Fig. 2: Biochemical evidence for binding of RUFY3 and RUFY4 to ARL8, and dissection of RUFY3 domains required for ARL8 binding.

a GST-ARL8B-Q75L and GST-ARL8B-T34N were used to pull down the indicated RUFY-FLAG proteins expressed by transfection in HEK293T cells. FLAG-tagged proteins were identified by immunoblotting (IB) and GST proteins by Ponceau S staining. b Extracts of HEK293T cells transfected with plasmids encoding the indicated FLAG- or FOS (FLAG-one-strep)-tagged proteins were immunoprecipitated (IP) with anti-FLAG, and immunoblotted (IB) for endogenous ARL8A and ARL8B and the FLAG tag. Red asterisks indicate the positions of the different FLAG- or FOS-tagged proteins (expected molecular masses: RUFY1-FLAG, 80.8 kDa; RUFY2-FLAG, 71 kDa; RUFY3.1-FLAG, 71.1 kDa; RUFY3.2-FLAG, 54 kDa; RUFY4-FLAG, 65 kDa; FLAG-HOOK1, 86 kDa; SKIP-FOS, 116 kDa). Data in (a) and (b) are representative of 2 experiments with similar results. c Schematic representation of RUFY3 deletion constructs. Domain organization is as depicted in Fig. 1b. Amino-acid numbers are indicated. Δ stands for deletion. Constructs were tagged with GFP or the FLAG epitope. d Immunofluorescence microscopy of HeLa cells expressing GFP or the RUFY3-GFP deletion constructs shown in panel c (green) together with MTS-BioID2-ALR8B-Q75L. Cells were fixed and stained as described in Fig. 1e. Scale bars: 10 μm. e Quantification of the percentage of cells in which RUFY-GFP proteins were re-localized to mitochondria from experiments such as that shown in panel (d). Values are the mean ± SD from a minimum of three independent experiments, each scoring a minimum of 300 cells per condition. Statistical significance compared to cells expressing GFP was calculated using one-way ANOVA with multiple comparisons with Dunnett’s test. ****p < 0.0001. f, g GST-ARL8B-Q75L and GST-ARL8B-T34N were used to pull down the indicated RUFY3-FLAG deletion constructs expressed by transfection in HEK293T cells. FLAG-tagged proteins (f, g) were detected by immunoblotting (IB) for the FLAG epitope. Input GST-ARL8B-Q75L and GST-ARL8B-T34N were detected by Ponceau-S staining. h, i GST-ARL8B-Q75L and GST-ARL8B-T34N were used to pull down the indicated RUFY3-GFP deletion constructs expressed by transfection in HEK293T cells. GFP-tagged proteins (h, i) were detected by immunoblotting (IB) for GFP. Input GST-ARL8B-Q75L and GST-ARL8B-T34N (i) were detected by Ponceau-S staining. The experiments in (f–i) are representative of 2 experiments with similar results. Mr represents molecular mass (kDa).