Glucose uptake mutants have not been previously obtained in Saccharomyces cerevisiae, possibly because there seem to be at least two transport systems, of low and high affinities. We showed that snf3 (sucrose nonfermenting) mutants did not express high-affinity glucose uptake. Furthermore, their growth was completely impaired on low concentrations of glucose in the presence of antimycin A (which blocks respiration).Several genes which complemented the original snJ3 gene were obtained on multicopy plasmids. Some of them, as well as plasmid-carried SNF3 itself, conferred a substantial increase in high-affinity glucose uptake in both snf3 and wild-type hosts. The effects of glucose on the expression of such a plasmid-determined high-affinity uptake resembled those in the wild type. Other genes complementing snJ3 seemed to cause an increase in low-affinity glucose uptake. We suggest that SNF3 may function specifically in high-affinity glucose uptake, which is needed under some conditions of growth on low glucose concentrations. SNF3 itself or the other complementing genes may specify components of the glucose uptake system.The uptake of glucose and fructose in Saccharomyces cerevisiae is mediated by both high (Km = 1 to 2 mM for glucose and 5 to 7 mM for fructose)-and low (Km = 15 to 20 mM for glucose and 50 to 100 mM for fructose)-affinity transport systems (1). Low-affinity uptake appears to occur by constitutive facilitated diffusion. High-affinity uptake, on the other hand, is not expressed in cells grown in the presence of high glucose concentrations (3), and mutant studies revealed that this transport system is under glucose catabolite repression control (L. Bisson, unpublished data).The activity of the high-affinity hexose transport system is dependent upon the activity of glucose-phosphorylating enzymes within the cell. In S. cerevisiae, there are three known enzymes catalyzing the phosphorylation of glucose at the six position: hexokinase A (PI), hexokinase B (Pll), and glucokinase; the hexokinases also catalyze the phosphorylation of fructose (11,12). While triple kinase mutants (hxkl hxk2 glk) lacking all three kinase activities do not display high-affinity uptake of either glucose or fructose (1), a double kinase mutant (hxkl hxk2) lacking both hexokinases A and B but retaining glucokinase lacks high-affinity uptake of fructose but retains high-affinity uptake of glucose (1). Transformation of a triple kinase mutant with a plasmid carrying a gene encoding any one of the kinases (pHXKI, pHXK2, or pGLK) restored high-affinity uptake of glucose; only pHXKI and pHXK2 were able to restore high-affinity uptake of both glucose and fructose (1). High-affinity uptake of a nonphosphorylatable glucose analog, 6-deoxyglucose, was also found to be "kinase dependent," suggesting some role of the kinases in high-affinity uptake other than simple phosphorylation of the substrate (2).Understanding the role of the kinases in kinase-dependent transport as well as the physiological roles of both low-and high-affinity uptake...

Two signals are required for meiosis and spore formation in the yeast Saccharomyces cerevisiae: starvation and the MAT products al and a2, which determine the a/a cell type. These signals lead to increased expression of the IMEI (inducer of meiosis) gene, which is required for sporulation and sporulation-specific gene expression. We report here the sequence of the IME1 gene and the consequences of IMEI expression from the GAL] promoter. The deduced IMEI product is a 360-amino-acid protein with a tyrosine-rich C-terminal region. Expression Sporulation of the yeast Saccharomyces cerevisiae is a cellular differentiation pathway (reviewed in references 12 and 26). It is normally restricted to one type of cell, the a/a cell, and is induced by nitrogen starvation. These circumstances lead to arrest of the mitotic cell cycle, to expression of sporulation-specific genes, and to initiation of the sporulation program. Cells engage in meiotic DNA synthesis, recombination, and two meiotic divisions. Each of the four meiotic products is packaged into a spore, and the four spores of the cell are encased in a sac, the ascus. Sporulation thus includes meiosis and spore formation.One of the earliest unique events in starved a/a cells is elevated accumulation of IME1 RNA (22,28,38; reviewed in reference 27). The IME1 product is thought to play a pivotal role in activating meiosis, because multicopy IME1 plasmids permit sporulation in cells that lack the determinants of a/a cell type, the MATal and MATa2 gene products, and also permit meiotic recombination in the absence of nitrogen starvation (22,38 Sporulation is accompanied by express'ion of a unique set of genes, the sporulation-specific genes. Some of these genes are essential for particular meiotic events, and others have no essential role in sporulation under laboratory conditions (1,6,11,14,17,21,25,26,33,34,42,45,47). These genes fall into early, middle, and late expression classes (1,23,25,26 The imel-12, ime2-2, gal80, ho::LYS2, his4-G, and his4-N mutations have previously been described, as have the auxotrophic markers in these strains (28,38). We note that the ho::LYS2 insertion confers a weak Lys' phenotype.The a/a diploid (strain 545) was derived from an a/a diploid (strain 537) after mild UV irradiation by screening colonies for mating-factor production through a halo assay (46). Engebrecht and Roeder observed that a/a and a/a diploids in the SK1 background were able to sporulate at a low level (strains J254 and J256 [11]). In side-by-side comparisons, we confirmed that J254 and J256 were able to sporulate and that our strain 545 was unable to sporulate. Fourteen four-spored tetrads were analyzed from an a/a/a/a tetraploid derived from crossing strains 545 and J256. Nine segregants were able to mate and able to sporulate weakly; 25 segregants were able to mate but unable to sporulate. These observations indicate that the difference in sporulation abilities 6103 on May 10, 2018 by guest

