Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause a different phenotype than do previously isolated missense mutations.

Abstract

Missense mutations in the SNF3 gene of Sacharomyces cerevisiae were previously found to cause defects in both glucose repression and derepression of the SUC2 (invertase) gene. In addition, the growth properties of snf3 mutants suggested that they were defective in uptake of glucose and fructose. Glucose repression, or carbon catabolite repression, is a global regulatory system affecting the expression of many genes. Our studies of glucose repression in Saccharomyces cerevisiae have focused on the SUC2 gene. Expression of SUC2 is regulated only by glucose repression and is modulated over a greater than 200-fold range. The SUC2 gene encodes both secreted and intracellular forms of invertase via two mRNAs with different 5' ends (4, 7, 18). Secreted invertase is encoded by a glucose-repressible 1.9-kilobase (kb) mRNA. This secreted enzyme is responsible for the extracellular hydrolysis of sucrose and raffinose. The intracellular invertase is encoded by a constitutive 1.8-kb mRNA and has no obvious physiological function (22).We previously isolated mutations in six genes, SNFI through SNF6 (sucrose nonfermenting), essential for regulated SUC2 expression (6, 17). The snJf3 mutants were unable to grow on raffinose and were defective in growth on sucrose, but none showed pleiotropic defects in utilization of galactose or nonfermentable carbon sources. Our 11 snf3 mutants displayed a range of phenotypes with respect to regulation of SUC2 expression: derepressed secreted invertase activity ranged from 10 to 35% of the wild-type level. Moreover, all showed constitutive (glucoseinsensitive) synthesis of secreted invertase, ranging up to 20% of the derepressed wild-type level (17; P.S., unpublished results). Further studies suggested that regulation of SUC2 transcription was aberrant; some snJ3 mutations affected expression of a gene fusion in which the LEU2 promoter is controlled by the SUC2 upstream regulatory region (23).A puzzling phenotype of the snJ3 mutants was that all were more defective in growth on sucrose and raffinose than would have been predicted from their invertase activity. We suggested previously (17) that the snf3 mutants might be defective in uptake of glucose and fructose, which are released by the extracellular hydrolysis of sucrose and raffinose. Although one can imagine that such a defect in glucose uptake could account for the invertase constitutivity * Corresponding author. of snf3 mutants, it is not obvious how such a defect per se could also impair derepression of secreted invertase.We report here the cloning of the SNF3 gene. A 3-kb mRNA encoded by SNF3 was identified, and its level was shown to be regulated by glucose repression. The gene was disrupted at its chromosomal locus by several methods. Disruption resulted in phenotypes consistent with a defect in glucose uptake, but surprisingly did not cause the aberrant regulation of invertase expression that was observed with the missense mutations. MATERIALS AND METHODSStrains and genetic methods. Table 1 lists the S. cerevisiae strains used in t...

Three Different Regulatory Mechanisms Enable Yeast Hexose Transporter (HXT) Genes To Be Induced by Different Levels of Glucose

“…There must be another glucose transporter(s) that functions as a sensor of high glucose levels, since induction of HXT1 and HXT3 expression by high levels of glucose is not significantly affected in snf3 mutants. The finding that Snf3p is not involved in sensing high levels of glucose is not surprising, given the fact that expression of the SNF3 gene itself is about 5-to 10-fold repressible by glucose (Table 4) (33).…”

Characterization of HIR1 and HIR2, two genes required for regulation of histone gene transcription in Saccharomyces cerevisiae.

“…In this regard, it is intriguing that the predicted Hirl protein contains three copies of the ,B-transducin repeat, a motif found in another transcriptional repressor, Tupl (40). Tupl also does not bind directly to DNA but has been shown to act with another protein, Ssn6 (20,23,32), to represses specific groups of genes in response to signals which determine either the mating type of the cell or the utilization of carbon sources (20,40). The Tupl/Ssn6 repressor is apparently targeted to particular promoters by its association with additional, site-specific DNA binding proteins.…”

Std1 and Mth1 Proteins Interact with the Glucose Sensors To Control Glucose-Regulated Gene Expression inSaccharomyces cerevisiae

“…Many of the mutations which produce a Snf Ϫ phenotype show large defects in the derepression of SUC2 (25). In contrast, disruption of the SNF3 gene causes a Snf Ϫ phenotype with relatively little effect on SUC2 expression (26). Since mutations in MTH1 suppress the Snf Ϫ phenotype in a snf3 disruption, we analyzed invertase expression in cells lacking different combinations of SNF3, STD1, and MTH1.…”

“…Second, the cells must also express sufficient high-affinity hexose transporter proteins for the import of hydrolyzed raffinose. Null mutations in the SNF3 gene have little effect on invertase expression (26), yet they generate a Snf Ϫ phenotype due to impaired expression of the high-affinity hexose transporters (28,29).…”