Calcium-sensitive cls mutants of Saccharomyces cerevisiae showing a Pet- phenotype are ascribable to defects of vacuolar membrane H(+)-ATPase activity.

Ca(2+)-sensitive mutants of the yeast Saccharomyces cerevisiae showing a Pet- phenotype (cls7-cls11) have lesions in a system for maintaining intracellular Ca2+ homeostasis (Ohya, Y., Ohsumi, Y., and Anraku, Y. (1986) J. Gen. Microbiol. 132, 979-988). Genetic and biochemical studies have demonstrated that these Pet- cls mutants are related to defects in vacuolar membrane H(+)-ATPase. CLS7 and CLS8 were found to be identical with the structural genes encoding subunit c (VMA3) and subunit a (VMA1), respectively, of the enzyme. In addition, these five mutants all had vma defects; no vacuolar membrane ATPase activity was detected in the cls cells, and the cls mutants showed a loss of ability to acidify the vacuole in vivo. Measurements of the cytosolic free Ca2+ concentration [( Ca2+]i) in individual cells showed that the average [Ca2+]i in wild-type cells was 150 +/- 80 nM, whereas that in five Pet- cls cells was 900 +/- 100 nM. These data are consistent with the observation that vacuolar membrane vesicles prepared from the Pet- cls cells have lost ATP-dependent Ca2+ uptake activities. The cls defects of vacuolar membrane H(+)-ATPase resulted in pleiotropic effects on several cellular activities, including Ca2+ homeostasis, glycerol metabolism, and phospholipid metabolism. The mutants showed an inositol-dependent phenotype, possibly due to alteration in regulation of phospholipid biosynthesis; the phosphatidylserine decarboxylase activities of the mutants were 15-50% of that of the wild-type cells and were not repressed by the addition of inositol. In contrast to the majority of previously isolated pet mutants (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225), the Pet- cls mutants showed no detectable mitochondrial defects. Taking all these findings into account, we suggest that at least six genes, VMA1 (CLS8, subunit a), VMA2 (subunit b), VMA3 (CLS7, subunit c), VMA11 (CLS9), VMA12 (CLS10), and VMA13 (CLS11), are required for expression of the vacuolar membrane H(+)-ATPase activity.     

A multinuclear magnetic resonance study of a cls11 mutant showing the Pet- phenotype of Saccharomyces cerevisiae

Energetic and intermediary metabolism was studied in a Pet- mutant of Saccharomyces cerevisiae with a calcium-sensitive phenotype that shows an inability to grow when cultured in a medium containing non-fermentable substrates. The perchloric acid extracts were prepared from suspensions of cls11 mutant and wild-type cells incubated with [1,3-13C]glycerol or [2-13]acetate, and analyzed by 31P, 13C and 1H NMR. 31P- and 1H-NMR spectra showed significant differences between cls11 and wild-type cells at the level of amino acids, the storage carbohydrate trehalose (higher in mutant cells), and sugar phosphates (higher in wild-type cells). 13C-NMR spectra revealed major differences in the steady-state labelling of glutamate carbons. For incubations with [1,3-13C]glycerol, we estimated from the relative 13C enrichment of glutamate carbons that acetyl-CoA C2 is 43% C13 labelled in wild-type and 10% 13C labelled in mutant cells, respectively. For incubations with [2-13C]acetate, we calculated that the ratio of the relative flux through the glyoxylate shunt versus oxidative reactions is 58% in wild-type cells and 44% in the cls11 mutant cells. Again, a dilution of the relative enrichment of C2 of acetyl-CoA was observed in the mutant cells (89%) compared to the wild-type cells (97%). These results are discussed in terms of pleiotropic defects in non-fermentable carbon metabolism in mutant cells.     

Glucose transport in a kinaseless Saccharomyces cerevisiae mutant.

Wild-type Saccharomyces cerevisiae organisms contain three kinases which catalyze the phosphorylation of glucose: two hexokinase isozymes (PI and PII) and one glucokinase. Glucose transport measurements for triple-kinaseless mutants, which lack all three of these kinases, confirm that the kinases are involved in the low apparent Km transport process observed in metabolizing cells. Thus kinase-positive cells containing one or more of the three kinases exhibit biphasic transport kinetics with a low apparent Km (1 to 2 mM) and high apparent Km (40 to 50 mM) component. Triple-kinaseless cells, however, exhibit only the high apparent Km component of kinase-positive cells (60 mM). Kinetic analysis of glucose transport in the triple-kinaseless cells shows that glucose is transported by a facilitated diffusion process which exhibits trans-stimulated equilibrium exchange and influx counterflow.     

Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis.

A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme. This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis. Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors. The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase. Phosphatidylinositol synthase was not affected in the cdg1 mutant.     

Genetic studies with a phosphoglucose isomerase mutant of Saccharomyces cerevisiae.

