Figure 2.
Figure 2. Ppz phosphatases are required for Mup1 endocytic trafficking in response to methionine. (A) WT (expressing Vph1-mCherry) or Δppz1Δppz2 mutant (lacking mCherry expression) yeast cells (SEY6210 strain background) expressing Mup1-GFP were cultured to mid-log phase, mixed, stimulated by addition of methionine for 30 min, and imaged by fluorescence deconvolution microscopy. (B) Mup1-GFP signal intensity was measured at the PM and the vacuole lumen, and a Vac:PM ratio was calculated for many individual yeast cells (n ≥ 40). Statistical significance was calculated using a two-way ANOVA test (P values indicated in red). (C) A methionine stimulation time course was performed on WT and ppz mutant yeast cells expressing Mup1-FLAG. At the indicated time points after stimulation, cell lysates were analyzed by SDS-PAGE and immunoblot. (D) Flow cytometry analysis of yeast cells expressing Mup1-pHluorin grown in the absence of methionine and then stimulated by addition of methionine. Fluorescence of pHluorin is pH sensitive and lost during endocytic trafficking when the cargo encounters an acidic environment. Plot represents average of multiple biological replicate experiments (n ≥ 3), and error bars indicate standard deviation. Statistical significance was calculated using a Student’s t test (P < 0.005). (E) WT (expressing Vph1-mCherry) or Δppz1Δppz2 mutant (lacking mCherry expression) yeast cells (SEY6210 strain background) expressing Fur4-GFP were cultured to mid-log phase, mixed, stimulated by addition of methionine for 30 min, and imaged by fluorescence deconvolution microscopy. (F) Fur4-GFP signal intensity was measured at the PM and the vacuole lumen, and a Vac:PM ratio was calculated for many individual yeast cells (n ≥ 30). Statistical significance was calculated using a two-way ANOVA test, but no statistically significant difference between WT and Δppz1Δppz2 mutant cells was observed. (G and H) Flow cytometry analysis of yeast cells expressing Mup1-pHluorin grown in the absence of methionine and then stimulated by addition of methionine. Fluorescence of pHluorin is pH sensitive and lost during endocytic trafficking when the cargo encounters an acidic environment. Plot represents average of multiple biological replicate experiments (n ≥ 3), and error bars indicate standard deviation. Statistical significance was calculated using a Student’s t test (P < 0.005). Vac, vacuole lumen.

Ppz phosphatases are required for Mup1 endocytic trafficking in response to methionine. (A) WT (expressing Vph1-mCherry) or Δppz1Δppz2 mutant (lacking mCherry expression) yeast cells (SEY6210 strain background) expressing Mup1-GFP were cultured to mid-log phase, mixed, stimulated by addition of methionine for 30 min, and imaged by fluorescence deconvolution microscopy. (B) Mup1-GFP signal intensity was measured at the PM and the vacuole lumen, and a Vac:PM ratio was calculated for many individual yeast cells (n ≥ 40). Statistical significance was calculated using a two-way ANOVA test (P values indicated in red). (C) A methionine stimulation time course was performed on WT and ppz mutant yeast cells expressing Mup1-FLAG. At the indicated time points after stimulation, cell lysates were analyzed by SDS-PAGE and immunoblot. (D) Flow cytometry analysis of yeast cells expressing Mup1-pHluorin grown in the absence of methionine and then stimulated by addition of methionine. Fluorescence of pHluorin is pH sensitive and lost during endocytic trafficking when the cargo encounters an acidic environment. Plot represents average of multiple biological replicate experiments (n ≥ 3), and error bars indicate standard deviation. Statistical significance was calculated using a Student’s t test (P < 0.005). (E) WT (expressing Vph1-mCherry) or Δppz1Δppz2 mutant (lacking mCherry expression) yeast cells (SEY6210 strain background) expressing Fur4-GFP were cultured to mid-log phase, mixed, stimulated by addition of methionine for 30 min, and imaged by fluorescence deconvolution microscopy. (F) Fur4-GFP signal intensity was measured at the PM and the vacuole lumen, and a Vac:PM ratio was calculated for many individual yeast cells (n ≥ 30). Statistical significance was calculated using a two-way ANOVA test, but no statistically significant difference between WT and Δppz1Δppz2 mutant cells was observed. (G and H) Flow cytometry analysis of yeast cells expressing Mup1-pHluorin grown in the absence of methionine and then stimulated by addition of methionine. Fluorescence of pHluorin is pH sensitive and lost during endocytic trafficking when the cargo encounters an acidic environment. Plot represents average of multiple biological replicate experiments (n ≥ 3), and error bars indicate standard deviation. Statistical significance was calculated using a Student’s t test (P < 0.005). Vac, vacuole lumen.

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