The low-affinity component of the glucose transport system in Saccharomyces cerevisiae is not due to passive diffusion

It has been claimed that the low-affinity component of glucose transport in Saccharomyces cerevisiae is due to passive diffusion of the sugar across the plasma membrane. We have investigated this possibility. For this purpose we have measured the permeability coefficient of hexoses in this organism. We have found that this coefficient is at least two to three orders of magnitude lower than required to account for the low-affinity component of glucose transport, and have concluded that this component is not due to passive diffusion.     

The SKS1 protein kinase is a multicopy suppressor of the snf3 mutation of Saccharomyces cerevisiae

Saccharomyces cerevisiae strains carrying snf3 are defective in high affinity glucose transport, and thus are unable to grow fermentatively on media with low concentrations of glucose. A multicopy suppressor of the snf3 growth defect, SKS1 (suppressor kinase of snf3), was found to encode a putative ser/thr protein kinase homologous to Ran1p, a kinase that regulates the switch between meiosis and vegetative growth in Schizosaccharomyces pombe. Overexpression of the SKS1 open reading frame is sufficient for suppression of the growth defects of snf3 mutants. Disruption of the open reading frame eliminates this suppression; as does the mutation of the consensus ATP binding site of Sks1p. A DDSE (DNA dependent snf3 suppressor element) was found to be present in the SKS1 promoter region. The suppression by this DDSE occurs in the absence of SKS1 coding region, that is, the DDSE can suppress a snf3 sks1 double null mutant which fails to grow fermentatively on low glucose as a snf3 mutant does. Both SKS1 and its DDSE can additionally suppress the growth defects of grr1 mutants, which are also impaired in high affinity glucose transport. The snf3 genomic suppressors, rgt1, RGT2 and ssn6, are also capable of suppressing snf3 associated growth defects in a strain lacking sks1.     

High-affinity glucose uptake in Saccharomyces cerevisiae is not dependent on the presence of glucose-phosphorylating enzymes

Glucose uptake in Saccharomyces cerevisiae is believed to consist of two kinetically distinguishable components, the affinity of which is modulated during growth on glucose. It has been reported that triple hexose-kinase deletion mutants do not exhibit high-affinity glucose uptake. This raises the question of whether and how high-affinity glucose uptake is related to the presence of glucose-phosphorylating enzymes. In this study the kinetics of glucose uptake in both wild-type cells and cells of hexose-kinase deletion mutants, grown on either glycerol or galactose, were determined using a rapid-uptake method. In wild-type cells glucose uptake measured over either 5 s or 200 ms exhibited high affinity. In contrast, in cells of hexose-kinase deletion mutants the apparent affinity of glucose uptake was dependent on the time scale during which uptake was measured. Measurements on the 5-s scale showed apparent low-affinity uptake whereas measurements on the 200-ms scale showed high-affinity uptake. The affinity and maximal rate of the latter were comparable to those in wild-type cells. Using a simple model for a symmetrical facilitator, it was possible to simulate the experimentally determined relation between apparent affinity and the time scale used. The results suggest that high-affinity glucose transport is not necessarily dependent on the presence of glucose-phosphorylating enzymes. Apparent low-affinity uptake kinetics can arise as a consequence of an insufficient rate of removal of intracellular free glucose by phosphorylation. This study underlines the need to differentiate between influences of the translocator and of metabolism on the apparent kinetics of sugar uptake in yeast.     

I. Yeast sequencing reports. Isolation and characterization of the LEU2 gene of Hansenula polymorpha

A DNA fragment carrying the LEU2 gene of methylotrophic yeast Hansenula polymorpha was isolated by complementation of the leuB mutation of Escherichia coli. The nucleotide sequence of the isolated DNA fragment contains an open reading frame of 363 codons, coding for a protein 80% identical to the LEU2 gene product of Saccharomyces cerevisiae. Further downstream, there is a partial reading frame with no obvious similarity to known proteins. The LEU2 gene of H. polymorpha cannot complement the leu2 mutation of S. cerevisiae. The sequence has been entered in the EMBL data library under the Accession Number U00889.     

Properties and Heterologous Expression of the Glucose Transporter GHT1 from Schizosaccharomyces pombe

Genomic DNA of the Schizosaccharomyces pombe glucose transporter, GHT1, was obtained by complementation of the glucose transport deficient Sz. pombe strain YGS-5. Here we describe the GHT1 gene that encodes a protein of 565 amino acids with a corresponding molecular mass of 62·5 kDa. This eukaryotic glucose transporter contains 12 putative transmembrane segments and is homologous to the HXT multigene family of S. cerevisiae with several amino acid motifs of this sugar transporter family. It is also homologous to other sugar carriers from human, mouse and Escherichia coli. The function of the Ght1 protein as a glucose transporter was proved both by homologous and heterologous expression in the Sz. pombe mutant YGS-5 and in the S. cerevisiae hxt mutant RE700A, respectively. Both transformed yeast strains transported d -glucose with substrate specificity similar to that in Sz. pombe wild-type cells. Moreover, the cells of the two transformed yeast strains accumulated 2-deoxy-d -glucose, a non-metabolizable d -glucose analogue, with an efficiency similar to Sz. pombe wild-type cells. The ability of the S. cerevisiae mutant RE700A to accumulate 2DG in an Δμdependent manner after transformation with GHT1 provides evidence that the Sz. pombe transporter catalyses an energy-dependent uptake of glucose. The sequence of GHT1 was deposited at EMBL, Outstation EBI, Accession Number X91218. ©1997 John Wiley & Sons, Ltd.     

