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. 2020 Aug 3;39(15):e103649.
doi: 10.15252/embj.2019103649. Epub 2020 Jun 11.

CALCOCO1 acts with VAMP-associated proteins to mediate ER-phagy

Thaddaeus Mutugi Nthiga  1 , Birendra Kumar Shrestha  1 , Eva Sjøttem  1 , Jack-Ansgar Bruun  1 , Kenneth Bowitz Larsen  1 , Zambarlal Bhujabal  1 , Trond Lamark  1 , Terje Johansen  1
Affiliations

CALCOCO1 acts with VAMP-associated proteins to mediate ER-phagy

Thaddaeus Mutugi Nthiga et al. EMBO J. .

Abstract

The endoplasmic reticulum (ER) plays important roles in protein synthesis and folding, and calcium storage. The volume of the ER and expression of its resident proteins are increased in response to nutrient stress. ER-phagy, a selective form of autophagy, is involved in the degradation of the excess components of the ER to restore homeostasis. Six ER-resident proteins have been identified as ER-phagy receptors so far. In this study, we have identified CALCOCO1 as a novel ER-phagy receptor for the degradation of the tubular ER in response to proteotoxic and nutrient stress. CALCOCO1 is a homomeric protein that binds directly to ATG8 proteins via LIR- and UDS-interacting region (UIR) motifs acting co-dependently. CALCOCO1-mediated ER-phagy requires interaction with VAMP-associated proteins VAPA and VAPB on the ER membranes via a conserved FFAT-like motif. Depletion of CALCOCO1 causes expansion of the ER and inefficient basal autophagy flux. Unlike the other ER-phagy receptors, CALCOCO1 is peripherally associated with the ER. Therefore, we define CALCOCO1 as a soluble ER-phagy receptor.

Keywords: FFAT; VAPA; Autophagy; CALCOCO1; ER-phagy.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. CALCOCO1 is degraded by macro‐autophagy
  1. A

    Domain architecture of CALCOCO paralogs showing the SKICH domain, a conserved LIR motif (LVV), coiled‐coli regions (CC), and zinc finger domains (ZF).

  2. B–E

    Immunoblot analysis of indicated cell lines, starved for 6 h (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In (C, E), endogenous CALCOCO1 is analyzed and the bars represent the mean ± SD of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test and significance displayed as ***P ˂ 0.001, **P ˂ 0.005, *P ˂ 0.01; ns is not significant.

  3. F

    A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 and immunostained for endogenous GM130. Scale bars are 5 and 2 μm (zoomed inset).

  4. G

    Same cells as in (E) were left untreated or treated with Baf A1 for 6 h and then immunostained for endogenous p62 and LC3B. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.

  5. H

    CALCOCO1, p62, and LC3B puncta in the indicated conditions, counted using an automated system. The error bars represent mean ± SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***P < 0.001.

  6. I

    Percentage of co‐localization of CALCOCO1 puncta with p62 and/or LC3B in cells treated as indicated. The error bars represent mean ± SEM of three independent experiments per condition and over 250 cells per experiment. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***P < 0.001.

  7. J

    HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 were treated with Baf A1 for 6 h and immunostained for endogenous LAMP1. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.

Figure EV1
Figure EV1. CALCOCO1 homomerizes via coiled‐coil domains, but does not heterodimerize with TAX1BP1 or NDP52

  1. 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.

  2. 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.

  3. 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.

  4. 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.

Figure EV2
Figure EV2. CALCOCO1 is degraded by autophagy and co‐localizes in HBSS‐treated cells with p62, LC3B, and GABARAP
  1. A, B

    Immunoblot analysis of indicated cell lines, starved for 6 h (HBSS) as indicated, and treated with MG132 or Baf A1 as indicated.

  2. C

    Extension of Fig 1G. HeLa CALCOCO1 KO cells stably transfected with EGFP‐CALCOCO1 were induced with tetracycline for 24 h and then starved (HBSS) with or without Baf A1 treatment as indicated, before immunostaining for endogenous p62 and LC3B. Scale bars, 5 μm for confocal microscopy images, 2 μm for airyscans.

  3. D

    Extension of Fig 1J. Percentage of LAMP1 rings associated with a CALCOCO1 structure. The error bars represent mean ± SEM of three independent experiments per condition and 200 cells per experiment.

