Skip to main content
. 2020 May 11;9:e56090. doi: 10.7554/eLife.56090

Figure 1. Rab5 effect on Mon1-Ccz1 function.

(A) Vacuole morphology in Rab5 mutants. Cells with the indicated mutations were grown in the presence of 10 µM FM4-64 and analyzed by fluorescence microscopy. Size bar, 5 µm. (B) Quantification of vacuole morphology. Percentage of cells with less than four vacuoles is shown. Error bars represent standard deviation. (C) Model of cooperation of Rab5-GTP and PI-3-P for Mon1-Ccz1 recruitment to late endosomes. PI-3-P is indicated as red lipid. (D) Interaction of yeast Rab5-like proteins with Mon1-Ccz1. Purified GST-tagged Rab5 proteins (Vps21, Ypt10, Ypt52, and Ypt53) were loaded with GTP (T) or GDP (D) and incubated with purified Mon1-Ccz1 complex. Eluates were analyzed on SDS-PAGE by Western blotting with an antibody against the TAP-tag on Ccz1 (top) and Coomassie staining (bottom). For details see methods. (E) Localization of Ypt10 in yeast. Cells expressing endogenously GFP-tagged Ypt10 and mKate-tagged Ccz1 were analyzed by fluorescence microscopy. Size bar, 5 µm. (F) Analysis of Ypt7 localization and vacuole morphology in Rab5 deletion strains. mNeon-tagged Ypt7 was expressed under the control of the Ypt7 promoter in cells the indicated Rab5 proteins. Cells were stained with FM4-64, and analyzed by fluorescence microscopy. Size bar, 5 µm.

Figure 1—source data 1. Quantification of vacuole number in FM4-64 stained wild-type and mutant strains in Figure 1A.

Figure 1.

Figure 1—figure supplement 1. Vacuole morphology in Rab5 mutants.

Figure 1—figure supplement 1.

Cells with the indicated (A) single (B) double (C) triple and (D) quadruple mutations were grown in the presence of 10 µM FM4-64 and analyzed by fluorescence microscopy. Size bar, 5 µm.
Figure 1—figure supplement 2. Interaction of yeast Rab-GTPases with Mon1-Ccz1.

Figure 1—figure supplement 2.

(A) Purified GST-tagged Rab-GTPases were loaded with GTP (T) or GDP (D) and incubated with purified Mon1-Ccz1 complex. Eluates were analyzed on SDS-PAGE by Western blotting with an antibody against the TAP-tag on Ccz1 (top) and Coomassie staining (bottom). For details see methods.