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Characterization of Rab1 binding to the FHF complex <t>and</t> <t>CALCOCO1.</t> (A) Coomassie gel showing an in vitro–binding assay using beads coated with GST-Rab1A and purified FHF complex containing FHIP2A. Both GTP- and GDP-locked Rab1A proteins were used as indicated. (B) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-CALCOCO1. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. (C) GUV-binding assay using GTP- or GDP-locked Rab1A bound to GUVs before applying GFP-CALCOCO1 WT or Rab1-binding mutant. Each large (colored) datapoint in the graph depicts the average mean fluorescence intensity of GFP-CALCOCO1 WT or Rab1-binding mutant on a selection of GUV membrane and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (D) Sulfo-NHS-diazarine (SDA) intra-protein and dimer cross-links (purple and orange lines) mapped onto the AlphaFold 3 predicted structure of the <t>OPTN</t> homodimer. Intra-protein cross-links mapped onto the structure are pictured as a green dashed line; dimer cross-links are pictured as a cyan dashed line. (E) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-OPTN. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. Source data are available for this figure: .
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Characterization of Rab1 binding to the FHF complex <t>and</t> <t>CALCOCO1.</t> (A) Coomassie gel showing an in vitro–binding assay using beads coated with GST-Rab1A and purified FHF complex containing FHIP2A. Both GTP- and GDP-locked Rab1A proteins were used as indicated. (B) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-CALCOCO1. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. (C) GUV-binding assay using GTP- or GDP-locked Rab1A bound to GUVs before applying GFP-CALCOCO1 WT or Rab1-binding mutant. Each large (colored) datapoint in the graph depicts the average mean fluorescence intensity of GFP-CALCOCO1 WT or Rab1-binding mutant on a selection of GUV membrane and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (D) Sulfo-NHS-diazarine (SDA) intra-protein and dimer cross-links (purple and orange lines) mapped onto the AlphaFold 3 predicted structure of the <t>OPTN</t> homodimer. Intra-protein cross-links mapped onto the structure are pictured as a green dashed line; dimer cross-links are pictured as a cyan dashed line. (E) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-OPTN. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. Source data are available for this figure: .
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Characterization of Rab1 binding to the FHF complex <t>and</t> <t>CALCOCO1.</t> (A) Coomassie gel showing an in vitro–binding assay using beads coated with GST-Rab1A and purified FHF complex containing FHIP2A. Both GTP- and GDP-locked Rab1A proteins were used as indicated. (B) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-CALCOCO1. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. (C) GUV-binding assay using GTP- or GDP-locked Rab1A bound to GUVs before applying GFP-CALCOCO1 WT or Rab1-binding mutant. Each large (colored) datapoint in the graph depicts the average mean fluorescence intensity of GFP-CALCOCO1 WT or Rab1-binding mutant on a selection of GUV membrane and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (D) Sulfo-NHS-diazarine (SDA) intra-protein and dimer cross-links (purple and orange lines) mapped onto the AlphaFold 3 predicted structure of the <t>OPTN</t> homodimer. Intra-protein cross-links mapped onto the structure are pictured as a green dashed line; dimer cross-links are pictured as a cyan dashed line. (E) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-OPTN. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. Source data are available for this figure: .
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Characterization of Rab1 binding to the FHF complex <t>and</t> <t>CALCOCO1.</t> (A) Coomassie gel showing an in vitro–binding assay using beads coated with GST-Rab1A and purified FHF complex containing FHIP2A. Both GTP- and GDP-locked Rab1A proteins were used as indicated. (B) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-CALCOCO1. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. (C) GUV-binding assay using GTP- or GDP-locked Rab1A bound to GUVs before applying GFP-CALCOCO1 WT or Rab1-binding mutant. Each large (colored) datapoint in the graph depicts the average mean fluorescence intensity of GFP-CALCOCO1 WT or Rab1-binding mutant on a selection of GUV membrane and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (D) Sulfo-NHS-diazarine (SDA) intra-protein and dimer cross-links (purple and orange lines) mapped onto the AlphaFold 3 predicted structure of the <t>OPTN</t> homodimer. Intra-protein cross-links mapped onto the structure are pictured as a green dashed line; dimer cross-links are pictured as a cyan dashed line. (E) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-OPTN. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. Source data are available for this figure: .
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Image Search Results


