Figure 4.

Uptake of cytosolic proteins depends on protease-sensitive component(s) on the surface of vacuoles. Vacuoles were isolated from yeast cells grown in nitrogen starvation medium and treated with increasing concentrations of trypsin for 20 min on ice. Trypsin was inhibited with a fivefold molar excess of soybean trypsin inhibitor for 10 min on ice. Uptake experiments (lanes 3–5) as well as the appropriate controls (lanes 1 and 6) were performed as described in the legend to Figure 2A. To determine whether trypsin and soybean trypsin inhibitor interfere with the integrity of the vacuoles and the uptake process, vacuoles were treated with 100 μg/ml trypsin in the presence of a fivefold molar excess of soybean trypsin inhibitor for 20 min on ice (lane 2, Mock Treatment). The uptake assay was performed as described in Figure 2A. Data and SD were calculated from two independent experiments performed in triplicate.