Fig 2: TAX1BP1 recruits FIP200 to induce local autophagosome formation Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. P-TBK1, magenta; NBR1, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm.Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm.Representative confocal micrographs (as maximum intensity projections) from wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 and transduced with indicated sgRNA. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm.Quantitation of colocalization between NBR1 with FIP200 in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C) (see Materials and Methods for details of quantitation). Bar graphs represent mean ± SD for three independently generated deletion cell lines (dots). sgATG9A samples were compared using a one-way ANOVA (P < 0.0001) with Tukey's HSD post-test. ***P < 0.001. n > 200 NBR1 puncta for each biological replicate.Quantitation of FIP200 intensity at NBR1 puncta in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C). Bar graphs represent mean ± SD for three independently generated deletion cell lines (dots). n > 80 FIP200-positive puncta for each biological replicate. Samples were compared using a one-way ANOVA (P < 0.0001) with Tukey's HSD post-test. ***P < 0.001. a.u., arbitrary intensity units.TAX1BP1 drives local autophagosome formation in the context of NBR1 puncta. This activity is independent of LC3 or the ubiquitin-binding domains of TAX1BP1. Rather, a newly defined NBR-interacting domain (or N domain, not shown) drives the association of TAX1BP1 (red) with NBR1 puncta. Subsequent recruitment and clustering of FIP200 (blue) by TAX1BP1 induces local autophagosome formation and enforces cargo specificity, thereby replacing the requirement for lipidated LC3. Data information: See also Fig EV5. Source data are available online for this figure.

Fig 3: G-TPP leads to recruitment of autophagy adapters and degradation of mitochondria(A) HeLa cells stably expressing untagged Parkin were treated with 10 µM G-TPP for 8 h. Western blots were prepared from cell lysates and probed with antibodies against LC3, phospho-TBK1 (Ser172) and TBK1. GAPDH was used as a loading control. Upon 8 h the levels of LC3-I and LC3-II were both increased. At 8 h after treatment with G-TPP but not at 4 or 24 h, TBK1 was phosphorylated. (B) HeLa cells stably expressing EGFP-Parkin were treated with 10 µM G-TPP and fixed 8 h after treatment. Cells were stained with antibodies against the autophagy adapter proteins NBR1, NDP52, OPTN, p62, and TAX1BP1 (red). Mitochondria were counterstained with TOM20 antibodies (cyan), nuclei with Hoechst (blue). EGFP-Parkin epifluorescence is shown in green. Scale bar corresponds to 10 µM. (C) HeLa cells stably expressing EGFP-Parkin and the reporter protein mitoKeima were treated with 10 µM CCCP or G-TPP and imaged over time. The ratio of ‘neutral’ mitoKeima to ‘acidic’ mitoKeima was calculated as readout for mitophagy. Parkin translocation was monitored at the same time. Values for Parkin translocation and mitophagy were normalized to 12 h treatment with 10 µM CCCP as positive control and DMSO as negative control (two-way ANOVA with Tukey’s post-hoc test, **p < 0.005, ***p < 0.0005).

Fig 4: The N-terminal SKICH domain and NBR1-binding domain of TAX1BP1 are required for ATG7-independent flux of NBR1 (related to Fig 6) HEK293T cells were transfected with myc-TAX1BP1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP-conjugated dynabeads. Input and eluates were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies.Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. Ubiquitin, magenta; NBR1, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm.Wild-type K562 cells were nucleofected with tf-NBR1 (isoform 1) or tf-NBR1 (isoform 2; lacking the UBA domain) and analyzed for red:green ratio at 18 h post-nucleofection. Median values for each sample are identified by a black line within each violin. The red dotted line (max value) corresponds to the red:green ratio of tf-NBR1 isoform 1. The solid red line (min value) corresponds to the red:green ratio of tf-NBR1 isoform 1 upon Bafilomycin A1 (BafA1) treatment. n > 2,000 cells for each sample.Representative confocal micrographs (as maximum intensity projections) of ATG9AKO/TAX1BP1KO cells expressing tf-NBR1. Cells were transduced with the indicated BFP-tagged TAX1BP1 variants. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. BFP-TAX1BP1, magenta; NBR1, green; Scale bars: large panels, 5 µm; small panels, 1 µm.K562-derived extracts prepared from wild-type (WT) and clonal deletion isolates were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies. All samples were normalized by total protein using a BCA assay prior to loading.Clonal isolates of indicated knockout cells lines (from (E)) were treated with Bafilomycin A1 (BafA1) or DMSO for 18 h and analyzed for tf-NBR1 flux by red:green ratio as assessed by flow cytometry. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio of WT cells (normal flux). The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n > 5,000 cells.HEK293T cells were transfected with indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP conjugated dynabeads. Input and eluates were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies.HEK293T cells were transfected with HA-FIP200 and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP conjugated dynabeads. Input and eluates were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies. Source data are available online for this figure.

Fig 5: A heterotypic receptor complex defines an ATG7-independent substrate Extracts derived from wild-type and deletion K562 cells expressing tf-NBR1 were normalized for total protein by BCA. NBR1 was immunoprecipitated using GFP-Trap dynabeads. Input and eluate were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies.Representative confocal micrographs (as maximum intensity projections) of wild-type K562 and ATG9AKO cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. In conditions where puncta were not observed, representative cytoplasmic regions were selected to showcase diffuseness of signal. TAX1BP1, SQSTM1, or NDP52, magenta; NBR1, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm.Shown are representative confocal micrographs (as maximum intensity projections) of K562 cells expressing tf-NDP52 (or tf-TAX1BP1) and transduced with sgRNAs targeting ATG9A (sgATG9A) or a negative control (sgControl). Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. See Fig EV3D and E for confirmation of ATG9A deletion. Data information: See also Fig EV3. Source data are available online for this figure.