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-bisphospha-tase. 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. Site-directed 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.
Subject(s)
Fructose-Bisphosphatase/metabolism , Peptide Hydrolases/metabolism , Proline/metabolism , Proteasome Endopeptidase Complex , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Base Sequence , Biopolymers/metabolism , Catalysis , DNA Primers , Electrophoresis, Gel, Pulsed-Field , Fructose-Bisphosphatase/genetics , Hydrolysis , Isocitrate Lyase/antagonists & inhibitors , Kinetics , Malate Dehydrogenase/antagonists & inhibitors , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphoenolpyruvate Carboxykinase (ATP)/antagonists & inhibitors , Phosphorylation , Polyubiquitin , Proline/genetics , Substrate Specificity , Ubiquitins/metabolismABSTRACT
In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.
Subject(s)
Chromosome Mapping/methods , Chromosomes, Fungal/genetics , DNA, Fungal/genetics , Genes, Fungal/genetics , Saccharomyces cerevisiae/genetics , Base Composition , Base Sequence , Cloning, Molecular , Cosmids/genetics , Molecular Sequence Data , Open Reading Frames , Quality Control , Repetitive Sequences, Nucleic Acid , Reproducibility of Results , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Telomere/geneticsABSTRACT
The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.