Characterization of Rab1 binding to the FHF complex and CALCOCO1. (A) Coomassie gel showing an in vitro–binding assay using beads coated with GST-Rab1A and purified FHF complex containing FHIP2A. Both GTP- and GDP-locked Rab1A proteins were used as indicated. (B) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-CALCOCO1. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. (C) GUV-binding assay using GTP- or GDP-locked Rab1A bound to GUVs before applying GFP-CALCOCO1 WT or Rab1-binding mutant. Each large (colored) datapoint in the graph depicts the average mean fluorescence intensity of GFP-CALCOCO1 WT or Rab1-binding mutant on a selection of GUV membrane and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (D) Sulfo-NHS-diazarine (SDA) intra-protein and dimer cross-links (purple and orange lines) mapped onto the AlphaFold 3 predicted structure of the OPTN homodimer. Intra-protein cross-links mapped onto the structure are pictured as a green dashed line; dimer cross-links are pictured as a cyan dashed line. (E) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-OPTN. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: A Rab1 interactome illuminates a dual role in autophagy and membrane trafficking

doi: 10.1083/jcb.202507084

Figure Lengend Snippet: Characterization of Rab1 binding to the FHF complex and CALCOCO1. (A) Coomassie gel showing an in vitro–binding assay using beads coated with GST-Rab1A and purified FHF complex containing FHIP2A. Both GTP- and GDP-locked Rab1A proteins were used as indicated. (B) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-CALCOCO1. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. (C) GUV-binding assay using GTP- or GDP-locked Rab1A bound to GUVs before applying GFP-CALCOCO1 WT or Rab1-binding mutant. Each large (colored) datapoint in the graph depicts the average mean fluorescence intensity of GFP-CALCOCO1 WT or Rab1-binding mutant on a selection of GUV membrane and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (D) Sulfo-NHS-diazarine (SDA) intra-protein and dimer cross-links (purple and orange lines) mapped onto the AlphaFold 3 predicted structure of the OPTN homodimer. Intra-protein cross-links mapped onto the structure are pictured as a green dashed line; dimer cross-links are pictured as a cyan dashed line. (E) Representative Coomassie blue–stained gel showing expression of either WT or Rab1-binding mutant GFP-OPTN. Proteins were purified from the same amount of starting material, and equal protein amounts were loaded on the gel. Source data are available for this figure: .

Article Snippet: Antibodies used in this study were RABEP1 (610676; BD Transduction Laboratories, RRID: AB_398003), PPP1R37 (HPA041500; Atlas Antibodies, RRID: AB_10795122), CLEC16A (26257-1-AP; Proteintech, RRID: AB_2880449), Rab1A (13075; Cell Signaling Technologies, RRID: AB_2665537), HA (3F10; Roche, RRID: AB_2314622), TOM20 (ab56783; Abcam, RRID: AB_945896), Flag M2 (F1804; Sigma-Aldrich, RRID), CALCOCO1 (HPA038313; Atlas Antibodies, RRID: AB_10675794), GAPDH (60004-1; Proteintech, RRID: AB_2107436), OPTN (70928; Cell Signaling Technologies, RRID: AB_3073769), and α-tubulin (YL1/2, RRID: AB_305328).

Techniques: Binding Assay, In Vitro, Purification, Staining, Expressing, Mutagenesis, Fluorescence, Selection, Membrane

Rab1A-GTP binds directly to CALCOCO1 and OPTN. (A and B) Coomassie gels showing in vitro binding to GST-Rab1A–coated beads of either purified GFP-CALCOCO1 (A) or GFP-OPTN (B), with GFP as a negative control. Rab1A was in GTP- or GDP-locked forms as indicated. (C and D) GUV-binding assay using GTP- or GDP-locked Rab1A on the GUV with applied GFP-CALCOCO1 (C) or GFP-OPTN (D). Each large datapoint in the graph depicts the average mean fluorescence intensity of GFP-CALCOCO1 or GFP-OPTN on a selection of GUV membrane and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. **P < 0.01 (unpaired t test). Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: A Rab1 interactome illuminates a dual role in autophagy and membrane trafficking

doi: 10.1083/jcb.202507084

Figure Lengend Snippet: Rab1A-GTP binds directly to CALCOCO1 and OPTN. (A and B) Coomassie gels showing in vitro binding to GST-Rab1A–coated beads of either purified GFP-CALCOCO1 (A) or GFP-OPTN (B), with GFP as a negative control. Rab1A was in GTP- or GDP-locked forms as indicated. (C and D) GUV-binding assay using GTP- or GDP-locked Rab1A on the GUV with applied GFP-CALCOCO1 (C) or GFP-OPTN (D). Each large datapoint in the graph depicts the average mean fluorescence intensity of GFP-CALCOCO1 or GFP-OPTN on a selection of GUV membrane and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. **P < 0.01 (unpaired t test). Source data are available for this figure: .

