Metabolic adaptation of Saccharomyces cerevisiae cells from a nonfermentable carbon source to glucose induces selective, rapid breakdown of the gluconeogenetic key enzyme fructose-1,6-bisphosphatase (FBPase), a process called catabolite degradation. Herein, we identify eight novel GID genes required for proteasome-dependent catabolite degradation of FBPase. Four yeast proteins contain the CTLH domain of unknown function. All of them are Gid proteins. The site of catabolite degradation has been controversial until now. Two FBPase degradation pathways have been described, one dependent on the cytosolic ubiquitin-proteasome machinery, and the other dependent on vacuolar proteolysis. Interestingly, three of the novel Gid proteins involved in ubiquitin-proteasome–dependent degradation have also been reported by others to affect the vacuolar degradation pathway. As shown herein, additional genes suggested to be essential for vacuolar degradation are unnecessary for proteasome-dependent degradation. These data raise the question as to whether two FBPase degradation pathways exist that share components. Detailed characterization of Gid2p demonstrates that it is part of a soluble, cytosolic protein complex of at least 600 kDa. Gid2p is necessary for FBPase ubiquitination. Our studies have not revealed any involvement of vesicular intermediates in proteasome-dependent FBPase degradation. The influence of Ubp14p, a deubiquitinating enzyme, on proteasome-dependent catabolite degradation was further uncovered

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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 glucoseinduced inactivation.In the yeast Saccharomyces cerevisiae, galactose transport proceeds via a rather specific transporter-mediated process of facilitated diffusion. The GAL2 gene product, which is inducible by its own substrate, is thought to be the major component of transport (11,40). The Gal2 transporter was predicted to be a 63.7-kDa integral plasma membrane protein containing 12 putative membrane-spanning helices connected by hydrophilic charged loops (37,53). A limited sequence of 101 amino acid residues that consists of membrane-spanning segments 10 to 12 and about 18 amino acids of the hydrophilic carboxy terminus was shown to be important for substrate recognition (38). Gal2p belongs to a superfamily of sugar transporters of prokaryotes and eukaryotes, sharing high similarity and several motifs of potential structural and/or functional importance (4). An analysis of Gal2p in temperature-conditional sec mutants showed that its delivery to the cell surface requires a functional secretory pathway (55). In contrast to the inducibility of Gal2p by galactose, glucose as a readily fermentable carbon source was shown to negatively influence its transport activity in galactose-grown cells at two different levels. One is represented by repression of GAL2 gene transcription, and the second occurs at the posttranslational level by a mechanism called glucose-induced inactivation or catabolite inactivation (25). Earlier experiments with galactose (11, 35), maltose (5), and unspecified high-affinity glucose (6) transport systems have revealed their inactivation to be a relatively rapid and irreversible process that requires arrest of cytosolic protein synthesis either by nitrogen source deprivation or cycloheximide addition to the medium and the presenc...