Fig. 6. Cells with the pI^-/II^-/scar^- triple mutation are severely defective in fluid phase pinocytosis, exocytosis and lysosomal enzyme secretion. Cells were incubated with FITC-dextran and, at various times, the intracellular fluorescence was calculated as described in Materials and Methods; the averages of four independent experiments are shown (A). Alternatively, cells were loaded with FITC-dextran for 3 hours, washed and placed back in fresh growth medium. At various times, cells were collected, washed and the remaining intracellular FITC-dextran was measured (B). Finally, cells growing exponentially were collected by centrifugation and the intracellular and extracellular levels of -mannosidase were measured (C). (A) pI^-/II^-/scar^- cells displayed a defect in pinocytosis compared with control cells, and pI^-/II^-/scar^- cells showed an additive defect compared with Scar and profilin null mutants alone, supporting the hypothesis that these two proteins interact to regulate fluid internalization. (B) pI^-/II^- cells displayed an exocytic defect: 50% of the fluid phase remained inside the cell after 90 minutes post-chase (compared with 45 minutes for release of one half of the fluid phase from control cells). Cells with the pI^-/II^-/scar^- triple mutation showed an additive exocytic defect: 50% of the fluid phase remained inside the cell after 150 minutes into the chase period. After 3 hours into the chase period, none of the fluid phase remained in control cells, whereas 20% remained in the pI^-/II^- cells and 40% remained inside the pI^-/II^-/scar^- cells. (C) The steady state secretion rate of -mannosidase was calculated by comparing the extracellular enzymatic activity with the total enzymatic activity of the cells and supernatant from pelleted cells. At steady state, only 20% of the -mannosidase activity remained inside the cell, whereas 80% of the activity resided in the supernatant, owing to secretion of the lysosomal hydrolase. In scar^- cells, 40% of the -mannosidase activity remained inside the cell, and for the pI^-/II^- cells, 30% of the enzymatic activity remained intracellular, indicating that there was a secretion defect in both of these strains. The pI^-/II^-/scar^- cells displayed an additive secretion defect: only 15% of the -mannosidase activity was found in the supernatant.