EMBO/soda-nel huggingface dataset:
CALCOCO1 acts with VAMP-Associated Proteins to mediate ER-phagy. (2020)
Figure 3

Figure 3. CALCOCO1 binds both to LDS and UDS of ATG8 family proteins.

A, B GST pulldowns 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).

C GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 and 35S-Myc-p62 with recombinant GST-GABARAPL2 (WT and indicated mutants).

D GST pulldown assays of in vitro transcribed/translated 35S-Myc-TAX1BP1 (WT and indicated mutants) with recombinant GST-GABARAP (WT and indicated mutants).

E, F Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691. Cells were induced with tetracycline for 24 hours 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.

G HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B. Scale bars, 5 μm.
CALCOCO1 acts with VAMP-Associated Proteins to mediate ER-phagy. (2020)
Figure 4

Figure 4. CALCOCO1 promotes basal autophagic flux but not bulk autophagy.

A, B HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were starved for 4 hours 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 and100-200 cells per each experiment.

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.
CALCOCO1 acts with VAMP-Associated Proteins to mediate ER-phagy. (2020)
Figure 5

Figure 5. CALCOCO1 interacts with VAP-A/B via a FFAT-like motif.

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.

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

C, D GST pulldown 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.

E HeLa cells transiently co-transfected with EGFP-CALCOCO1 and Myc-VAPA or -VAPB were immunostained with anti-Myc antibody. Scale bars, 5 μm.
CALCOCO1 acts with VAMP-Associated Proteins to mediate ER-phagy. (2020)
Figure 6

Figure 6. VAP proteins promote autophagy and starvation-induced degradation of tubular ER.

A, B Immunoblot analysis of indicated cell lines, starved for 6 hours (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.

C Immunoblot analysis of HeLa cells transfected with the indicated siRNAs.

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.

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.
CALCOCO1 acts with VAMP-Associated Proteins to mediate ER-phagy. (2020)
Figure 7

Figure 7. CALCOCO1 is a soluble ER-phagy receptor.

A, B Immunoblot analysis of HeLa WT and HeLa CALCOCO1 KO cells. The cells were starved for 6 hours (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, 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.

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.

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.

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.
CALCOCO1 acts with VAMP-Associated Proteins to mediate ER-phagy. (2020)
Figure 8

Figure 8. Interaction with ATG8 proteins is required for CALCOCO1-mediated ER-phagy.

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.

B Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 mLIR+∆623-691 for 24 hours or not, and then treated as indicated.

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 anti-body. 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.

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.
PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1 (2010)
figf1

(a) Schematic view of human Parkin structure (UBL, ubiquitin-like domain; R0, RING0/unique Parkin domain; R1/2, RING finger domains; IBR, in-between-ring domain). Parkinson's disease-associated mutations and the Parkin fragments used are indicated. (b) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy. HeLa cells transfected with HA–Parkin were treated with CCCP for the indicated times. Mitochondria were stained by anti-TOM20 (pseudo coloured; blue) and a Δ&PSgr;m-dependent MitoTracker (red). Parkin was stained with anti-HA (green). Without treatment, mitochondria are intact and stained by both mitochondrial markers, whereas Parkin is equally distributed in the cytoplasm. After 2 h of CCCP treatment, mitochondria are depolarized as shown by the loss of MitoTracker staining. Parkin completely translocates to mitochondria clustering at perinuclear regions. After 24 h of CCCP treatment, massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker. Only Parkin-positive cells show mitochondrial clustering and clearance, in contrast to adjacent untransfected cells. Scale bars, 10 μm. (c) Pathogenic Parkin mutations abrogate mitochondrial translocation and/or clearance. HeLa cells transfected with Flag–Parkin wild-type or pathogenic mutants were treated with CCCP and stained with anti-Flag (green), anti-TOM20 (red) and Hoechst33342 (blue). Representative merged images are shown. Scale bars, 10 μm. Immunostaining of cells transfected with non-functional cysteine mutants and functional Parkin variants is shown in Supplementary Information, Fig. S1a and S1b, respectively. (d, e) Based on the mitochondrial effects, Parkin mutants can be classified into functional and non-functional classes. (d) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment, as defined by complete colocalization of Parkin with the mitochondrial marker (n = 3). (e) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment, as shown by complete loss of mitochondrial staining (n = 3). Data represent the mean ± s.d., *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 compared with wild-type. WT, wild-type.
PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1 (2010)
figf2

