CATABOLITE INACTIVATION OF THE GALACTOSE TRANSPORTER IN THE YEAST SACCHAROMYCES-CEREVISIAE - UBIQUITINATION, ENDOCYTOSIS, AND DEGRADATION IN THE VACUOLE

When Saccharomyces cerevisiae cells growing on galactose are transferr ed onto glucose medium containing cycloheximide, an inhibitor of prote in synthesis, a rapid reduction of Gal2p-mediated galactose uptake is observed, We show that glucose-induced inactivation of Gal2p is due to its degradation, Stabilization of Gal2p in pra1 mutant cells devoid o f vacuolar proteinase activity is observed. Subcellular fractionation and indirect immunofluorescence showed that the Gal2 transporter accum ulates in the vacuole of the mutant cells, directly demonstrating that its degradation requires vacuolar proteolysis, In contrast, Gal2p deg radation is proteasome independent since its half-life is unaffected i n pre1-1 pre2-2, cim3-1, and cim5-1 mutants defective in several subun its of the protease complex, In addition, vacuolar delivery of Gal2p w as shown to be blocked in conditional end3 and end4 mutants at the non permissive temperature, indicating that delivery of Gal2p to the vacuo le occurs via the endocytic pathway, Taken together, the results prese nted here demonstrate that glucose-induced proteolysis of Gal2p is dep endent on endocytosis and vacuolar proteolysis and is independent of t he functional proteasome, Moreover, we show that Gal2p is ubiquitinate d under conditions of glucose-induced inactivation.