-way ANOVA. (D) Relative ATP levels from WT and SAMM50 KD cells grown in either glucose or galactose media were measured with an ATP determination kit. Values are mean ± SD based on three independent experiments. ***, P < 0.001; *, P < 0.01; one-way ANOVA. (E) Extracts from HeLa cells transiently transfected with 3x-FLAG, 3x-FLAG-LC3B, and 3x-FLAG-p62 expression vectors and treated with CCCP, hypoxia (1% oxygen), and HBSS for 6h were immunoprecipitated with FLAG resin. Co-immunoprecipitation of endogenous NIPSNAP1 and SAMM50 was analyzed by immunoblotting. (F and G) Mapping of the SAMM50 binding site on p62. Myc-SAMM50 in vitro translated in the presence of radioactive methionine was incubated with recombinant GST, GST-p62 WT, and indicated deletion constructs (F) or with recombinant MBP, MBP-p62, and indicated mutants (G). Bound SAMM50 was analyzed by AR, and GST-tagged proteins were stained with CBB. The graphs in F represent percentage binding of in vitro translated Myc-SAMM50 to recombinant GST proteins. Values are mean ± SD based on three independent experiments.