Stable Parkin wild-type or pathogenic mutants expressing SH-SY5Y cell lines were induced with doxycycline (DOX) for 72 h and treated with CCCP for the indicated times. Cells were stained with anti-Parkin (green), anti-TOM20 (red) and Hoechst33342 (blue). (a–b) CCCP treatment of stable SH-SY5Y cells for 0 h. Unstimulated control cells (– DOX) show relatively low levels of endogenous Parkin, compared to cells with induced expression of Parkin wild-type or K161N, R275W and G430D mutants (+ DOX). Immunostaining (a) and western blot analysis (b) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones. β-Actin served as a loading control. (c, d) After 2 h of CCCP treatment, massive colocalization of wild-type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls. Similarly, Parkin K161N and R275W mutants translocate to mitochondria, whereas G430D does not. (e, f) After 24 h of CCCP treatment, induced wild-type Parkin-expressing cells show full clearance of damaged mitochondria. In contrast, the pathogenic mutants analysed fail to execute mitophagy and, therefore, damaged mitochondria remain uncleared. Representative images of three independent experiments are shown. Scale bars, 10 μm (a, c and e). (d, f) Quantifications of the observed mitochondrial effects in Parkin-expressing SH-SY5Y cells (n = 3). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with induced wild-type Parkin. WT, wild-type.
PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1 (2010)
figf3

(a) Schematic representation of human PINK1 (MTS, mitochondrial targeting sequence; TM, transmembrane domain; Ser/Thr-Kinase, Serine/threonine kinase domain). Mutations of an artificial triple kinase-dead (3×KD) PINK1 variant are indicated. (b) Co-immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization. Endogenous Parkin and PINK1 were co-immunoprecipitated from neuronal SH-SY5Y cells after CCCP treatment with specific antibodies. (c) Western blot of PINK1 siRNA-transfected HeLa cells used in d–h demonstrates the knockdown efficiency and specificity of PINK1 silencing. (d–h) PINK1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. This can be rescued by re-transfection with wild-type PINK1, but not with a kinase-dead mutant or a PINK1 variant lacking the MTS. Control siRNA- or PINK1 siRNA-silenced HeLa cells were transfected with EGFP–Parkin wild-type and PINK1–V5 wild-type, 3×KD or ΔMTS mutants. Use of appropriate empty vector controls is depicted. Cells were treated with CCCP and stained with anti-TOM20 (pseudo coloured; blue) and anti-V5 (red) to visualize mitochondrial morphology and re-transfected PINK1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative images of four independent experiments are shown. Scale bars, 10 μm. (e) Wild-type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed (n = 4). (f) Uncleared mitochondria were analysed after 24 h of CCCP treatment (n = 4). (g) Quantification of results from cells in e. (h) Quantification of results from cells in f. Data represent the mean ± s.d., **P ≤ 0.005, ***P ≤ 0.0005 compared with control siRNA-transfected cells. Immunostaining of control siRNA-transfected cells at all analysed time points is shown in Supplementary Information, Fig. S2. WT, wild-type.
PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1 (2010)
figf4

(a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag–Parkin wild-type or pathogenic mutants. Cells were treated with CCCP before immunostaining of Parkin with anti-Flag (green) and endogenous poly-ubiquitin with a specific antibody FK1 (red). (a) In contrast to pathogenic mutations, wild-type Parkin shows colocalization with poly-ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria. (b) After 24 h of CCCP treatment, K161N and R275W Parkin mutants colocalize with both poly-ubiquitin and mitochondria. In contrast, translocation-deficient Parkin mutants K211N, T240R and G430D lack the mitochondria-associated poly-ubiquitin signal. Representative images are shown. Scale bars, 10 μm. Complementary immunostaining of untreated cells and quantification of colocalization of Parkin, mitochondria and poly-ubiquitin upon CCCP treatment are shown in Supplementary Information, Fig. S3a, b and c, respectively. (c, d) Parkin-directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63. HeLa cells were transfected with EGFP–Parkin wild-type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti-HtrA2/Omi, as a mitochondrial marker (pseudo coloured; blue) and anti-His (red) to detect ectopic ubiquitin. Parkin was visualized by its fusion to EGFP (green). (c) Note that only wild-type, K27 and K63 6×His-tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP–Parkin after 6 h of CCCP treatment. All other ubiquitin mutants interfere with Parkin translocation to mitochondria. (d) Only co-expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6×His-tagged wild-type ubiquitin after 24 h of CCCP treatment. Representative images are shown. Scale bars, 10 μm. Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information, Fig. S4a and b, c, respectively. WT, wild-type; PolyUb, poly-ubiquitin.
PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1 (2010)
figf5

