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: SourceData FS2.