Volume 68, Issue 4, Pages 786-796.e6 (November 2017) Autophagosomal Content Profiling Reveals an LC3C-Dependent Piecemeal Mitophagy Pathway  François Le Guerroué, Franziska Eck, Jennifer Jung, Tatjana Starzetz, Michel Mittelbronn, Manuel Kaulich, Christian Behrends  Molecular Cell  Volume 68, Issue 4, Pages 786-796.e6 (November 2017) DOI: 10.1016/j.molcel.2017.10.029 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 68, 786-796.e6DOI: (10.1016/j.molcel.2017.10.029) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Autophagosome Targeting of APEX2 (A) Schematic overview of autophagosome targeting and labeling using APEX2 (AP2) N-terminally fused to hATG8s. (B) Homogenates from AP2-hATG8s chimera expressing HeLa cells grown in the presence of BafA1 (2 hr), BP (30 min), and H2O2 (1 min) were left untreated or incubated with protK, Triton-X-100, or both followed by immunoblotting. (C) HeLa cells expressing AP2-hATG8s were grown in the presence of BafA1 (2 hr), BP (30 min), and H2O2 (1 min) followed by fixation and immunolabeling with the indicated antibodies. Scale bars, 5 μm. Yellow and blue arrows indicate colocalization events. Magnifications are provided for blue-arrow-labeled structures in Figure S1. See also Figure S1. Molecular Cell 2017 68, 786-796.e6DOI: (10.1016/j.molcel.2017.10.029) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Identification of Luminal Autophagosome Proteins by Protease-Protection Proximity Proteomics (A) Proteomic workflow for autophagosomal content profiling. (B) Overlap of total (left) and BafA1 upregulated (right) protK-protected, biotinylated proteins identified across biological replicates of hATG8s. (C) Scatterplot analysis of proteins identified by MS in pull-downs from protK-protected, AP2-labeled autophagosomes. R, Pearson’s correlation of two biological replicates. (D) Comparison of BafA1 upregulated proteins across biological duplicates of hATG8s. (E) Heatmap representation of known autophagy receptors and substrates found by Mancias et al. (2014) (italic), Gao et al. (2010) (underline), and Dengjel et al. (2012) (regular). (F) Overlap of BafA1 upregulated proteins between GABARAP (top) and LC3 (middle) subfamily members as well as between GABARAPs and LC3s (bottom). Notably, for the latter, proteins were required to be identified by at least two members of each hATG8 subfamily. (G) BafA1 upregulated proteins found in two biological replicates. See also Figure S2 and Table S1. Molecular Cell 2017 68, 786-796.e6DOI: (10.1016/j.molcel.2017.10.029) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 LC3C Is Required for Basal Autophagosomal Degradation of MTX1 (A) HeLa cells inducibly expressing HA-MTX1 for 3 hr were transiently transfected with AP2-LC3C and grown in the presence of BP (30 min) and H2O2 (1 min) followed by fixation and immunolabeling with the indicated antibodies. Scale bars, 5 μm. Yellow arrow indicates a colocalization event. (B) Homogenates from BafA1-treated (2 hr) HeLa cells inducibly expressing HA-MTX1 for 3 hr were left untreated or incubated with protK, Triton X-100, or both followed by immunoblotting. (C–G) HeLa cells inducibly expressing HA-MTX1 (C, F, and G), HA-SAMM50 (D), or HA-MTX2 (E) were left untreated, transfected with the indicated small interfering RNAs (siRNAs) for 72 hr (C–E and G), or treated with PepA/E64d (G) for 4 hr followed by immunoblotting. Data represent mean ± SEM. Statistical analysis (n = 3) of the HA-tagged protein/actin ratio was performed (∗p < 0.05, unpaired t test). (H and I) HeLa cells harboring endogenously FLAG-tagged MTX1 were transfected with non-targeting control siRNA (siCtrl), siLC3C, or siMTX1 (H) and with siCtrl, siLC3C, siLC3B, or siGABARAP (I) for 72 hr followed by immunoblotting. Data represent mean ± SEM. Statistical analysis (n = 3) of the MTX1/actin ratio was performed (∗p < 0.05, unpaired t test). (J) HeLa cells harboring endogenously HA-tagged LC3C and overexpressing parkin were transfected with the indicated siRNAs and treated with O/A for 24 hr prior to lysis and immunoblot analysis. (K) Top: HeLa cells inducibly expressing HA-MTX1 for 3 hr were transiently transfected with GFP-LC3C followed by fixation and immunolabeling with indicated antibodies. Scale bars, 5 μm. Blue arrow indicates colocalization event. Bottom: magnifications of blue arrow-labeled structures. Scale bars, 2.5 μm. (L and M) Homogenates derived from PepA/E64d-treated (4 hr) HeLa cells inducibly expressing HA-MTX1 (L) or HA-SAMM50 (M) for 3 hr were subjected to density gradient ultracentrifugation. Selected fractions corresponding to bands formed in the gradient were analyzed by immunoblotting. (N and O) Homogenates from wild-type (WT) or ATG7-knockout (KO) HeLa cells were processed and analyzed as in B (N) and L (O), respectively. Dotted line represents removal of the marker lane. See also Figure S3. Molecular Cell 2017 68, 786-796.e6DOI: (10.1016/j.molcel.2017.10.029) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 LC3C Mediates Lysosomal Delivery of Selective Mitochondrial Proteins and Is Required for Maintaining Mitochondrial Morphology (A) Overlap between protK-protected, biotinylated proteins found in duplicate AP2-LC3B and AP2-MTX1 experiments and annotated with the gene ontology term mitochondrion. Inset shows numbers of redundant proteins in distinct mitochondrial compartments. (B) HeLa cells transiently transfected with Myc-BirA∗-LC3B and Myc-BirA∗-LC3C for 48 hr were treated with biotin for 16 hr followed by lysis and streptavidin pull-down. DNP-derivatized protein samples were analyzed by immunoblotting. (C–F) HeLa cells expressing HA-MTX1 were transfected with an siRNA targeting DRP1 for 72 hr followed by fixation and immunolabeling with anti-HA antibody together with anti-TOM20 and -LAMP1 (C), anti-PDH and -LAMP1 (D), anti-PDH and -TOM20 (E), or anti-TOM20 and -IMMT (F) antibodies. Top: scale bars, 5 μm; yellow and blue arrows indicate colocalization events. Bottom: magnifications of blue-arrow-labeled structures; scale bars, 2.5 μm. (G) HeLa cells grown on galactose were transfected with the indicated siRNAs for 72 hr and then fixed and subjected to immunolabeling with the indicated antibodies. Scale bars, 2.5 μm. (H) Quantification of mitochondrial morphologies from (G). ∗p < 0.05 (unpaired t test). (I) Lysates form HeLa cells harboring endogenously FLAG-tagged MTX1 were grown in glucose- or galactose-containing media, treated with DMSO or PepA/E64d, and analyzed by immunoblotting. See also Figure S4. Molecular Cell 2017 68, 786-796.e6DOI: (10.1016/j.molcel.2017.10.029) Copyright © 2017 Elsevier Inc. Terms and Conditions