Fig 2: OPTN and NDP52 Fluoppi foci contain ATG9A and ATG13, respectively. (A) HA-Ash-6Ub– and hAG-tagged OPTN or NDP52 were transiently expressed in Penta KO HeLa cells. The cells were immunostained with anti-HA and anti-ATG9A or anti-ATG13 antibodies. Scale bars, 10 µm; insets, 2 µm. (B) HA-Ash-6Ub and hAG alone or hAG-tagged autophagy adaptors were transiently expressed in ATG9A KO HeLa cells. The cells were immunostained with anti-HA and anti-ATG13 antibodies. Scale bars, 10 µm; insets, 2 µm.

Fig 3: OPTN–ATG9A interaction is critical for efficient mitophagy. (A) Penta KO HeLa cells stably expressing YFP-Parkin and mt-Keima with or without the indicated 3FLAG-OPTN were treated with AO and analyzed by FACS. Representative FACS plots with the mt-Keima 561/488-nm ratio are indicated. (B) Quantification of FACS-based mitophagy in A. Error bars represent mean ± SD in three independent experiments. (C) Quantification of GFP-Parkin translocation to mitochondria in Penta KO HeLa cells with or without the indicated 3FLAG-OPTN after treatment for 3 h with AO. Error bars represent mean ± SD, with >100 cells counted in each of three independent experiments. (D) Immunoblots of Penta KO cells stably expressing untagged Parkin with or without the indicated 3FLAG-OPTN following AO treatment. (E) Penta KO HeLa cells stably expressing untagged Parkin and the indicated 3FLAG-OPTN or NDP52 were treated with valinomycin for 3 h and then immunostained. HSP60 was used as a mitochondrial marker. Scale bars, 10 µm; insets, 2 µm. (F) Quantification of ATG9A recruitment to mitochondria in cells in E. Error bars represent mean ± SD, with 50 Fluoppi foci quantified in two independent experiments. (G) HeLa cells stably expressing untagged Parkin and the indicated 3FLAG-OPTN were treated with valinomycin for 3 h and then immunostained. Line scan plots of the fluorescent signals in the merged images (indicated by the white arrows) are shown in the lower panel. Scale bars, 10 µm; insets, 2 µm. (H) Quantification of cells with either a heterogeneous or a homogeneous OPTN distribution on mitochondria after treatment with valinomycin for 3 h. Representative images are shown in the upper panel. Error bars represent mean ± SD, with >100 cells counted in each of three independent experiments. Scale bars, 10 µm. ***, P < 0.001.

Fig 4: Heterogeneous mitochondrial localization of OPTN and NDP52 during Parkin-mediated mitophagy. (A) Immunoblots of HeLa cells stably expressing untagged Parkin with or without 3FLAG-tagged autophagy adaptors (OPTN, NDP52, p62, and NBR1) following valinomycin treatment for 3 h. 3FLAG-tagged autophagy adaptors and those with ubiquitination are indicated by the blue and red dots, respectively. I and II denote unmodified and lipidated LC3B, respectively. ~Ub1 and ~Ub2 denote ubiquitinated forms of TOMM20. (B) HeLa cells in A were treated with valinomycin for 3 h and then immunostained. (C) The cells prepared in B were immunostained with anti-FLAG and anti-p62 antibodies. (D) HeLa cells stably expressing untagged Parkin were treated with valinomycin for 3 h and then immunostained. (E) The cells prepared in B were immunostained with anti-FLAG and anti-LC3B antibodies. Magnified images in B–E are shown. Scale bars, 10 µm; insets, 2 µm.

Fig 5: Engineered LC3B and GATE-16 variants co-immunoprecipitate endogenous OPTN, p62 or NDP52. (A–C) Co-immunoprecipitation (Co-IP) of (A) OPTN, (B) NDP52, or (C) p62 binding variants and corresponding wt controls expressed as SGFP2 fusion proteins in stable inducible HeLa cells. Western blots were probed with indicated antibodies. (D–F) Mass spectrometry of Co-IPs using anti-GFP beads from stable HeLa cells expressing inducibly SGFP2 fusion proteins of LC3Bwt, GATE-16wt, OPTN.LC3Bv or p62.GATE-16v. In case of NDP52.LC3Bv, HeLa cells were transiently transfected with SGFP2 fusion constructs either expressing LC3Bwt or NDP52.LC3Bv. Detected proteins (gray) were plotted as log2 enrichment vs log10 intensity. Endogenous OPTN (blue), p62 (orange), NDP52 (green) and other LIR containing proteins (black) are shown as indicated.