Article Snippet: Antibodies used in this study were RABEP1 (610676; BD Transduction Laboratories, RRID: AB_398003), PPP1R37 (HPA041500; Atlas Antibodies, RRID: AB_10795122), CLEC16A (26257-1-AP; Proteintech, RRID: AB_2880449), Rab1A (13075; Cell Signaling Technologies, RRID: AB_2665537), HA (3F10; Roche, RRID: AB_2314622), TOM20 (ab56783; Abcam, RRID: AB_945896), Flag M2 (F1804; Sigma-Aldrich, RRID), CALCOCO1 (HPA038313; Atlas Antibodies, RRID: AB_10675794), GAPDH (60004-1; Proteintech, RRID: AB_2107436), OPTN (70928; Cell Signaling Technologies, RRID: AB_3073769), and α-tubulin (YL1/2, RRID: AB_305328).

Techniques: In Vitro, Binding Assay, Purification, Negative Control, Fluorescence, Selection, Membrane

Molecular characterization of the Rab1A-binding site on CALCOCO1 and OPTN. (A) Structure of the Rab1A:GTP:Mg 2+ :CALCOCO1 complex formed by two copies of CALCOCO1 and one copy of Rab1A as predicted by AlphaFold 3, with accompanying PAE plot. (B) Sulfo-NHS-diazirine (SDA) interprotein cross-links (green dashed lines) mapped onto the predicted structure of the Rab1A–CALCOCO1 complex. (C) The contact interface between Rab1A and the CALCOCO1 dimer, showing ChimeraX calculated contact residues of CALCOCO1 mediating the binding interface (red labels). (D) Structure of the Rab1A:GTP:Mg 2+ :OPTN complex formed by two copies of OPTN and one copy of Rab1A as predicted by AlphaFold 3, with accompanying PAE plot. (E) SDA interprotein cross-links (green dashed lines) mapped onto the predicted structure of the Rab1A–OPTN complex. (F) Magnified image of the contact interface between Rab1A and the OPTN dimer, showing ChimeraX calculated contact residues of OPTN mediating the binding interface (orange labels).

Journal: The Journal of Cell Biology

Article Title: A Rab1 interactome illuminates a dual role in autophagy and membrane trafficking

doi: 10.1083/jcb.202507084

Figure Lengend Snippet: Molecular characterization of the Rab1A-binding site on CALCOCO1 and OPTN. (A) Structure of the Rab1A:GTP:Mg 2+ :CALCOCO1 complex formed by two copies of CALCOCO1 and one copy of Rab1A as predicted by AlphaFold 3, with accompanying PAE plot. (B) Sulfo-NHS-diazirine (SDA) interprotein cross-links (green dashed lines) mapped onto the predicted structure of the Rab1A–CALCOCO1 complex. (C) The contact interface between Rab1A and the CALCOCO1 dimer, showing ChimeraX calculated contact residues of CALCOCO1 mediating the binding interface (red labels). (D) Structure of the Rab1A:GTP:Mg 2+ :OPTN complex formed by two copies of OPTN and one copy of Rab1A as predicted by AlphaFold 3, with accompanying PAE plot. (E) SDA interprotein cross-links (green dashed lines) mapped onto the predicted structure of the Rab1A–OPTN complex. (F) Magnified image of the contact interface between Rab1A and the OPTN dimer, showing ChimeraX calculated contact residues of OPTN mediating the binding interface (orange labels).

Article Snippet: Antibodies used in this study were RABEP1 (610676; BD Transduction Laboratories, RRID: AB_398003), PPP1R37 (HPA041500; Atlas Antibodies, RRID: AB_10795122), CLEC16A (26257-1-AP; Proteintech, RRID: AB_2880449), Rab1A (13075; Cell Signaling Technologies, RRID: AB_2665537), HA (3F10; Roche, RRID: AB_2314622), TOM20 (ab56783; Abcam, RRID: AB_945896), Flag M2 (F1804; Sigma-Aldrich, RRID), CALCOCO1 (HPA038313; Atlas Antibodies, RRID: AB_10675794), GAPDH (60004-1; Proteintech, RRID: AB_2107436), OPTN (70928; Cell Signaling Technologies, RRID: AB_3073769), and α-tubulin (YL1/2, RRID: AB_305328).

