Proteins of Newly Isolated Mutants and the Amino-terminal Proline Are Essential for Ubiquitin-Proteasome-catalyzed Catabolite Degradation of Fructose-1,6-bisphosphatase of Saccharomyces cerevisiae

Abstract

Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a non-fermentable carbon source leads to selective and rapid degradation of fructose-1,6-bisphosphatase. This so called catabolite inactivation of the enzyme is brought about by the ubiquitin-proteasome system. To identify additional components of the catabolite inactivation machinery, we isolated three mutant strains, gid1, gid2, and gid3, defective in glucose-induced degradation of fructose-1,6-bisphosphatase. All mutant strains show in addition a defect in catabolite inactivation of three other gluconeogenic enzymes: cytosolic malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase. These findings indicate a common mechanism for the inactivation of all four enzymes. The mutants were also impaired in degradation of short-lived N-end rule substrates, which are degraded via the ubiquitin-proteasome system. Sitedirected mutagenesis of the amino-terminal proline residue yielded fructose-1,6-bisphosphatase forms that were no longer degraded via the ubiquitin-proteasome pathway. All amino termini other than proline made fructose-1,6-bisphosphatase inaccessible to degradation. However, the exchange of the amino-terminal proline had no effect on the phosphorylation of the mutated enzyme. Our findings suggest an essential function of the amino-terminal proline residue for the degradation process of fructose-1,6-bisphosphatase. Phosphorylation of the enzyme was not necessary for degradation to occur.

Catabolite Degradation of Fructose-1,6-bisphosphatase in the YeastSaccharomyces cerevisiae: A Genome-wide Screen Identifies Eight NovelGIDGenes and Indicates the Existence of Two Degradation Pathways

“…Previous experiments suggested an increased level of Arg-␤-gal in gid2-1 mutant cells compared with wild type (Hämmerle et al, 1998). Following the fate of Arg-␤-gal in gid2⌬ cells via pulse-chase measurements did not uncover an altered degradation of the substrate ( Figure 3B).…”

“…The linkage of polyubiquitin chains onto proteins via an enzyme cascade consisting of an ubiquitin activating enzyme (E1), ubiquitin conjugating enzymes (E2), and ubiquitin-protein ligases (E3) marks these proteins for degradation via the cytoplasmic and nuclear proteolytic nanocompartment, the proteasome Wolf, 1996, 2000;Baumeister et al, 1998). We furthermore showed that FBPase degradation is independent of the phosphorylation of the protein (Hämmerle et al, 1998). In a genetic approach isolating mutants defective in the glucose-induced degradation process of FBPase (gid-mutants) (Hämmerle et al, 1998;Schüle et al, 2000), we uncovered the essential function of the ubiquitin-conjugating enzyme Ubc8p (Gid3p) for glucoseinduced proteasome-dependent degradation of FBPase (Schüle et al, 2000).…”

“…Although we find a cytosolic, ubiquitin-proteasome-dependent catabolite degradation of FBPase (Schork et al, 1994a(Schork et al, ,b, 1995Hämmerle et al, 1998;Schü le et al, 2000), vacuolar degradation of FBPase (Chiang and Schekman, 1991) and the involvement of vesicular intermediates in this process has also been suggested (Huang and Chiang, 1997;Chiang and Chiang, 1998). We therefore were interested in whether some FBPase became membrane protected during the catabolite degradation process in gid2⌬ cells.…”

“…Using a fusion protein consisting of the amino-terminal part of FBPase fused to the marker ␤-galactosidase, we previously isolated three mutants defective in the GID1, GID2, and GID3 genes (gid1-3, gid2-1, and gid3-1) necessary for glucose-induced degradation of FBPase (Hämmerle et al, 1998). We uncovered GID3 as the gene encoding the ubiquitin-conjugating enzyme Ubc8p as a crucial component involved in catabolite degradation of FBPase (Schü le et al, 2000).…”

“…We furthermore showed that FBPase degradation is independent of the phosphorylation of the protein (Hämmerle et al, 1998). In a genetic approach isolating mutants defective in the glucose-induced degradation process of FBPase (gid-mutants) (Hämmerle et al, 1998;Schüle et al, 2000), we uncovered the essential function of the ubiquitin-conjugating enzyme Ubc8p (Gid3p) for glucoseinduced proteasome-dependent degradation of FBPase (Schüle et al, 2000).…”