Fig 1: Interaction with ATG8 proteins is required for CALCOCO1‐mediated ER‐phagy AImmunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 in fed or starved conditions and treated as indicated with Baf A1 or PI3KC3 inhibitor SAR405.BImmunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 mLIR + Δ623–691 for 24 h or not, and then treated as indicated.C, DRepresentative confocal images of HeLa CALCOCO1 KO cells transiently co‐transfected with mCherry‐CALCOCO1, Myc‐VAPA, and EGFP‐LAMP1, and then treated as indicated before immunostaining with anti‐Myc antibody. Arrows indicate co‐localization. Scale bars in (C), 5 μm for large merged images and 1 μm for zoomed images. In (D), data are presented as mean ± SD. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test and significance displayed as ***P ˂ 0.001; ns is not significant, n = 3.EModel of ER‐phagy mediated by CALCOCO1 dimers illustrating the dual LIR‐LDS and UIR‐UDS interaction with lipidated GABARAP subfamily proteins on the phagophore, and the FFAT‐like motif‐mediated interaction with MSP domains of VAP proteins on tubular ER. Source data are available online for this figure.
Fig 2: CALCOCO1 homomerizes via coiled-coil domains, but does not heterodimerize with TAX1BP1 or NDP52 Co-immunoprecipitation (co-IP) of Myc-CALCOCO1 with EGFP-CALCOCO1, following transient co-transfection of EGFP-CALCOCO1 and indicated Myc-CALCOCO1 constructs in HEK293 cells.Co-IP of Myc-CALCOCO1 with EGFP-CALCOCO1. 35S-GFP-CALCOCO1 (upper panels) or 35S-GFP (lower panels) were in vitro co-transcribed/translated with indicated 35S-Myc-CALCOCO1 constructs. GFP-CALCOCO1 or GFP, respectively, were immunoprecipitated with GFP-TRAP and the immunoprecipitates then resolved by SDS–PAGE. The resolved immunoprecipitates were detected by autoradiography.GFP-CALCOCO1 or GFP were in vitro co-transcribed/translated with Myc-CALCOCO1, Myc-NDP52, or Myc-TAX1BP1 and then immunoprecipitated with GFP-TRAP. The analysis of the immunoprecipitates was done as in B. GFP construct is indicated with a circle and Myc-constructs with a star.GST pull-down analyses of binding of in vitro transcribed/translated 35S-Myc-CALCOCO1 with recombinant GST-tagged Galectin-3 and-8. In vitro transcribed/translated Myc-NDP52 and Myc-TAX1BP1 were included as positive controls.
Fig 3: The C-terminal parts of CALCOCO1 and TAX1BP1 contribute to their interaction with ATG8 family proteins A, BGST pull-down binding of the indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with recombinant GST-tagged ATG8 family proteins.CGST pull-down analyses of binding of in vitro transcribed/translated WT or LIR-mutated (LVV/AAA) 35S-Myc-NDP52 with recombinant GST-tagged ATG8 family proteins.D, EGST pull-down analyses of binding of indicated in vitro transcribed/translated 35S-Myc-TAX1BP1 constructs with indicated recombinant GST-tagged ATG8 family proteins.
Fig 4: CALCOCO1 is a soluble ER‐phagy receptor A, BImmunoblot analysis of HeLa WT and HeLa CALCOCO1 KO cells. The cells were starved for 6 h (HBSS) as indicated and treated with Baf A1 as indicated. Numbers below the blots in (A) represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In (A), the panels are collected from more than one Western blot experiment but for clarity, only a single GAPDH loading control is shown. In (B), the bars represent the mean ± SD of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test and significance displayed as **P ˂ 0.005, *P ˂ 0.01; ns is not significant.C, DImmunoblot analysis of HeLa CALCOCO1 KO cell lines reconstituted with EGFP‐CALCOCO1. Expression of EGFP‐CALCOCO1 was induced or not with tetracycline, and the cells were treated with MG132 or Baf A1 as indicated. Numbers below the blots in (C) represent relative intensity of the bands in the shown blots normalized against actin loading control. In (C), the panels are collected from more than one Western blot experiment but only a single actin loading control is shown. In (D), the bars represent the mean ± SD of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed as in B and significance displayed as **P ˂ 0.005, *P ˂ 0.01; ns is not significant.EHeLa CALCOCO1 KO cell lines reconstituted with EGFP‐CALCOCO1 were treated with tetracycline or not to induce expression of EGFP‐CALCOCO1. Abundance of the ER was quantified from widefield fluorescence images of endogenous RTN3 staining (see Materials and Methods). Data are presented as mean ± SD of three independent experiments. Statistical comparison was analyzed as in (B) and significance displayed as ***P ˂ 0.001, **P ˂ 0.005, *P ˂ 0.01; ns is not significant.F, GImmunoblot analysis of cells analyzed in (C). The bars in (G) represent the mean ± SD of band intensities relative to the loading control from three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test. Significance is displayed as **P ˂ 0.005, *P ˂ 0.01; ns is not significant.H, IImmunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 in fed or starved conditions and transfected with the indicated VAPA/B siRNAs. Source data are available online for this figure.
Fig 5: CALCOCO1 binds directly to ATG8 family proteins with preference for the GABARAP subfamily GST pull-down binding assay of in vitro transcribed/translated 35S-Myc-CALCOCO1 with recombinant GST-tagged ATG8 family proteins. GST and GST fusion proteins were visualized by Coomassie Brilliant Blue staining (bottom panel), and the co-precipitated Myc-CALCOCO1 was detected by autoradiography (upper panel). The numbers below the AR represent % binding in the shown AR.GST pull-down assay of transiently transfected Myc-CALCOCO1 from HEK293 cell extracts with recombinant GST-tagged ATG8 family proteins. GST and GST fusions were visualized by Ponceau S staining (bottom panel), and co-precipitated Myc-CALCOCO1 detected by immunoblotting with anti-Myc antibody (upper panel).CALCOCO1 deletion constructs used to map the ATG8 interactions. The red X indicate a point mutation or deletion of the LIR motif. Constructs with no or very weak interaction are indicated in orange.D-H GST pull-down assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in (A).GST pull-down assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST-tagged CALCOCO1 constructs. GST and GST fusions were visualized as in A, and co-precipitated GABARAP with anti-GABARAP antibody (upper panel).
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