  4. E

    HeLa cells stably transfected with EGFP‐CALCOCO1 were treated with tetracycline for 24 h to induce expression of EGFP‐CALCOCO1. Cells were then starved or not and immunostained with anti‐p62, anti‐LC3, and anti‐GABARAP antibodies as indicated. Co‐localization in dots is indicated by circles and supported using line plots shown below the micrographs. Scale bars, 10 μm.

Figure 2
Figure 2. CALCOCO1 binds directly to ATG8 family proteins with preference for the GABARAP subfamily

  1. 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.

  2. 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).

  3. 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.

  4. 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).

  5. 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).

Figure EV3
Figure EV3. The C‐terminal parts of CALCOCO1 and TAX1BP1 contribute to their interaction with ATG8 family proteins
  1. A, B

    GST pull‐down binding of the indicated in vitro transcribed/translated 35S‐Myc‐CALCOCO1 constructs with recombinant GST‐tagged ATG8 family proteins.

  2. C

    GST 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.

  3. D, E

    GST pull‐down analyses of binding of indicated in vitro transcribed/translated 35S‐Myc‐TAX1BP1 constructs with indicated recombinant GST‐tagged ATG8 family proteins.

Figure 3
Figure 3. CALCOCO1 binds both to LDS and UDS of ATG8 family proteins
  1. A, B

    GST pull‐down testing binding of indicated in vitro transcribed/translated 35S‐Myc‐CALCOCO1 constructs with indicated recombinant GST‐tagged ATG8 family proteins (left). Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs. The presence of two well separated binding surfaces on ATG8 proteins binding to LIR (LDS) and UIR (UDS) is indicated (right).

  2. C

    GST pull‐down assays of in vitro transcribed/translated 35S‐Myc‐CALCOCO1 and 35S‐Myc‐p62 with recombinant GST‐GABARAPL2 (WT and indicated mutants).

  3. D

    GST pull‐down assays of in vitro transcribed/translated 35S‐Myc‐TAX1BP1 (WT and indicated mutants) with recombinant GST‐GABARAP (WT and indicated mutants).

  4. E, F

    Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP‐CALCOCO1 or EGFP‐CALCOCO1 mLIR + Δ623–691. Cells were induced with tetracycline for 24 h and then starved or treated with MG132 or Baf A1 as indicated. The blot panels are from more than one Western blot experiment but for clarity, only a single actin/GAPDH loading control is shown. In (F), the bars represent the mean ± SD of band intensities relative to the actin or GAPDH loading controls of three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test and significance displayed as ***P ˂ 0.001, *P ˂ 0.01; ns is not significant.

  5. G

    HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 or EGFP‐CALCOCO1 mLIR + Δ623–691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B. Scale bars, 5 μm.

Source data are available online for this figure.
Figure 4
Figure 4. CALCOCO1 promotes basal autophagic flux but not bulk autophagy
  1. A, B

    HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 were starved for 4 h and then immunostained with anti‐ATG13 and anti‐WIPI2 antibodies. Scale bars in (A) are 5 μm for the confocal microscopy images and 2 μm for the airyscans. In (B), the error bars represent mean ± SD of puncta per cell from three independent experiments and 100–200 cells per each experiment.

  2. C–L

    Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH or actin). The asterisk in (K) indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP‐CALCOCO1 in cells extracts from the rescued cells. In (D, F, H, I, and L), the bars represent the mean ± SD of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ, n = 5 in (I), n = 3 in others. Statistical comparison was analyzed by one‐way ANOVA and significance displayed as ***P ˂ 0.001, **P ˂ 0.005, *P ˂ 0.01; ns is not significant.

Figure EV4
Figure EV4. CALCOCO1 KO inhibits basal autophagy
  1. A, B

    WT or CALCOCO1 KO HeLa cells were transiently transfected with mCherry‐EGFP‐LC3B and 24 h after transfection, the cells were treated or not with HBSS as indicated. In (A), representative confocal images are shown, and in (B), the fraction of red‐only puncta is counted and shown as a percentage. The error bars represent mean ± SD of red‐only puncta percentages of three independent experiments. 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. The arrows in FM show either red‐only or red + green puncta while in the HBSS‐treated cells, the arrows show red‐only puncta.

