Figure 8.
Figure 8. Localization of the ESCRT subunits Snf7 and Vps4 to APs is regulated by the Rab5 GTPase Vps21. (A–C) During starvation, Snf7 colocalizes with Atg8-marked APs and Pho8-marked class E compartment in a Vps21-dependent manner. (A) WT and vps21Δ mutant cells expressing GFP-Atg8, Snf7-mCherry, and 3×tagBFP-Pho8 were grown in YPD (top), shifted to SD-N (30 min, bottom), and visualized by live-cell fluorescence microscopy (right, number of dots counted for the quantification in B and C). Arrows point to Atg8-Snf7 colocalization; arrowheads indicate Snf7-Pho8 colocalization. Scale bar, 2 µm. (B) During starvation, Snf7 localization to APs is lower in vps21Δ mutant cells when compared with WT cells. Bar graph showing percentage of colocalization of Atg8 with Snf7 (black bars) and Snf7 with Atg8 (gray bars). (C) During starvation, the majority of Snf7 colocalize with Pho8. This colocalization is also lower in vps21Δ mutant cells when compared with WT cells. Bar graph showing percentage of colocalization of Pho8 with Snf7 (dark blue bars) and Snf7 with Pho8 (light blue bars). More than 100 GFP-Atg8, Snf7-mCherry, or 3×tagBFP-Pho8 dots per strain were examined in B and C. (D) During starvation, Snf7 localizes to APs in vps21Δ mutant cells and for about one third of the time it does so in WT cells. Quantification of time-lapse video microscopy (6-s intervals during 25 min) showing time points (%) in which the two proteins colocalized (Videos 3 and 4). 30 cells per strain were quantified. (E and F) During starvation, Vps4 colocalizes with the Atg8-marked APs in a Vps21-dependent manner. (E) WT and vps21Δ mutant cells expressing endogenously tagged Vps4-NG and mCherry-Atg8 were grown in YPD (top) or SD-N (bottom) and visualized as described for A (right, number of dots counted for the quantification in F). Arrows point to Atg8-Vps4 colocalization. Scale bar, 2 µm. (F) During starvation, Vps4 localization to APs is lower in vps21Δ mutant cells when compared with WT cells. Bar graph showing percentage of colocalization of Atg8 with Vps4 (black bars) and Vps4 with Atg8 (gray bars). Columns in B–D and F represent mean; error bars represent SD. Results represent three independent experiments. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Localization of the ESCRT subunits Snf7 and Vps4 to APs is regulated by the Rab5 GTPase Vps21. (A–C) During starvation, Snf7 colocalizes with Atg8-marked APs and Pho8-marked class E compartment in a Vps21-dependent manner. (A) WT and vps21Δ mutant cells expressing GFP-Atg8, Snf7-mCherry, and 3×tagBFP-Pho8 were grown in YPD (top), shifted to SD-N (30 min, bottom), and visualized by live-cell fluorescence microscopy (right, number of dots counted for the quantification in B and C). Arrows point to Atg8-Snf7 colocalization; arrowheads indicate Snf7-Pho8 colocalization. Scale bar, 2 µm. (B) During starvation, Snf7 localization to APs is lower in vps21Δ mutant cells when compared with WT cells. Bar graph showing percentage of colocalization of Atg8 with Snf7 (black bars) and Snf7 with Atg8 (gray bars). (C) During starvation, the majority of Snf7 colocalize with Pho8. This colocalization is also lower in vps21Δ mutant cells when compared with WT cells. Bar graph showing percentage of colocalization of Pho8 with Snf7 (dark blue bars) and Snf7 with Pho8 (light blue bars). More than 100 GFP-Atg8, Snf7-mCherry, or 3×tagBFP-Pho8 dots per strain were examined in B and C. (D) During starvation, Snf7 localizes to APs in vps21Δ mutant cells and for about one third of the time it does so in WT cells. Quantification of time-lapse video microscopy (6-s intervals during 25 min) showing time points (%) in which the two proteins colocalized (Videos 3 and 4). 30 cells per strain were quantified. (E and F) During starvation, Vps4 colocalizes with the Atg8-marked APs in a Vps21-dependent manner. (E) WT and vps21Δ mutant cells expressing endogenously tagged Vps4-NG and mCherry-Atg8 were grown in YPD (top) or SD-N (bottom) and visualized as described for A (right, number of dots counted for the quantification in F). Arrows point to Atg8-Vps4 colocalization. Scale bar, 2 µm. (F) During starvation, Vps4 localization to APs is lower in vps21Δ mutant cells when compared with WT cells. Bar graph showing percentage of colocalization of Atg8 with Vps4 (black bars) and Vps4 with Atg8 (gray bars). Columns in B–D and F represent mean; error bars represent SD. Results represent three independent experiments. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

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