Figure 5.
Figure 5. WDR91 interacts with and inhibits the activity of the Rab7-associated PI3K complex. (A) IP of endogenous Beclin1, Rab7, and WDR91 from HeLa cells using their cognate antibodies. Coprecipitated proteins were detected with antibodies against the indicated proteins. (B) Loss of WDR91 enhances the activity of the Rab7-associated PI3K complex. Endogenous Rab7 was immunoprecipitated from cell lysates of Ctrl and KO-91 HeLa cells. Precipitated proteins were detected with antibodies to the indicated proteins (bottom). Equal amounts of precipitated Vps34 from each genotype were examined for PI3K activity by measuring the relative units of luminescence (RLU) of ADP converted from ATP. (C) Rescuing effects of Flag-tagged WDR91(WT), WDR91(3A), and WDR91(Δ5) on the activity of the Rab7-associated PI3K complex in KO-91 cells. Control and KO-91 HeLa cells were cotransfected with vectors expressing GFP-Rab7(Q67L) and the indicated WDR91 proteins. 36 h later, cell lysates were subjected to IP using GFP-trap beads. Precipitates were detected with antibodies against the indicated proteins (bottom) and analyzed for PI3K activity as in B. (B and C) Data representing mean ± SEM are from three independent experiments and are normalized to PI3K activity in Ctrl cells (top). (D and E) Immunostaining (D) and quantification (E) of PtdIns3P levels in control and KO-91 HeLa cells transfected with the indicated vectors expressing Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) for 24 h. Bars, 5 µm. (F) Rescuing effects of Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) on defective endosomal EGFR trafficking in KO-91 cells. Control or KO-91 HeLa cells were transfected with vectors expressing the indicated Flag-WDR91 proteins for 24 h and were stimulated with 2 µg/ml Alexa Fluor 647–conjugated EGF. 60 min after stimulation, cells were fixed and stained with EEA1 and Flag antibodies. Solid lines mark the cells expressing the indicated proteins. Dashed lines mark cells that do not express WT or mutant WDR91. Zoom images represent magnified views of boxed areas. Bars, 5 µm. (G and H) Quantification of the intensity of EEA1 staining (G) and the colocalization of EEA1 with EGFR (H) as shown in F. Data represent mean ± SEM in G and Pearson’s correlation coefficient in H. ≥50 cells were scored. ***, P < 0.001.

WDR91 interacts with and inhibits the activity of the Rab7-associated PI3K complex. (A) IP of endogenous Beclin1, Rab7, and WDR91 from HeLa cells using their cognate antibodies. Coprecipitated proteins were detected with antibodies against the indicated proteins. (B) Loss of WDR91 enhances the activity of the Rab7-associated PI3K complex. Endogenous Rab7 was immunoprecipitated from cell lysates of Ctrl and KO-91 HeLa cells. Precipitated proteins were detected with antibodies to the indicated proteins (bottom). Equal amounts of precipitated Vps34 from each genotype were examined for PI3K activity by measuring the relative units of luminescence (RLU) of ADP converted from ATP. (C) Rescuing effects of Flag-tagged WDR91(WT), WDR91(3A), and WDR91(Δ5) on the activity of the Rab7-associated PI3K complex in KO-91 cells. Control and KO-91 HeLa cells were cotransfected with vectors expressing GFP-Rab7(Q67L) and the indicated WDR91 proteins. 36 h later, cell lysates were subjected to IP using GFP-trap beads. Precipitates were detected with antibodies against the indicated proteins (bottom) and analyzed for PI3K activity as in B. (B and C) Data representing mean ± SEM are from three independent experiments and are normalized to PI3K activity in Ctrl cells (top). (D and E) Immunostaining (D) and quantification (E) of PtdIns3P levels in control and KO-91 HeLa cells transfected with the indicated vectors expressing Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) for 24 h. Bars, 5 µm. (F) Rescuing effects of Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) on defective endosomal EGFR trafficking in KO-91 cells. Control or KO-91 HeLa cells were transfected with vectors expressing the indicated Flag-WDR91 proteins for 24 h and were stimulated with 2 µg/ml Alexa Fluor 647–conjugated EGF. 60 min after stimulation, cells were fixed and stained with EEA1 and Flag antibodies. Solid lines mark the cells expressing the indicated proteins. Dashed lines mark cells that do not express WT or mutant WDR91. Zoom images represent magnified views of boxed areas. Bars, 5 µm. (G and H) Quantification of the intensity of EEA1 staining (G) and the colocalization of EEA1 with EGFR (H) as shown in F. Data represent mean ± SEM in G and Pearson’s correlation coefficient in H. ≥50 cells were scored. ***, P < 0.001.

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