Fig 1.
Vacuolar morphology of wild-type and mutant cells.
(A) Model of Rab5 to Rab7 conversion during endosome maturation in budding yeast. Yeast Rab5 Vps21p is activated by Vps9p, a GEF for Rab5, on early endosome (E.E.). Activated Vps21p recruits Ccz1p/Mon1p, a GEF for Rab7, by biding GFP-bound Vps21p, and enhances dissociation of Vps21p form the early endosome. Ccz1p/Mon1p also binds to the HOPS complex and activates yeast Rab7 Ypt7p directly, thereby promoting endosome maturation and fusion of mutivesicular body/late endosome (MVB/L.E.) to the vacuole. Modified from Nordmann et al., 2010. (B, E, G, I) Vacuolar morphology in wild-type and indicated mutant cells. Vacuolar morphology was assessed by FM4-64 staining and different interference contrast (DIC) in the following strains: yeast Rab5-disrupted mutant (rab5Δ) (B), yeast Rab5 (vps21Δ), Rab7 (ypt7Δ) and Rab7-GEF mutants (mon1Δ)(E), CORVET and HOPS mutants (G), and SNARE mutants (I). (C) Locaization of GFP-Ypt7p, and -Vps41p in wild-type and rab5Δ cells. (D) Locaization of GFP-Mon1p in wild-type and rab5Δ cells. Cells were labeled with FM4-64. (F, H, J) Quantification of number (black bar) and size (gray bar) of vacuole in wild-type and mutant cells. Numbers were analyzed by counting ring-like compartments stained by FM4-64. Vacuolar size was measured inside diameter of the largest compartment stained by FM4-64 in cell. Data show the mean ± SD, with > 50 cells counted for each strain. Different letters indicate significant difference at p < 0.05 (S2 Table) (One-way ANOVA with Tukey’s post-hoc test). Scale bars, 2.5 μm.
Fig 2.
Localization of yeast Rab7 in wild-type and mutant cells.
(A) Localization of putative GTP- or GDP-locked mutant of Ypy7p in living cells. Cells were grown to early to mid-logarithmic phase in YPD medium at 25°C and observed by fluorescence microscopy and DIC (upper panels). Colocalization of GFP-Ypt7(T22N) with mCherry-fused Sec63p (lower paneles). Each image pair was acquired at successive 2 sec (s) intervals. (B) Localization of GFP-Ypt7p in wild-type or vps21Δ cells. Cells were labeled with 200 μM FM4-64. Cells expressing Ypt7p from a single copy plasmid were grown to mid-logarithmic phase in selective synthetic rich medium at 25°C. (C) Localization of GFP-Ypt7p in wild-type and mutant cells. All cells express Ypt7p from a single copy plasmid. Cells were labeled with FM4-64 and observed as described above. (D) The graph shows quantification of the fluorescence intensity of GFP-Ypt7p in the cytosol. Error bars represent the SEM from at least three experiments (n > 50 cells for each strain per experiment). Different letters indicate significant difference at p < 0.05 (S2 Table) (One-way ANOVA with Tukey’s post-hoc test). (E) Localization of GFP-fused FYVE domain (EEA1) in wild-type and mutant cells. (F) Localization of GFP-Vps21p in wild-type and mutant cells. Cells were labeled with FM4-64. (G) Higher magnification views of the boxed areas in (F). Scale bars, 2.5 μm.
Fig 3.
Convergence of the CPY pathway with the AP-3 pathway in vps21Δ ypt7Δ cells.
(A) Localization of Pho8-GFP and Pep4-mCherry in vps21Δ and ypt7Δ cells. Cells expressing Pho8-GFP and Pep4-mCherry were grown to early to mid-logarithmic phase in YPD medium at 25°C and observed by fluorescence microscopy. Merged images of GFP and mCherry channels are shown in the lower panel. (B) Localization of Pho8-GFP in wild-type and vps21Δ ypt7Δ cells. Cells were labeled with FM4-64 and observed as described above. (C) Convergence of the endocytic pathway with the CPY pathway in wild-type and vps21Δ ypt7Δ cells. The images were acquired at 4h after labeling with 200 μM FM4-64. (D) Localization of Hse1-tdTomato and GFP-fused FYVE domain (EEA1) in wild-type and vps21Δ ypt7Δ cells. (E, F) Ultrastructure of vacuole(s) observed in wild-type and vps21Δ ypt7Δ cells. Cells were grown at 25°C, fixed using propane jet freezing method and processed for electron microscopic analysis. (G, H) Higher magnification views of the boxed areas in wild-type (E) and vps21Δ ypt7Δ (F) cells. Scale bars: 2.5 μm (A-D), 1 μm (E, F), 0.5 μm (G, H).
Fig 4.
Defect of the AP-3 pathway in vps21Δ ypt7Δ mutant.
(A-D) Analysis of the AP-3 pathway in wild-type and mutant cells. Cells expressing GFP–Nyv1–Snc1-TMD (GNS) fusion protein were grown to early to mid-logarithmic phase at 25°C and observed by fluorescence microscopy and DIC. (E, F) Localization of Apl5-GFP in wild-type and mutant cells. (G) Quantification of the number of Apl5-GFP positive vesicles in indicated cells displayed in (E) and (F). Data show the mean ± SEM of three experiments, with 50 cells per experiment. Different letters indicate significant difference at p < 0.05 (S2 Table) (One-way ANOVA with Tukey’s post-hoc test). Scale bars, 2.5 μm.
Fig 5.
Model of vacuole delivery pathways in wild-type, vps21Δ and ypt7Δ cells.
Convergence of the endocytic and CPY pathways occur at an early stage of endocytosis, independently of Rab5. vps21Δ ypt7Δ double mutant has defect in the trafficking of the AP-3 and CPY pathways although the AP3 pathway is mostly intact in each vps21Δ or ypt7Δ single mutant. See details in the text.