Techniques: Binding Assay

Mutation of the Rab1-binding site in OPTN does not affect its dimerization or binding to LC3 or ubiquitin. (A) Micrographs of beads coated with GST-4×ubiquitin (GST-4xUb) and incubated with either 1 μM GFP-OPTN (WT) or versions with mutations in the Rab1 or ubiquitin-binding sites. Each large datapoint in the bar graph depicts the average mean fluorescence intensity on a selection of beads and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (B) Micrographs of beads coated with GST-LC3B and incubated with either 1 μM GFP-OPTN (WT) or versions with mutations in the Rab1 or LC3 (LIR)-binding sites. Each large datapoint in the bar graph depicts the average mean fluorescence intensity on a selection of beads and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (C) Immunoblot of cell lysates of pentaKO HeLa cells stably expressing WT OPTN or the OPTN Rab1-binding mutant (representative of three repeats). Lysates were incubated with increasing concentrations of the cross-linker bis(sulfosuccinimidyl)suberate (BS3) at RT and quenched at 50 mM Tris-HCl prior to SDS-PAGE and blotting. With cross-linker, OPTN dimers are readily detected. GAPDH is a loading control and a negative control as it does not readily dimerize. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: A Rab1 interactome illuminates a dual role in autophagy and membrane trafficking

doi: 10.1083/jcb.202507084

Figure Lengend Snippet: Mutation of the Rab1-binding site in OPTN does not affect its dimerization or binding to LC3 or ubiquitin. (A) Micrographs of beads coated with GST-4×ubiquitin (GST-4xUb) and incubated with either 1 μM GFP-OPTN (WT) or versions with mutations in the Rab1 or ubiquitin-binding sites. Each large datapoint in the bar graph depicts the average mean fluorescence intensity on a selection of beads and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (B) Micrographs of beads coated with GST-LC3B and incubated with either 1 μM GFP-OPTN (WT) or versions with mutations in the Rab1 or LC3 (LIR)-binding sites. Each large datapoint in the bar graph depicts the average mean fluorescence intensity on a selection of beads and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (C) Immunoblot of cell lysates of pentaKO HeLa cells stably expressing WT OPTN or the OPTN Rab1-binding mutant (representative of three repeats). Lysates were incubated with increasing concentrations of the cross-linker bis(sulfosuccinimidyl)suberate (BS3) at RT and quenched at 50 mM Tris-HCl prior to SDS-PAGE and blotting. With cross-linker, OPTN dimers are readily detected. GAPDH is a loading control and a negative control as it does not readily dimerize. Source data are available for this figure: .

Article Snippet: Antibodies used in this study were RABEP1 (610676; BD Transduction Laboratories, RRID: AB_398003), PPP1R37 (HPA041500; Atlas Antibodies, RRID: AB_10795122), CLEC16A (26257-1-AP; Proteintech, RRID: AB_2880449), Rab1A (13075; Cell Signaling Technologies, RRID: AB_2665537), HA (3F10; Roche, RRID: AB_2314622), TOM20 (ab56783; Abcam, RRID: AB_945896), Flag M2 (F1804; Sigma-Aldrich, RRID), CALCOCO1 (HPA038313; Atlas Antibodies, RRID: AB_10675794), GAPDH (60004-1; Proteintech, RRID: AB_2107436), OPTN (70928; Cell Signaling Technologies, RRID: AB_3073769), and α-tubulin (YL1/2, RRID: AB_305328).

Techniques: Mutagenesis, Binding Assay, Ubiquitin Proteomics, Incubation, Fluorescence, Selection, Western Blot, Stable Transfection, Expressing, SDS Page, Control, Negative Control

