Fig. 1: Identification of RUFY3 and RUFY4 as ARL8 effectors.
From: RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin
a Schematic representation of control and Mito-ARL8 constructs used in MitoID. Mito-ARL8 constructs comprise the mitochondrial targeting sequence (MTS) from TOM2038, followed by the BioID2 biotin ligase39, and ARL8A and ARL8B lacking the N-terminal α-helix and harboring the activating Q75L or inactivating T34N mutations. Data from 3 biological replicates were used for the assay. b Graph showing the relative abundance of hits identified by mass spectrometry for MTS-BioID2-ARL8A-Q75L/MTS-BioID2 control vs. MTS-BioID2-ARL8A-T34N/MTS-BioID2 control using MitoID. c Same as (b) for MTS-BioID2-ARL8B-Q75L/MTS-BioID2 control vs. MTS-BioID2-ARL8B-T34N/MTS-BioID2 control. Hits of interest in panels (b) and (c) are highlighted. d Domain organization of RUFY proteins in N- to C-terminal direction. RUN: RPIP8, UNC-14 and NESCA domain, CC1: coiled-coil 1 domain, CC2: coiled-coil 2 domain, FYVE: Fab1, YOTB, Vac1 and EEA1 domain. Amino-acid numbers are indicated. RUFY3.1 and RUFY3.2 are two spliceforms of RUFY3. e Immunofluorescence microscopy of HeLa cells co-expressing GFP or RUFY-GFP fusion proteins (green) with MTS-BioID2-ALR8B-Q75L or MTS-BioID2-ALR8B-T34N. Fixed cells were stained with antibody to BioID2 (magenta) and imaged by confocal microscopy. Single channels are shown in grayscale. Scale bars: 10 μm. f Quantification of the percentage of cells in which RUFY proteins were re-localized to mitochondria in experiments such as that in panel (e). Values are the mean ± SD from three independent experiments (minimum of 300 cells per condition). Statistical significance was calculated using one-way ANOVA with multiple comparisons between groups using Tukey’s test. **** p < 0.0001. See also Supplementary Fig. 1 and Supplementary Data 1.
