Figure 2.

Localization of Fab1 mutants. (A) Wild-type 6210 cells or vps34Δ cells expressing GFP-tagged Fab1 and its mutant forms at endogenous protein levels. Vacuoles were labeled with FM4-64. FM4-64 and DIC images were merged. Line plot fluorescence measurement of the GFP and FM4-64 signal is shown for each panel. A line 25–40 pixels in length and 5 pixels wide was drawn from the cytosol to the lumen. FM4-64 peak demarcates the vacuole limiting membrane. Fluorescence is normalized for both channels against the highest fluorescence value acquired. C, cytosolic side; V, vacuolar lumen side; dotted line, vacuolar membrane. Bar, 5 μm. (B) Quantification of the vacuole-to-cytosol ratio (V/C) of the Fab1-GFP fluorescence signal. Fluorescence intensity readings were taken by averaging the signal on >25% of the cytosolic area and >50% of the vacuole membrane for each cell. The V/C ratio for n > 20 and its SD are illustrated. Statistical analysis was performed using unpaired, two-tailed Student's t test.