Fig. 7: Initiation hubs establish contact sites with the ER in mammalian cells.

a, Schematic representation of the proximity biotinylation setup. FKBP-APEX2-ULK1 is tethered to mitochondrial FIS1–FRB by rapalogue addition. FIP200 connects this mitochondrial assembly to the ER. Proximity biotinylation is induced by the addition of biotin–phenol and H₂O₂. Biotinylated proteins are isolated by affinity purification with streptavidin beads and analysed by mass spectrometry. FKBP–APEX2–ULK1 was compared with the unspecific control (FKBP–APEX) in each experiment. b, Mass spectrometry analysis of APEX2-based proximity labelling in HEK293 cells stably expressing 2×FKBP–APEX2–ULK1 or 2×FKBP–APEX2. Cells were grown under nutrient-rich conditions and rapalogue. Proximity labelling was induced by the addition of biotin–phenol and a short pulse of H₂O₂. The volcano plot shows the enrichment of biotinylated proteins in 2×FKBP–APEX2–ULK1 compared with 2×FKBP–APEX2. Dashed purple lines indicate the cutoffs used to identify ULK1-specific proteins (log₂ ratio >1 and adjusted P < 0.01). Known autophagy proteins enriched in 2×FKBP–APEX2–ULK1 are highlighted in cyan. Ratios are calculated using mean values of three biological replicates. Statistical analyses carried out were moderated t-statistics using the limma-trend method and multiple testing correction with the Benjamini–Hochberg procedure. c, Mass spectrometry data from b. The kernel density estimate (KDE) plot compares all proteins against those listed under the Gene Ontology (GO) term ‘autophagy’ (GO:0006914) using the FKBP–APEX2–ULK1 versus FKBP–APEX2 ratio dataset. d, Heatmap displaying ULK1-specific ER membrane proteins with a significantly reduced signal upon knockdown of FIP200 (adjusted P value < 0.05). The enrichment of protein signal in FKBP–APEX2–ULK1 over FKBP-APEX2 is shown for three biological replicates of wild-type and FIP200 knockdown cells. Statistical analysis: two-tailed unpaired t-test, multiple testing correction with the Benjamini–Hochberg procedure. e, Mass spectrometry data from d. The KDE plot shows the comparison FKBP–APEX2–ULK1 WT versus FKBP–APEX2–ULK1 siFIP200 of all proteins that are positive for the GO term ‘ER membrane’ (GO:0005789). The samples were control-corrected before the analysis. f, U2OS cells transfected with FKBP–GFP–FIP200, mCherry–p62 and BFP–Sec61β, were cultured in nutrient-rich medium. Intensity profiles were calculated for the 405 nm, 488 nm and 561 nm channels along the indicated line. A representative image from one out of three biological replicates is shown. Scale bar, 1 µm. The KDE plot shows the difference in ER proximity between FIP200 and p62 structures, calculated from 47 structures. The 3D surfaces of BFP–Sec61β (cyan), FKBP–GFP–FIP200 (green) and mCherry–p62 (magenta) were rendered with the Imaris software using the machine-learning tool for surface segmentation. A z-stack of 0.125 µm was taken to define the borders in z. Further examples are shown in Extended Data Fig. 7c,d. Source numerical data are available in Source data.