Figure 7. Ubiquitin is degraded in the vacuole via the ESCRT pathway.

(A) Analysis of ubiquitin half-life following galactose induction/glucose repression in wild-type or pup1pre3 mutant yeast cells (strain background WCG4A) that have been mock-treated or treated with 50 µM MG-132, a proteasome inhibitor. (B) Analysis of ubiquitin half-life following galactose induction/glucose repression in wild-type or vacuole protease mutant yeast cells (strain background BY4742). (C) SDS-PAGE and immunoblot analysis of lysates collected from yeast cells following a galactose induction/glucose repression experiment to measure ubiquitin half-life. (D) Quantitation of slot blot analysis of FLAG-ubiquitin levels following a galactose induction/glucose repression (n = 4). Error bars indicate standard deviation. (E) Fluorescence microscopy imaging of GFP-tagged ubiquitin (green) in cells where FM4-64 (red) has been chased to label the vacuole membrane. Wild-type and Δsnf7 cells were mixed prior to imaging, with the field showing both wild-type cells and Δsnf7 cells, which are distinguished by the presence of an E-compartment.

Figure 7—figure supplement 1. Slot blot analysis of FLAG-ubiquitin levels following a galactose induction/glucose repression.

Results shown represent a single biological replicate. Results from triplicate experiments were quantified in ImageJ and used to generate the plots, as shown in Figure 7A, B and D.

Figure 7—figure supplement 2. Quantitation of slot blot analysis of FLAG-ubiquitin levels following a galactose induction/glucose repression.

Results plotted are the average values (n = 3) for each time point with error bars indicating standard deviation.

Figure 7—figure supplement 3. Quantitation of slot blot analysis of FLAG-ubiquitin levels following a galactose induction/glucose repression.

Results are shown are averaged (n = 4) with error bars indicating standard deviation.