  2. C

    GST pull‐down analyses of binding of different in vitro transcribed/translated 35S‐Myc‐tagged ER proteins to recombinant GST‐tagged CALCOCO1. GST is included as a negative control.

  3. D

    CALCOCO1 KO HeLa cells were transiently transfected with the indicated EGFP‐CALCOCO1 constructs and immunostained for endogenous VAPA. Scale bars, 10 μm.

Source data are available online for this figure.
Figure 5
Figure 5. CALCOCO1 interacts with VAPA/B via a FFAT‐like motif
  1. A

    Stably expressed EGFP or EGFP‐CALCOCO1 in HEK293 cells were immunoprecipitated from the cell lysates followed by mass spectrometry identification of interacting proteins. Only some of the identified proteins are shown.

  2. B

    Co‐IP of Myc‐VAPA or Myc‐VAPB with EGFP‐CALCOCO1 from transiently transfected HEK293 cells. The asterisks (*) indicate Myc‐VAP previously detected on the same membrane.

  3. C, D

    GST pull‐down assays of in vitro transcribed/translated 35S‐Myc‐CALCOCO1 constructs with indicated recombinant GST‐VAPA or GST‐VAPB constructs. The scheme is a representation of CALCOCO1 domains and motifs.

  4. E

    HeLa cells transiently co‐transfected with EGFP‐CALCOCO1 and Myc‐VAPA or—VAPB were immunostained with anti‐Myc antibody. Scale bars, 5 μm.

Figure 6
Figure 6. VAP proteins promote autophagy and starvation‐induced degradation of tubular ER
  1. A, B

    Immunoblot analysis of indicated cell lines, starved for 6 h (HBSS) as indicated, and treated with MG132 or 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 (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 and significance displayed as **P ˂ 0.005, *P ˂ 0.01; ns is not significant.

  2. C

    Immunoblot analysis of HeLa cells transfected with the indicated siRNAs.

  3. D, E

    Immunoblot analysis of HeLa cells transfected with the indicated siRNAs and treated with Baf A1 as indicated. In (D), the panels are from more than one Western blot experiment but only a single GAPDH loading control is shown. In (E), 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 ˂ 0.001, **P ˂ 0.005; ns is not significant.

  4. F

    HeLa KO CALCOCO1 cells were transiently co‐transfected with EGFP‐CALCOCO1 and Myc‐VAPA or Myc‐VAPB, and then immunostained with anti‐Myc and anti‐LC3B antibodies. Arrows indicate dots of co‐localization of all three proteins. Scale bars, 5 μm for large merged images and 1 μm for zoomed images.

Source data are available online for this figure.
Figure 7
Figure 7. CALCOCO1 is a soluble ER‐phagy receptor
  1. A, B

    Immunoblot 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.

  2. C, D

    Immunoblot 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.

  3. E

    HeLa 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.

  4. F, G

    Immunoblot 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.

  5. H, I

    Immunoblot 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.
Figure EV5
Figure EV5. CALCOCO1 KO inhibits autophagic degradation of VAPA under starvation
  1. A, B

    WT or CALCOCO1 KO HeLa cells were transiently transfected with mCherry‐EGFP‐VAPA or mCherry‐EGFP‐FAM134B as indicated and 24 h after transfection, the cells were treated or not with HBSS as indicated. In (A), representative confocal images are shown, and in (B), the fraction of red‐only puncta is counted and shown as a percentage. The error bars represent mean ± SD of red‐only puncta percentages of three independent experiments. 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. The arrows in FM show either red‐only (WT cells) or red + green puncta (CALCOCO1 KO cells) while in the HBSS‐treated cells, the arrows show red‐only puncta.

  2. C

    Immunoblot analysis of HeLa CALCOCO1 KO cell lines reconstituted with EGFP‐CALCOCO1 Δ145–513. Expression of EGFP‐CALCOCO1 Δ145–513 was induced or not with tetracycline, and the cells were treated with MG132, HBSS, or Baf A1 as indicated.

Source data are available online for this figure.
Figure 8
Figure 8. Interaction with ATG8 proteins is required for CALCOCO1‐mediated ER‐phagy
  1. A

    Immunoblot 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.

  2. B

    Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 mLIR + Δ623–691 for 24 h or not, and then treated as indicated.

  3. C, D

    Representative 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.

  4. E

    Model 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.
See this image and copyright information in PMC

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