Fig 2: 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 3: Domain analysis of TAX1BP1 function distinguishes canonical and non-canonical autophagy functions Domain organization of TAX1BP1 (NP_006015.4) and derived variants. Coiled-coil regions were defined by PCOILS using a 28 amino acid window and cutoff of 0.5. SKICH, SKIP carboxyl homology domain; CC, coiled-coil; Z1 and Z2, ubiquitin-binding zinc finger (UBZ) domains; N, NBR-interacting domain.A summary of TAX1BP1 variants' activity in complementation and flux analyses. For complementation analysis, ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 were electroporated with BFP-TAX1BP1 constructs, and BFP-positive cells were analyzed for red:green ratio (for underlying data, see Appendix Fig S2A and B). For flux analysis, tf-TAX1BP1 vari ants were transfected into TAX1BP1KO K562 cells and assessed for red:green ratio by flow cytometry (for underlying data, see Appendix Fig S2C and D). In all experiments, median red:green ratios were used to compare populations.HEK293T cells were transfected with either myc-TAX1BP1WT or myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies.HEK293T cells were transfected with myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies. Data information: See also Fig EV4G and H. Source data are available online for this figure.

Fig 4: TAX1BP1 is required for NBR1 flux in lipidation-deficient cells (related to Figs 3 and 4) 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. ATG7KO/TAX1BP1KO cells expressing tf-NBR1 were transfected with TagBFP-V5-TAX1BP1 and analyzed for red:green ratio and BFP expression. Plotted are the median red:green ratios of BFP-negative and BFP-positive cells relative to ATG7KO cells alone. Bar graphs represent mean ± SD from three independently generated ATG7KO/TAX1BP1KO cell lines. P values were determined using a ratio paired t-test. **P < 0.01; ns, not significant. n = 10,000 cells per sample. TAX1BP1KO and ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 were nucleofected with TagBFP, TagBFP-ATG7, or TagBFP-ATG7C572S and analyzed for red:green ratio and BFP expression at 8 h post-nucleofection. Median values for each sample are identified by a black line within each violin. The red dotted line corresponds to the red:green ratio of TAX1BP1KO cells expressing TagBFP. The solid red line corresponds to the red:green ratio of ATG7KO/TAX1BP1KO cells expressing TagBFP. n > 5,000 cells for each sample.Wild-type K562 cells expressing tf-LC3 or tf-NDP52 were transfected with TagBFP or TagBFP-RavZ. BFP-positive cells were analyzed for red:green ratio. The median red:green ratio of each sample was used to calculate flux relative to WT + TagBFP. Bar graphs represent mean ± SD from three independent experiments. n > 10,000 cells per sample. P values were determined using a ratio paired t-test. *P < 0.05; **P < 0.005; ns, not significant.Representative confocal micrographs (as maximum intensity projections) of indicated K562 genotypes expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. LC3, magenta; NBR1, green; Hoechst, blue. 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.Total RNA was extracted from the indicated cell lines. Equal amounts of subsequently generated cDNA were analyzed for NBR1 expression levels. All values were normalized to GAPDH expression levels using the ??C method. RPL0 was included as an unchanged control. Bar graphs represent mean ± SD. n = 3 technical replicates.HEK293T cells with indicated genotypes were treated with Bafilomycin A1 (BafA1) or DMSO for 18h. Extracts prepared from wild-type (WT) and deletion cell lines 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.Quantitation of protease protection data for tubulin from experiments in Fig 4E. Bar graphs show the mean ± SD of each sample from four independent experiments. Source data are available online for this figure.

Fig 5: Lysosomal degradation of NBR1 persists in an ATG7-independent manner Schematic depicting the tf-Reporter system. Both RFP and GFP fluorescence are observed throughout autophagosome formation. Upon autolysosome formation, GFP fluorescence is quenched due to the low pH of the lysosomal lumen. Corresponding changes in red:green ratio are reflective of reporter delivery to autolysosomes.Dot plot indicating fraction of tf-Reporters delivered to the lysosome by ATG7-independent mechanisms. The median red:green ratio of each population was used to calculate fractional delivery according to the following formula: (ratiosgATG7-ratiosgATG9A)/(ratioNegativeCtl1-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Mean values for each reporter are indicated by a black bar. n = 3 for each reporter. P values were determined using a one-way ANOVA (P < 0.0001) with Dunnett's multiple comparison test to compare each sample to the tf-LC3 control. *P < 0.05; **P < 0.005; ***P < 0.001; ns, not significant.Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. 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 in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n = 10,000 cells. See Fig EV1A for validation of genotype.Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion isolates expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. Red fluorescent protein (RFP), magenta; Green fluorescent protein (GFP), green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. All images scaled equally. See Fig EV1C for related images.Correlative light and electron microscopy (CLEM) of K562 ATG7KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrows indicate representative structures of interest. White boxes demarcate zoomed areas in subsequent images. NBR1, green; Hoechst, blue. Scale bar (small images), 2.5 µm. Scale bar (large images), 250 nm. See Fig EV1E for images of additional structures.Presumptive model for ATG7-independent autophagy. Delivery of NBR1 to the lysosome is dependent on FIP200 and ATG9A (not shown). In the presence of LC3-lipidation, NBR1 incorporation into autophagosomes is driven by receptor interactions with LC3. In the absence of lipidated LC3, NBR1 is selectively delivered to the lysosome by a largely unknown mechanism. Data information: See also Fig EV1. Source data are available online for this figure.