(a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag–Parkin wild-type or pathogenic mutants. Cells were treated with CCCP for the indicated times before immunostaining with anti-Flag (green) and anti-p62 (red). (a) In contrast to the pathogenic mutants, wild-type Parkin colocalizes with p62 at the site of clustered mitochondria, after 6 h of CCCP treatment. (b) After 24 h of CCCP treatment, wild-type Parkin-transfected cells show complete loss of mitochondria, whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria. In contrast, translocation-compromised mutants K211N, T240R and G430D, lacking the mitochondria-associated poly-ubiquitin signal (Fig. 4b), did not colocalize with p62. Representative images of four independent experiments are shown. Data from untreated control cells and quantification of colocalization of Parkin, mitochondria and p62 upon CCCP treatment are shown in Supplementary Information, Fig. 5a and b, c, respectively. (c, d) Control siRNA- or p62 siRNA-silenced HeLa cells were transfected with EGFP–Parkin wild-type. Cells were treated with CCCP for the indicated times and stained with anti-TOM20 (pseudo coloured; blue) to visualize mitochondrial morphology and anti-p62 (red) to monitor silencing efficiency. Parkin wild-type was visualized by EGFP epifluorescence (green). Parkin-positive cells with cleared mitochondria are surrounded by a line. (c–f) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment (c–e), but completely blocks final clearance after 24 h (c, d, f). (e, f) Relative parkin translocation (e) and mitochondrial clearance (f) after p62 knockdown and CCCP treatment (n = 4). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with control siRNA-transfected cells. Representative images of four independent experiments are shown. Scale bars, 10 μm. Immunostainings of control cells are shown in Supplementary Information, Fig. S5d, e. (g) Western blot of siRNA-transfected HeLa cells used in c–f demonstrates the efficiency of p62 silencing. β-Actin probing served as a loading control. WT, wild-type.
PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1 (2010)
figf6

(a) Time-course experiment of HeLa cells transfected with Flag–Parkin wild-type and treated with CCCP for the indicated periods. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. β-Actin probing served as a loading control. All of the proteins detected are endogenously expressed, except Flag–Parkin. (b) Pathogenic Parkin mutants fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. HeLa cells were transfected with empty vector (-), Flag–Parkin wild-type or pathogenic mutants along with 6×His-tagged ubiquitin wild-type for 24 h. Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer. Denaturing conditions of Ni-NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis-tagged ubiquitin. Western blots of protein lysates and purified Ni-NTA agarose elutions were probed with anti-VDAC1, anti-TOM20 and anti-Parkin antibodies. Modification of VDAC1 was observed in cells expressing Parkin wild-type and the functional mutant G328E, whereas non-functional mutants did not have this effect. (c) In response to CCCP treatment, Parkin catalyses predominantly K27-linked poly-ubiquitin chains on endogenous VDAC1 and is itself strongly poly-ubiquitylated. HeLa cells were transfected with an empty vector (-) or Flag–Parkin wild-type along with 6×His-tagged ubiquitin variants for 24 h. Treatment of cells, preparation of lysates and Ni-NTA purification were performed as described in c and probed for Parkin, VDAC1 and β-actin. WT, wild-type; Ub, ubiquitin.
PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1 (2010)
figf7

(a) Western blot analysis of parental SH-SY5Y cells treated with CCCP for the indicated times. Time-course experiments of CCCP-treated neuronal cells reveal increasing modifications of VDAC1 over time. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. All proteins represent endogenous levels. (b) Immunoprecipitations of Parkin and VDAC1 from parental SH-SY5Y cells confirm ubiquitylation of both proteins. CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time. Using specific antibodies, Parkin and VDAC1 were immunoprecipitated from lysates of parental SH-SY5Y cells at different time points, western blotted and probed with anti-ubiquitin, anti-Parkin and anti-VDAC1 antibodies. (c) Pathogenic Parkin mutations fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. Western blot analysis of a time-course experiment with inducible Parkin SH-SY5Y cells after CCCP treatment. Total lysates were prepared, and western blots were probed with specific anti-Parkin and anti-VDAC1 antibodies. β-Actin probing served as a loading control in a–c. WT, wild-type; Ub, ubiquitin.
PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1 (2010)
figf8

VDAC1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. Both effects can be rescued by re-transfection with wild-type VDAC1. (a) Immunofluorescence and (b) western blot of siRNA-transfected HeLa cells used in c–g demonstrate partial silencing of endogenous VDAC1. (a) Silenced cells were stained with anti-HtrA2/Omi, a mitochondrial marker (pseudo coloured; blue), and anti-VDAC1 (red) antibodies. (b) Lysates from silenced cells were subjected to western blot analysis and probed with an anti-VDAC1 antibody, while TOM20 and β-actin probing served as loading controls. (c–e) Control siRNA- or VDAC1 siRNA-silenced HeLa cells were transfected with the indicated combinations of EGFP–Parkin wild-type, VDAC1–Flag wild-type or appropriate empty vector controls. Cells were treated with CCCP for 2 h and 24 h and stained with anti-HtrA2/Omi (pseudo coloured; blue) and anti-Flag (red) to visualize mitochondrial morphology and re-transfected VDAC1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative pictures of three independent experiments are shown. Scale bars, 10 μm. (f, g) Quantification of d (f) and e (g). Parkin wild-type colocalization with impaired mitochondria after 2 h (f) and uncleared mitochondria after 24 h (g) of CCCP treatment (n = 3). Data represent the mean ± s.d., *P ≤ 0.05 compared with control siRNA-transfected or with VDAC1–Flag wild-type-transfected cells.