Summary A mutation pgi1 in the yeast Saccharomyces cerevisiae conferring deficiency of the glycolytic enzyme glucose 6-phosphate isomerase is characterised genetically. The mutation segregates 2⁺:2^- in tetrads from diploids heterozygous for the mutant phenotype. The mutation is semi-dominant and is located on the right arm of chromosome II in the order: tsm134-lys2-pgi1-tyr1 approximately 15 map units from tyr1. The mutation pgi1 defines the structural gene of glucose 6-phosphate isomerase and can be suppressed intragenically giving revertants that have an unstable enzyme. In one temperature-sensitive revertant no enzyme activity in excess of the mutant level could be detected although fructose 6-phosphate was converted to glucose 6-phosphate in vivo. The suppressor locus in this revertant is dominant and is unlinked to the pgi1 locus.     

A glutamine synthetase mutant of Saccharomyces cerevisiae shows defect in cell wall

To study the organization and biosynthesis of the yeast cell wall, hypo-osmolarity-sensitive mutants of Saccharomyces cerevisiae were analyzed. Cells of JS4 were irregular in shape and fragile. Calcofluor staining and quantitative analysis indicated that the chitin content was reduced. By DNA cloning and genetic analysis, the mutation hpo1-1 was found to be allelic to GLN1 which encodes glutamine synthetase. The glutamine content was significantly low in JS4, and the mutant was recovered from the cell wall defect by supplying glutamine in the medium. Partial inhibition of glutamine synthetase by phosphinothricin also induced defects in the cell wall. These results indicate that the shortage of glutamine affects cell wall integrity prior to other cellular functions.     

酿酒酵母SNF4基因敲除缺失菌株的构建

构建一株酿酒酵母SNF4基因缺失菌株并研究其对乙醇产量的影响。扩增带有SNF4基因上下游同源序列和Kanr筛选标记的SNF4基因敲除组件,转化到酿酒酵母YS2获得阳性克隆子,然后将质粒pSH65转到阳性克隆子中,半乳糖诱导pSH65表达Cre酶切除Kanr筛选标记,获得SNF4等位基因完全缺失菌株YS2-△SNF4。发酵实验结果表明,缺失菌株YS2-△SNF4乙醇产量较出发菌株提高了7.57％。利用Cre-LoxP系统,成功构建了SNF4等位基因完全缺失菌株并提高乙醇产生量。     

A halotolerant mutant of Saccharomyces cerevisiae.

FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated. Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression.     

Characterization of a Saccharomyces cerevisiae mutant with oversecretion phenotype

An oversecreting mutant of Saccharomyces cerevisiae was obtained from about 400 meiotic segregants derived from thediploid cells made by crossing the HBsAg-induced mutant NI-C with the wild-type strain Sey6211. When transformed with a plasmid containing mouse alpha-amylase cDNA, the mutant (NI-C-D4) exhibited an increased capacity (up to 13-fold) for the secretion of mouse alpha-amylase, higher than the parental strains and other standard wild-type strains. It was also shown that alpha-amylase secreted by the oversecreting mutant had a higher activity and contained more of the non-glycosylated form than the glycosylated form. This isolated oversecreting, low-glycosylation mutant may prove to be a potential S. cerevisiae host for the production of foreign proteins. Further genetic analysis suggested that the mutation responsible for the mutant's oversecretion was partially dominant and that both the oversecretion and low-glycosylation phenotypes were governed by a single chromosome mutation. These pleiotrophic phenotypes may be attributed to a defect in the synthesis of an ER-resident chaperone.     

The respiratory chain in a ubiquinone-deficient mutant of Saccharomyces cerevisiae.

1. Two allelic mutants of Saccharomyces cerevisiae with a deficiency in the biosynthesis of ubiquinone have been isolated. The properties of one particular mutant strain were investigated. Submitochondrial particles of this strain contain maximally 3% of the amount of ubiquinone in wild-type particles; the amounts of other components of the respiratory chain are essentially normal. 2. The respiratory rates of mutant cells, mitochondria and submitochondrial particles are low with ubiquinone-dependent substrates, but are restored to normal levels by addition of Q-1; the restored respiration is antimycin sensitive. Intact cells and mitochondria show respiratory control both in the absence and presence of Q-1. 3. The NADH:Q-1 oxidoreductase of submitochondrial particles of the mutant followspseudo first-order kinetics in [Q-1]. QH2-1 inhibits competitively with respect to Q-1, the Ki for QH2-1 being equal to the Km for Q-1. 4. Succinate dehydrogenase in both wild-type and mutant submitochondrial particles can be activated by NADH. 5. The turnover number of succinate dehydrogenase in the mutant, measured with phenazine methosulphate as primary electron acceptor, is about one-half that of wild-type particles. The turnover numbers measured with Q-1 as electron acceptor are about the same in the two types of particles. 6. The kinetics of redox changes in cytochrome b, in the presence of antimycin and oxygen, are distinctly different in the mutant and wild-type particles. They indicate that ubiquinone plays an important role in the phenomenon of the increased reducibility of cytochrome b induced by antimycin plus oxygen.     