Candida albicans homologue of GGP1/GAS1 gene is functional in Saccharomyces cerevisiae and contains the determinants for glycosylphosphatidylinositol attachment

The GGP1/GAS1/CWH52 gene of Saccharomyces cerevisiae encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gp115 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. PHR1 gene isolated from Candida alibicans is homologous to the GGP1 gene. In this report we have analysed the ability of PHR1 to complement a ggp1Δ mutation in S. cerevisiae. The expression of PHR1 controlled by its natural promoter or by the GGP1 promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that PHR1 in budding yeast gives rise to a 75–80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. albicans gene product is functional in S. cerevisiae.     

XV. Yeast sequencing reports. Nucleotide sequence of the GDS1 gene of Saccharomyces cerevisiae

We have cloned and sequenced the GDS1 gene located on the right arm of chromosome XV of Saccharomyces cerevisiae. The gene codes for a 522 amino acid serine-rich protein with no obvious homology to proteins in the database. GDS1 gene was isolated as the multicopy suppressor of the glycerol-deficient phenotype caused by the nam9-1 mutation in the yeast nuclear gene encoding the mitochondrial ribosomal protein homologous to S4 proteins from various organisms. Disruption-deletion of the GDS1 open reading frame leads to a partial impairment of growth on medium containing glycerol as the carbon source, indicating mitochondrial function of the gene product. The sequence has been deposited in the GenBank data library under Accession Number U18262.     

Glutathione levels influence chronological life span of Saccharomyces cerevisiae in a glucose‐dependent manner

Diet plays a key role in determining the longevity of the organisms since it has been demonstrated that glucose restriction increases life span whereas a high‐glucose diet decreases it. However, the molecular basis of how diet leads to the aging process is currently unknown. We propose that the quantity of glucose that fuels respiration influences reactive oxygen species generation and glutathione levels, and both chemical species impact in the aging process. Herein, we provide evidence that mutation of the gene GSH1 in Saccharomyces cerevisiae diminishes glutathione levels. Moreover, glutathione levels were higher with 0.5% than in 10% glucose in the gsh1Δ and wild‐type strains. Interestingly, the chronological life span was lowered in the gsh1Δ strain cultured with 10% glucose but not under dietary restriction. The gsh1Δ strain also showed inhibition of the mitochondrial respiration in 0.5 and 10% glucose but only increased the H₂O₂ levels under dietary restriction. These results correlate well with the GSH/GSSG ratio, which showed a decrease in gsh1Δ strain cultured with 0.5% glucose. Together, these data indicate that glutathione exhaustion impact negatively both the electron transport chain function and the chronological life span of yeast, the latter occurring when a low threshold level of this antioxidant is reached, independently of the H₂O₂ levels.     

Identification and characterization of the KlCMD1 gene encoding Kluyveromyces lactis calmodulin

The KlCMD1 gene was isolated from a Kluyveromyces lactis genomic library as a suppressor of the Saccharomyces cerevisiae temperature-sensitive mutant spc110-124, an allele previously shown to be suppressed by elevated copy number of the S. cerevisiae calmodulin gene CMD1. The KlCMD1 gene encodes a polypeptide which is 95% identical to S. cerevisiae calmodulin and 55% identical to calmodulin from Schizosaccharomyces pombe. Complementation of a S. cerevisiae cmd1 deletion mutant by KlCMD1 demonstrates that this gene encodes a functional calmodulin homologue. Multiple sequence alignment of calmodulins from yeast and multicellular eukaryotes shows that the K. lactis and S. cerevisiae calmodulins are considerably more closely related to each other than to other calmodulins, most of which have four functional Ca²⁺-binding EF hand domains. Thus like its S. cerevisiae counterpart Cmd1p, the KlCMD1 product is predicted to form only three Ca²⁺-binding motifs. The KlCMD1 sequence has been assigned Accession Number AJ002021 in the EMBL/GenBank database. © 1998 John Wiley & Sons, Ltd.     

A Candida albicans gene encoding a DNA ligase

A DNA ligase-encoding gene (Ca CDC9) was cloned from Candida albicans by complementation of an ime-1 mutation in Saccharomyces cerevisiae. In this system, IME1 function was assayed using a S. cerevisiae strain with a ime2-promoter-lacZ gene fusion such that following transformation with a C. albicans genomic library, the presence of positive clones was indicated upon the addition of X-gal to sporulation media. Transforming fragments were subcloned in pGEM7 and sequenced. Sequence homology with several ATP-dependent DNA ligases from viruses, fission yeast, human, baker yeast and bacteria was observed. The sequence has been deposited in the EMBL data bank under the Accession Number X95001.     

