Figure 3.
The RUN domain-containing region of RUFY1 is essential and sufficient for binding to Arl8b. (A) Schematic showing the Clustal Omega alignment of a short stretch of protein sequence within the RUN domain of PLEKHM2, RUFY1 (longer isoform), and PLEKHM1. The red arrows show conserved arginine residues (present in the context of the RxRAWL motif) in the RUN domains of all three proteins. In this study, R206 and R208 residues of RUFY1 were mutated to alanine. The color-coding of amino acids is based on their physiochemical properties, as listed on the Clustal Omega homepage. (B) HEK293T cell lysates expressing RUFY1 (WT)-FLAG or indicated RUFY1 mutants were incubated with recombinant GST and GST-Arl8b proteins immobilized on glutathione-coated-agarose beads. The precipitates were immunoblotted (IB) with anti-FLAG antibodies, and Ponceau S staining was done to visualize the purified proteins. (C) Densitometric analysis of band intensity of GST pulldown normalized to input RUFY1-FLAG signal (WT or mutants). The values plotted are the mean ± SD from four independent experiments (****P < 0.0001; ***P < 0.001; unpaired two-tailed t test). (D) Recombinant GST and GST-RUFY1 (RUN; 1–302 a.a.) proteins were immobilized on glutathione-coated-agarose beads and incubated with His-Arl8b (WT), His-Arl8b (Q75L), and His-Arl8b (T34N). The precipitates were IB with anti-His antibodies, and Ponceau S staining was done to visualize the purified proteins. Notably, His-Arl8b (Q75L) showed more non-specific binding (as observed in the GST lane) than His-Arl8b (WT). (E) Densitometric analysis of band intensity of GST pulldown normalized to input signal (His-Arl8b WT or mutants). The values plotted are the mean ± SD from three independent experiments (****P < 0.0001; unpaired two-tailed t test). (F–H) Representative confocal micrographs of HeLa cells co-transfected with Arl8b-HA and RUFY1 (WT)-FLAG (F), RUFY1 (ΔRUN)-FLAG (G) or RUFY1 (RR→A)-FLAG (H). The arrowhead marks the colocalized pixels. Bars: (main) 10 µm; (insets) 2 µm. (I and J) Pearson’s and Mander’s colocalization coefficient quantification of Arl8b-HA with indicated RUFY1 mutants. The values plotted are the mean ± SD from three independent experiments. Experiments are color-coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the top of each data set (****P < 0.0001; unpaired two-tailed t test). Source data are available for this figure: SourceData F3.

The RUN domain-containing region of RUFY1 is essential and sufficient for binding to Arl8b. (A) Schematic showing the Clustal Omega alignment of a short stretch of protein sequence within the RUN domain of PLEKHM2, RUFY1 (longer isoform), and PLEKHM1. The red arrows show conserved arginine residues (present in the context of the RxRAWL motif) in the RUN domains of all three proteins. In this study, R206 and R208 residues of RUFY1 were mutated to alanine. The color-coding of amino acids is based on their physiochemical properties, as listed on the Clustal Omega homepage. (B) HEK293T cell lysates expressing RUFY1 (WT)-FLAG or indicated RUFY1 mutants were incubated with recombinant GST and GST-Arl8b proteins immobilized on glutathione-coated-agarose beads. The precipitates were immunoblotted (IB) with anti-FLAG antibodies, and Ponceau S staining was done to visualize the purified proteins. (C) Densitometric analysis of band intensity of GST pulldown normalized to input RUFY1-FLAG signal (WT or mutants). The values plotted are the mean ± SD from four independent experiments (****P < 0.0001; ***P < 0.001; unpaired two-tailed t test). (D) Recombinant GST and GST-RUFY1 (RUN; 1–302 a.a.) proteins were immobilized on glutathione-coated-agarose beads and incubated with His-Arl8b (WT), His-Arl8b (Q75L), and His-Arl8b (T34N). The precipitates were IB with anti-His antibodies, and Ponceau S staining was done to visualize the purified proteins. Notably, His-Arl8b (Q75L) showed more non-specific binding (as observed in the GST lane) than His-Arl8b (WT). (E) Densitometric analysis of band intensity of GST pulldown normalized to input signal (His-Arl8b WT or mutants). The values plotted are the mean ± SD from three independent experiments (****P < 0.0001; unpaired two-tailed t test). (F–H) Representative confocal micrographs of HeLa cells co-transfected with Arl8b-HA and RUFY1 (WT)-FLAG (F), RUFY1 (ΔRUN)-FLAG (G) or RUFY1 (RR→A)-FLAG (H). The arrowhead marks the colocalized pixels. Bars: (main) 10 µm; (insets) 2 µm. (I and J) Pearson’s and Mander’s colocalization coefficient quantification of Arl8b-HA with indicated RUFY1 mutants. The values plotted are the mean ± SD from three independent experiments. Experiments are color-coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the top of each data set (****P < 0.0001; unpaired two-tailed t test). Source data are available for this figure: SourceData F3.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal