![Interaction with Rab7 is required for WDR91 to regulate SNX-retromer function. (A) Rescue of endosomal trapping of SNX27 by Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) in KO-91 cells. Ctrl and KO-91 cells were co-transfected with vectors expressing the indicated Flag-WDR91 proteins and mCh-CD63. 24 h later, the cells were fixed and stained with SNX27 and Flag antibodies. In the merged images, the boxed regions are magnified at the bottom left. Bars, 5 µm. (B) Quantification of the co-localization of SNX27 with mCh-CD63 as shown in A. n = 40 cells (Ctrl); n = 42 cells (KO-91, empty); n = 32 cells (KO-91, + WDR91[WT]); n = 35 cells (KO-91, + WDR91[3A]); n = 36 cells (KO-91, + WDR91[Δ5]). (C) Rescue of endosomal trapping of VPS35 by Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) in KO-91 cells. Ctrl and KO-91 cells were transfected with vectors expressing the indicated Flag-WDR91 proteins and mCh-CD63. 24 h later, the cells were fixed and stained with VPS35 and Flag antibodies. In the merged images, the boxed regions are magnified at the bottom left. Bars, 5 µm. (D) Quantification of the co-localization of VPS35 with mCh-CD63 as shown in C. n = 37 cells (Ctrl); n = 45 cells (KO-91, empty); n = 31 cells (KO-91, + WDR91[WT]); n = 36 cells (KO-91, + WDR91[3A]); n = 38 cells (KO-91, + WDR91[Δ5]). (E) Rescue of defective β2ARs recycling by Myc-WDR91, Myc-WDR91(3A), and Myc-WDR91(Δ5) in KO-91 cells. Ctrl and KO-91 cells were transfected with vectors expressing Flag-β2AR, Myc-WDR91 proteins and BFP (the marker for transfected cells). 24 h later, the cells were subjected to isoproterenol treatment and washout. 45 min after washout, the cells were fixed for immunostaining. Bars, 5 μm. (F) Quantification of the co-localization of Flag-β2ARs with EEA1 as shown in E. n = 44 cells (Ctrl); n = 42 cells (KO-91, empty); n = 47 cells (KO-91, + WDR91[WT]); n = 47 cells (KO-91, + WDR91[3A]); n = 46 cells (KO-91, + WDR91[Δ5]). (G) Rescue of defective CI-MPR trafficking by Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) in KO-91 cells. Ctrl and KO-91 cells were transfected with vectors expressing the indicated Flag-WDR91 proteins and BFP. 24 h later, the cells were fixed and stained with CI-MPR and EEA1 antibodies. Bars, 5 µm. (H) Quantification of the co-localization of CI-MPR with EEA1 as shown in G. n = 46 cells (Ctrl); n = 52 cells (KO-91, empty); n = 50 cells (KO-91, + WDR91[WT]); n = 48 cells (KO-91, + WDR91[3A]); n = 52 cells (KO-91, + WDR91[Δ5]). For all quantifications, error bars represent SEM. The y-axis shows the value of Pearson’s correlation coefficient. Data are from three independent experiments. Statistical analyses were performed with one-way ANOVA with Tukey’s post hoc test. ***, P < 0.001. NS, P > 0.05.](https://cdn.rupress.org/rup/content_public/journal/jcb/221/12/10.1083_jcb.202203013/2/jcb_202203013_fig4.png?Expires=1771195983&Signature=LoFtXqC-Vcl-jtdAiGrcOLxejHr~fWSaHz08SEjzLCdPEjy~u5VGI2ORQO~NWreEvzNPJ3dNVrZdTos64yR5Ac5no-kmWEOaDabo4tHIBIKkdYBNnlLHCQyNB8RBEhP7tx1QBn4MWVK14o0EAMQnp22S5aDtVpGqVhjISuS-wvALoSxEjGMXyvmYi0TjxBhLLALXHVih0SMgUncNojEIeijVQ9REWP1lUQN1HNlqHAt6ozuyD0iDT2GufLyrLZd8LetJ-f9a~Dhnt25PUAaT5t0FiGYRYTvUXOdjYswsadyIyq4N7RxhHggTaA-euNyWUMJdF2fkYc9~p8Bc9JRSqw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Interaction with Rab7 is required for WDR91 to regulate SNX-retromer function. (A) Rescue of endosomal trapping of SNX27 by Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) in KO-91 cells. Ctrl and KO-91 cells were co-transfected with vectors expressing the indicated Flag-WDR91 proteins and mCh-CD63. 24 h later, the cells were fixed and stained with SNX27 and Flag antibodies. In the merged images, the boxed regions are magnified at the bottom left. Bars, 5 µm. (B) Quantification of the co-localization of SNX27 with mCh-CD63 as shown in A. n = 40 cells (Ctrl); n = 42 cells (KO-91, empty); n = 32 cells (KO-91, + WDR91[WT]); n = 35 cells (KO-91, + WDR91[3A]); n = 36 cells (KO-91, + WDR91[Δ5]). (C) Rescue of endosomal trapping of VPS35 by Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) in KO-91 cells. Ctrl and KO-91 cells were transfected with vectors expressing the indicated Flag-WDR91 proteins and mCh-CD63. 24 h later, the cells were fixed and stained with VPS35 and Flag antibodies. In the merged images, the boxed regions are magnified at the bottom left. Bars, 5 µm. (D) Quantification of the co-localization of VPS35 with mCh-CD63 as shown in C. n = 37 cells (Ctrl); n = 45 cells (KO-91, empty); n = 31 cells (KO-91, + WDR91[WT]); n = 36 cells (KO-91, + WDR91[3A]); n = 38 cells (KO-91, + WDR91[Δ5]). (E) Rescue of defective β2ARs recycling by Myc-WDR91, Myc-WDR91(3A), and Myc-WDR91(Δ5) in KO-91 cells. Ctrl and KO-91 cells were transfected with vectors expressing Flag-β2AR, Myc-WDR91 proteins and BFP (the marker for transfected cells). 24 h later, the cells were subjected to isoproterenol treatment and washout. 45 min after washout, the cells were fixed for immunostaining. Bars, 5 μm. (F) Quantification of the co-localization of Flag-β2ARs with EEA1 as shown in E. n = 44 cells (Ctrl); n = 42 cells (KO-91, empty); n = 47 cells (KO-91, + WDR91[WT]); n = 47 cells (KO-91, + WDR91[3A]); n = 46 cells (KO-91, + WDR91[Δ5]). (G) Rescue of defective CI-MPR trafficking by Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) in KO-91 cells. Ctrl and KO-91 cells were transfected with vectors expressing the indicated Flag-WDR91 proteins and BFP. 24 h later, the cells were fixed and stained with CI-MPR and EEA1 antibodies. Bars, 5 µm. (H) Quantification of the co-localization of CI-MPR with EEA1 as shown in G. n = 46 cells (Ctrl); n = 52 cells (KO-91, empty); n = 50 cells (KO-91, + WDR91[WT]); n = 48 cells (KO-91, + WDR91[3A]); n = 52 cells (KO-91, + WDR91[Δ5]). For all quantifications, error bars represent SEM. The y-axis shows the value of Pearson’s correlation coefficient. Data are from three independent experiments. Statistical analyses were performed with one-way ANOVA with Tukey’s post hoc test. ***, P < 0.001. NS, P > 0.05.
