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.