Identification of the bud emergence gene BEM4 and its interactions with rho-type GTPases in Saccharomyces cerevisiae.

The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.     

A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase.

A novel, potent, semisynthetic pneumocandin, L-733,560, was used to isolate a resistant mutant in Saccharomyces cerevisiae. This compound, like other pneumocandins and echinocandins, inhibits 1,3-beta-D-glucan synthase from Candida albicans (F.A. Bouffard, R.A. Zambias, J. F. Dropinski, J.M. Balkovec, M.L. Hammond, G.K. Abruzzo, K.F. Bartizal, J.A. Marrinan, M. B. Kurtz, D.C. McFadden, K.H. Nollstadt, M.A. Powles, and D.M. Schmatz, J. Med. Chem. 37:222-225, 1994). Glucan synthesis catalyzed by a crude membrane fraction prepared from the S. cerevisiae mutant R560-1C was resistant to inhibition by L-733,560. The nearly 50-fold increase in the 50% inhibitory concentration against glucan synthase was commensurate with the increase in whole-cell resistance. R560-1C was cross-resistant to other inhibitors of C. albicans 1,3-beta-D-glucan synthase (aculeacin A, dihydropapulacandin, and others) but not to compounds with different modes of action. Genetic analysis revealed that enzyme and whole-cell pneumocandin resistance was due to a single mutant gene, designated etg1-1 (echinocandin target gene 1), which was semidominant in heterozygous diploids. The etg1-1 mutation did not confer enhanced ability to metabolize L-733,560 and had no effect on the membrane-bound enzymes chitin synthase I and squalene synthase. Alkali-soluble beta-glucan synthesized by crude microsomes from R560-1C was indistinguishable from the wild-type product. 1,3-beta-D-Glucan synthase activity from R560-1C was fractionated with NaCl and Tergitol NP-40; reconstitution with fractions from wild-type membranes revealed that drug resistance is associated with the insoluble membrane fraction. We propose that the etg1-1 mutant gene encodes a subunit of the 1,3-beta-D-glucan synthase complex.     

Properties of a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae

We characterized a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae, TFL20, that has a mutation in the LEU4 gene. We monitored the concentration of extracellular i-AmOH and intracellular amino acids, and compared the ratios of gene expression in TFL20 with the wild-type strain, K30. We found that the LEU1, LEU2, and BAT1 genes were up-regulated in TFL20 for metabolism, and that TFL20 simultaneously produced as much i-AmOH and leucine as K30 does.     

Plasmid-mediated complementation of a delta-aminolevulinic-acid-requiring Saccharomyces cerevisiae mutant

Recombinant plasmids able to complement the Saccharomyces cerevisiae ole3 mutation were isolated. The nucleotide sequence responsible for complementation was localized to a 3.5-kb region. The level of delta-aminolevulinate (ALV) synthase activity in wild-type cells was six-fold lower than in plasmid-transformed ole3 mutant cells. Certain clones secreted a compound that supported growth of a lawn of adjacent ole3 mutant cells.     

The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae.

We have constructed a dihydrofolate reductase mutant (dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfr1 mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfr1 mutants. The inability of the mutants to metabolize FA suggests that the DFR1 gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfr1 strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfr1 strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFR1 gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfr1 mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfr1 strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.     

Temperature-sensitive Saccharomyces cerevisiae mutant defective in lipid biosynthesis.

A temperature-sensitive mutant of Saccharomyces cerevisiae (DAM303) is described that exhibits an early defect in lipid biosynthesis at the restrictive growth temperature, 37 degrees C. This strain rapidly lost viability after 1 h of incubation at 37 degrees C, and this was accompanied by a significantly reduced incorporation of 32Pi into cellular lipid and an accumulation of [1-14C]acetate into the free fatty acid fraction. The temperature-sensitive DAM303 mutation failed to complement the sec13 mutation described by Novick et al. (Cell 21:205-215, 1980), and from analysis of invertase secretion in the temperature-sensitive DAM303 strain, it is clear that the loss of invertase secretion in the mutant occurs after the loss of phospholipid synthesis. Although the precise nature of the temperature-sensitive lesion in the DAM303 strain has still to be identified, the results from the study of this mutant indicate that a defect in lipid biosynthesis can be correlated with subsequent alterations in extracellular protein secretion and loss of other macromolecular functions including DNA, RNA, and protein syntheses. From studies of this mutant, two procedures of enriching for other temperature-sensitive mutants with defects in lipid biosynthesis have emerged: inositol overproduction and screening for increased buoyant densities.