We have identified a yeast gene, MCK1, that encodes a positive regulator of meiosis and spore formation. Sequence analysis revealed that MCK1 encodes a protein kinase homolog identical to YPK1, a phosphotyrosyl protein with demonstrated protein kinase activity. Increased MCK1 gene dosage accelerates the sporulation program; mckl mutations cause delayed and decreased levels of sporulation. MCK1 is required during sporulation for maximal transcript accumulation from IME1, which encodes a meiotic activator. MCK1 is required in vegetative cells for basal IME1 expression, as evidenced by functional assays of an imel-HIS3 fusion gene. MCK1 is also required for efficient ascus maturation. Although expression of IME1 from the GALl promoter restored high-level sporulation to mckl mutants, it did not correct the ascus-maturation defect. This observation indicates that MCK1 is required, independently, for both the activation of IME1 and subsequent ascus maturation. Expression of an mckl-lacZ fusion gene was not regulated by the signals that govern meiosis. This observation is consistent with evidence that MCK1 plays a role in governing centromere function during vegetative growth as well as sporulation.

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...

The SNF2 and SNF5 genes are required for derepression of SUC2 and other glucose-repressible genes of Saccharomyces cerevisiae in response to glucose deprivation. Previous (28). The SUC2 gene encodes these two invertases by producing two differently regulated mRNAs: a glucose-repressible, 1.9-kilobase (kb) mRNA that encodes secreted invertase, and a constitutive, 1.8-kb mRNA with a different 5' end that encodes an intracellular invertase (7,11,25). Previous studies have identified an upstream regulatory region that is required for transcription of the 1.9-kb mRNA (28) and able to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ gene fusion (29).We have previously isolated mutations in trans-acting genes necessary for sucrose utilization (9, 22). These recessive mutations fell into six complementation groups SNFJ through SNF6 (sucrose nonfermenting); a mutation in any one of these genes partially or completely blocks derepression of the SUC2 gene. Genetic analysis has suggested that the SNF2 and SNF5 genes play functionally related roles in derepression of SUC2. The interactions of these mutations with suppressor mutations at the SSN6 and SSN20 loci * Corresponding author.clearly distinguish snf2 and snf5 from snfl, snJ3, and snf4 (22,23). It is likely that SNF6 is also functionally related to SNF2 and SNF5, as judged by the interactions of our one, possibly leaky, snf6 mutation with ssn6 and ssn20.Two mutant alleles of both snJ2 and snpf have been isolated, including a nonsense allele of each (22). These snJ2 and snf5 mutants are unable to derepress secreted invertase to normal levels, but produce a small percentage of the wild-type activity under derepressing conditions. This defect results in an inability to utilize raffinose for growth; however, the low level of derepression is still sufficient to allow growth on sucrose. These results suggest that low-level regulated expression of SUC2 can occur in the absence of functional SNF2 or SNF5 gene product, but it is possible that neither nonsense mutation is truly a null mutation. The mutants also exhibit pleiotropic defects in utilization of galactose, maltose, and nonfermentable carbon sources, which are also regulated by glucose repression, and grow more slowly than the wild type on glucose. In addition, snf2 and snp homozygous diploids are unable to sporulate.In