Fig. 3. Biosynthetic and endocytic trafficking to the vacuole. (A) CPY transport was assayed by pulse chase as described previously (Peters et al., 1999), except that the pulse and chase were performed at 30°C. The strains used were OMY20 (vtc1::HIS3), OMY21 (vtc2::HIS3), OMY22 (vtc3::HIS3) and BY4727 (wt). The growth medium was supplemented with methionine (20 µg/ml). (B) Pulse-labeling with FM4-64: Cells were grown in YPD medium (12 hours, 25°C), labeled with 200 µM FM4-64 (2 minutes, 25°C), reisolated (30 seconds, 3000 g, 20°C), washed with YPD and reisolated as before. The cells were resuspended in YPD at OD₆₀₀=1 and chased at 25°C for various times. The cells were reisolated (30 seconds, 3000 g, 20°C) and resuspended in the supernatant at OD₆₀₀=10. 5 µl of the suspension were transferred to a microscopy slide and were quickly analyzed in a fluorescence microscope under minimal excitation.

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