The Rab1A – OPTN interaction is required for mitophagy. (A) GUV-binding assay using GTP- or GDP-locked Rab1A bound to GUVs before applying GFP-OPTN WT or Rab1-binding mutant. Each large datapoint in the graph depicts the average mean fluorescence intensity of GFP-OPTN WT or Rab1-binding mutant on a selection of GUV membrane and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (B) Representative immunoblot of Rab1 interactors following MitoID in HEK293A cells where Rab1A MitoID constructs (detected using anti HA) and 3xFlag-OPTN proteins (detected using anti Flag) were transiently expressed. Endogenous CALCOCO1 was used as a positive control. Each datapoint in the graph represents the normalized ratio between the OPTN and MitoID construct immunoblot intensities and depicts an independent experiment ( n = 3). The mean ± SD is indicated. ****P < 0.0001; (one-way ANOVA with Dunnett’s multiple comparisons test). (C) Schematic of the overall structure of OPTN, with different binding regions annotated (TBK1, TBK1-binding domain; Rab1, the Rab1-binding domain; LIR, LC3 interacting-region; UBAN, ubiquitin-binding domain in ABIN proteins and NEMO; ZF: zinc finger domain). The predicted coiled-coil regions of OPTN are also illustrated as predicted by MARCOIL . (D) Representative immunoblot and in-gel fluorescence analysis of cell lysates of pentaKO HeLa cells stably expressing pSu9-HaloTag-mGFP and Parkin and either mock transfected (/) or expressing OPTN WT, or the mutant constructs OPTN Rab1, OPTN Ub (unable to bind ubiquitin), or OPTN LIR (unable to bind ATG8/LC3 family proteins). To assay mitophagy, cells were pulse-labelled with 100 nM TMR HaloTag ligand and incubated in medium containing 1 μM oligomycin and 5 μM antimycin for 24 h to induce mitophagy (O + A). Each datapoint in the bar graph is an independent experiment representing the normalized ratio between the free HaloTag and the combined pSu9-HaloTag-mGFP + free HaloTag fluorescence intensities ( n = 3). The mean ± SD is indicated. ****P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: A Rab1 interactome illuminates a dual role in autophagy and membrane trafficking

doi: 10.1083/jcb.202507084

Figure Lengend Snippet: The Rab1A – OPTN interaction is required for mitophagy. (A) GUV-binding assay using GTP- or GDP-locked Rab1A bound to GUVs before applying GFP-OPTN WT or Rab1-binding mutant. Each large datapoint in the graph depicts the average mean fluorescence intensity of GFP-OPTN WT or Rab1-binding mutant on a selection of GUV membrane and represents an independent experiment ( n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (B) Representative immunoblot of Rab1 interactors following MitoID in HEK293A cells where Rab1A MitoID constructs (detected using anti HA) and 3xFlag-OPTN proteins (detected using anti Flag) were transiently expressed. Endogenous CALCOCO1 was used as a positive control. Each datapoint in the graph represents the normalized ratio between the OPTN and MitoID construct immunoblot intensities and depicts an independent experiment ( n = 3). The mean ± SD is indicated. ****P < 0.0001; (one-way ANOVA with Dunnett’s multiple comparisons test). (C) Schematic of the overall structure of OPTN, with different binding regions annotated (TBK1, TBK1-binding domain; Rab1, the Rab1-binding domain; LIR, LC3 interacting-region; UBAN, ubiquitin-binding domain in ABIN proteins and NEMO; ZF: zinc finger domain). The predicted coiled-coil regions of OPTN are also illustrated as predicted by MARCOIL . (D) Representative immunoblot and in-gel fluorescence analysis of cell lysates of pentaKO HeLa cells stably expressing pSu9-HaloTag-mGFP and Parkin and either mock transfected (/) or expressing OPTN WT, or the mutant constructs OPTN Rab1, OPTN Ub (unable to bind ubiquitin), or OPTN LIR (unable to bind ATG8/LC3 family proteins). To assay mitophagy, cells were pulse-labelled with 100 nM TMR HaloTag ligand and incubated in medium containing 1 μM oligomycin and 5 μM antimycin for 24 h to induce mitophagy (O + A). Each datapoint in the bar graph is an independent experiment representing the normalized ratio between the free HaloTag and the combined pSu9-HaloTag-mGFP + free HaloTag fluorescence intensities ( n = 3). The mean ± SD is indicated. ****P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). Source data are available for this figure: .

Article Snippet: Antibodies used in this study were RABEP1 (610676; BD Transduction Laboratories, RRID: AB_398003), PPP1R37 (HPA041500; Atlas Antibodies, RRID: AB_10795122), CLEC16A (26257-1-AP; Proteintech, RRID: AB_2880449), Rab1A (13075; Cell Signaling Technologies, RRID: AB_2665537), HA (3F10; Roche, RRID: AB_2314622), TOM20 (ab56783; Abcam, RRID: AB_945896), Flag M2 (F1804; Sigma-Aldrich, RRID), CALCOCO1 (HPA038313; Atlas Antibodies, RRID: AB_10675794), GAPDH (60004-1; Proteintech, RRID: AB_2107436), OPTN (70928; Cell Signaling Technologies, RRID: AB_3073769), and α-tubulin (YL1/2, RRID: AB_305328).

Techniques: Binding Assay, Mutagenesis, Fluorescence, Selection, Membrane, Western Blot, Construct, Positive Control, Ubiquitin Proteomics, Stable Transfection, Expressing, Transfection, Incubation