Figure 3.

Uptake and degradation of cytosolic proteins by isolated vacuoles show biphasic kinetics and are temperature dependent. (A) Vacuoles isolated from yeast cells grown in nitrogen starvation medium were incubated with radiolabeled cytosol for the indicated time period at 30°C (●). To control for the integrity of the isolated vacuoles during the incubation, vacuoles were incubated for the indicated time periods at 30°C and pelleted, and the supernatants were incubated for indicated time periods at 30°C with radiolabeled cytosol (○). All reactions were stopped with TCA. Radioactivity of the acid-soluble material was determined by liquid scintillation counting. Data and SD were calculated from two independent experiments performed in duplicate. (B) Vacuoles were isolated from yeast cells grown in nitrogen starvation medium. The experiment was performed as described in A except that uptake was done for 30 min at different temperatures. Background values attributable to lysis of vacuoles during the 30-min incubation were subtracted from the values obtained in the uptake measurements. Data and SD were calculated from three independent experiments performed in triplicate.