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 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.