FM 4-64 uptake assay

As a measure of bulk endocytosis, we observed uptake of the lipophilic dye N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl pyridinium dibromide (FM 4-64) (Molecular Probes, Eugene, OR/Invitrogen). To assess the effect of PP1 inhibition, wild-type, vma2Δ, glc7-12^ts, and glc7-12^tsvma2Δ cells were allowed to grow to midlog phase in YEPD pH 5. For each strain, three separate aliquots of cell suspension (equivalent to 1 OD₆₀₀ unit of cells) were pelleted and resuspended in ice-cold YEPD pH 5 containing 20 μM FM 4-64. To initially label only the PMs, cells were pulse labeled on ice with continuous shaking for 20 min (Vida and Emr 1995), washed with ice-cold fully supplemented minimal media to reduce background fluorescence, and imaged using a Texas Red filter. The remaining two tubes from each mutant were washed twice with YEPD pH 5 prewarmed to either 25° (permissive for glc7-12^ts) or 37° (nonpermissive for glc7-12^ts). After pelleting to remove excess dye, the pellets were resuspended in YEPD pH 5 and chased for 2 hr either at 25 or 37°, then washed with fully supplemented minimal media and imaged as above. To check the effects of CN inhibition, vma2Δ cells were grown to midlog phase and distributed across seven tubes (cell volumes equivalent to 1 OD₆₀₀ units in each). Pelleted cells were resuspended in 1 ml ice-cold YEPD pH 5 containing FM 4-64 as described above. After a 20 min pulse labeling, one tube of cells was washed and imaged as described above, and the other tubes were washed twice with room temperature YEPD pH 5 and resuspended in 1 ml of YEPD pH 5. FK-506 was added (final concentration 10 μg/ml) to three tubes and DMSO was added to the other three. Samples were chased at 30° for 2, 4, and 6 hr, then washed and imaged as described above.