Mapping of gene controlling thiamine transport in Saccharomyces cerevisiae

A recessive mutation leading to complete loss of thiamine uptake in Saccharomyces cerevisiae was mapped on the left arm of chromosome VII, approximately 56cM centromere-distal to trp5. As the analysed locus is relatively distant from its centromere and from the markers used, its attachment to chromosome VII was confirmed by chromosome loss methods.     

Identification of YHR019 in Saccharomyces cerevisiae chromosome VIII as the gene for the cytosolic asparaginyl-tRNA synthetase

Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae mutant strain 8556a, we have isolated the gene for the cytosolic asparaginyl-tRNA synthetase (AsnRS) of S. cerevisiae, by functional complementation of the mutation affecting this strain. The isolated gene could be identified to the open reading frame YHR019, called DED81, located on chromosome VIII. The mutant gene from the 8556a strain, asnrs⁻-1, was amplified from genomic DNA by PCR. This gene contains a point mutation, leading to the replacement of a glycine residue by a serine in a region of the protein probably important for the asparaginyl-adenylate recognition. The protein encoded by YHR019 is very similar to cytosolic AsnRS from other eukaryotic sources. In a phylogenetic analysis based on AsnRS sequences from various organisms, the eukaryotic sequences were clustered. Expression of YHR019 in Escherichia coli demonstrated that a yeast AsnRS activity was produced. The recombinant enzyme was purified to homogeneity in three chromatography steps. We showed that the recombinant S. cerevisiae AsnRS was able to charge unfractionated yeast tRNA, but not E. coli tRNA, with asparagine. © 1998 John Wiley & Sons, Ltd.     

Divergence and conservation of SUP2(SUP35) gene of yeasts Pichia pinus and Saccharomyces cerevisiae

SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF-1α-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82·4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part of a region homologous to EF-1α. The comparison of amino acid sequences of the Sup2 proteins reveals high conservations (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae where found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.     

α-GLUCOSIDASE, α-GLUCOSIDE PERMEASE,MALTOSE FERMENTATION AND LEAVENING ABILITY OF BAKER'S YEAST

α-Glucosidase and α-glucoside permease are synthesized simultaneously in two different strains of baker's yeast (Saccharomyces cerevisiae) when the cells are induced with maltose. α-Thioethyl D-glucopyranoside (α-TEG) inhibits the transport of maltose into the cells, and the transport of maltose and α-TEG appeared to be mediated by the same permease system. There is an obvious correlation between α-glucosidase, α-glucoside permease and maltose fermentation activities in the yeasts while no correlation between these and the leavening ability of the yeasts can be demonstrated. Apparently the glucose concentration in dough is high enough to inhibit the permease-mediated transport of maltose into the cells thus impairing leavening ability from the maltose fermenting system.     

Isolation of the Pichia pastoris PYC1 gene encoding pyruvate carboxylase and identification of a suppressor of the pyc phenotype

We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris. Disruption of this gene produced inability to grow in minimal medium with glucose as carbon source and ammonium as nitrogen source. Growth was possible with aspartate or glutamate as nitrogen source. The gene PpPYC1 expressd from its own promoter was able to rescue the phenotype of Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. In a P. pastoris strain carrying a disrupted PpPYC1 gene we have isolated spontaneous mutants able to grow in non-permissive conditions. In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepressed. The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions. The sequence of the PpPYC1 gene has been entered in the EMBL nucleotide sequence databank: Accession Number Y11106. © 1998 John Wiley & Sons, Ltd.     

Isolation of a CDC25 family gene,MSI2/LTE1, as a multicopy suppressor of ira1

We have identified MS12 as a gene of Saccharomyces cerevisiae which, when on a multicopy vector, suppresses the heat shock sensitivity caused by the loss of the IRA1 product, a negative regulator of the RAS protein. The multicopy MSI2 also suppresses the heat shock sensitivity of cells with the RAS2^val19 mutation but not those with the bcy1 mutation, suggesting that the MSI2 protein may interfere with the activity of the RAS protein. The sequence analysis of MSI2 reveals that it is identical to LTE1 belonging to the CDC25 family: CDC25, SCD25 and BUD5, each of which encodes a guanine nucleotide exchange factor for the ras superfamily gene products. Deletion of the entire MSI2 coding region reveals that MSI2 is not essential but the disruptant shows a cold-sensitive phenotype. Under the non-permissive conditions, more than 70% of the msi2 disruptants arrested at telophase as large budded cells with two nuclei divided completely and elongated spindles, indicating that the msi2 deletion is a cell division cycle mutation. These results suggest that MSI2 is involved in the termination of M phase and that this process is regulated by a ras superfamily gene product.