Figure 5.
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 (Delorenzi and Speed, 2002). (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: SourceData F5.

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 (Delorenzi and Speed, 2002). (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: SourceData F5.

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