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 transferred onto glucose medium containing cycloheximide, an inhibitor of protein 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 of vacuolar proteinase activity is observed. Subcellular fractionation and indirect immunofluorescence showed that the Gal2 transporter accumulates in the vacuole of the mutant cells, directly demonstrating that its degradation requires vacuolar proteolysis. In contrast, Gal2p degradation is proteasome independent since its half-life is unaffected in pre1-1 pre2-2, cim3-1, and cim5-1 mutants defective in several subunits of the protease complex. In addition, vacuolar delivery of Gal2p was shown to be blocked in conditional end3 and end4 mutants at the nonpermissive temperature, indicating that delivery of Gal2p to the vacuole occurs via the endocytic pathway. Taken together, the results presented here demonstrate that glucose-induced proteolysis of Gal2p is dependent on endocytosis and vacuolar proteolysis and is independent of the functional proteasome. Moreover, we show that Gal2p is ubiquitinated under conditions of glucose-induced inactivation.
Horak
J
J
Institute of Physiology, Department of Membrane Transport, Academy of Sciences of the Czech Republic, Prague.
Wolf
D H
DH
D001386:Azides / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins; D004705:Endocytosis; D005656:Fungal Proteins / Q000378:metabolism; D005690:Galactose / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D019810:Sodium Azide; D013696:Temperature; D014452:Ubiquitins / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism*
D001386:Azides; D003598:Cytoskeletal Proteins; C091908:END3 protein, S cerevisiae; D005656:Fungal Proteins; D009004:Monosaccharide Transport Proteins; D011500:Protein Synthesis Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D019810:Sodium Azide; D003513:Cycloheximide; D005947:Glucose; D005690:Galactose