Fig 2: Arl4A interacts with VPS36.a The yeast reporter strain L40 was transformed with a construct encoding Gal4 transcriptional activation domain-fused VPS36 (330–386) and the indicated wild-type and mutant constructs of Arl4A, Arl4C, and Arl4D fused to the LexA DNA-binding domain. Colonies were patched on selective plates and assayed for β-galactosidase activity. Lamin was included as a negative control. b Equal amounts of His-tagged Arl4A WT, Q79L, T51N, and Arl1 (negative control) were pulled down by either GST or GST-VPS36 (201–386). Inputs and pulled-downed Arl4 proteins were probed using an anti-His antibody. Equal inputs of GST fusion proteins used in the assay were visualized by Coomassie Blue staining as shown at the bottom of each figure. c VPS36 interacted with Arl4AWT, Arl4AQ79L, and Arl4AT51N in vivo. Lysates of HeLa cells transfected with the indicated plasmids were immunoprecipitated with HA beads, and the bound proteins were analyzed by Western blotting with anti-HA, anti-Arl4A, or anti-α-tubulin antibodies. The amounts of coimmunoprecipitated Arl4A were determined by densitometric quantification. The results represent the mean ± SD of six independent experiments and the p-values were assessed by one-way ANOVA with Tukey’s test. d HeLa cells were cotransfected with VPS36 WT-Myc and Arl4A WT, Q79L, T51N, or G2A (myristoylation-defective) constructs. The cells were stained for Arl4A (green), VPS36-Myc (red), and DAPI (blue). Scale bar, 10 µm. Quantification of VPS36 and Arl4A colocalization was obtained from three biological replicates and is shown as the colocalization coefficient by ZEN software. The results represent the mean ± SD of three independent experiments (total WT = 103, Q79L = 79, T51N = 60, and G2A = 105 cells; the p-values were assessed by one-way ANOVA with Tukey’s test). Source data are provided as a Source Data file.

Fig 3: Arl4A binding modulates the association of VPS36 with MVBs.a HeLa cells were transfected for 24 h with the indicated plasmid. Confocal sections of HeLa cells stained with anti-Myc (VPS36-WT-Myc or VPS36-A6-Myc) (green), anti-CD63 (red), and DAPI (blue). Scale bar, 10 µm. Quantification of VPS36-Myc and CD63 colocalization is shown as the Pearson coefficient. The results represent the mean ± SD of three independent experiments and the p-value was assessed by a two-sided t-test (VPS36-WT = 84 cells, VPS36-A6 = 104 cells). b HeLa cells were co-transfected with VPS36-WT-Myc or VPS36-A6-Myc and GFP-Rab5A Q79L for 24 h. The cells were fixed and then stained with anti-Myc (red) and anti-GFP (green) antibodies. The images were acquired using the Leica TCS SP8 STED microscope. Scale bar, 5 µm. Imaris software was used to quantify the 3D-colocalization coefficient values of VPS36 and Rab5 Q79L. The results represent the mean ± SD of three independent experiments and the p-value was assessed by a two-sided t-test (VPS36-WT = 16 cells, VPS36-A6 = 22 cells). c HeLa cells stably expressing control shRNA or VPS4A and VPS4B shRNA using shRNA–expressing lentivirus. After puromycin selection, the cells were transfected with the indicated plasmids. Confocal sections of HeLa cells stained with anti-Myc (VPS36-WT-Myc or VPS36-A6-Myc) (green), anti-CD63 (red), and DAPI (blue). Scale bar, 10 µm. Quantification of colocalization of VPS36-Myc and CD63 presented as colocalization coefficient using the software ZEN. Results represent the mean ± SD of three independent experiments, and the p values were determined by one-way ANOVA with Tukey’s test (shControl/VPS36- WT = 40 cells, shVPS36/VPS36- WT = 41 cells, shControl/VPS36-A6 = 52 cells, shVPS36/VPS36-A6 = 42 cells). Source data are provided as a Source Data file.