The C-terminal Domain of Snf3p is Sufficient to Complement the Growth Defect of snf3 Null Mutations in Saccharomyces cerevisiae: SNF3 Functions in Glucose Recognition

The SNF3 protein, Snf3p, of Saccharomyces cerevisiae was initially thought to be a high affinity glucose transporter required for efficient catabolism of low glucose concentrations. We now report evidence suggesting that Snf3p is a regulatory protein and not a catabolic transporter. The C-terminal domain of Snf3p is able to complement the growth defect on solid media of snf3 null mutants independent of attachment to the membrane-spanning domains. However, the C-terminal domain is unable to fully restore high affinity glucose transport to a snf3 null strain. Examination of deletions of the C-terminal domain of intact SNF3 demonstrates that this region is required for both the growth and transport functions of Snf3p. Loss of the SNF3 gene leads to a long-term adaptation phenotype for cells grown in liquid medium at low substrate concentrations in the presence of the respiratory inhibitor, antimycin A. The presence of the C-terminal domain shortens the time required for adaptation in a snf3 null strain. Thus, Snf3p appears to affect ability to adapt to low substrate conditions, but does not confer an absolute defect in uptake of substrate. Taken together, these data suggest that Snf3p is a regulatory protein likely functioning in the detection of glucose. © 1997 by John Wiley & Sons, Ltd.     

Saccharomyces cerevisiae SCY1发酵kefir的工艺研究

本文采用Saccharomyces cerevisiaeSCY1和乳酸菌混合发酵牛乳制备kefir,分别研究了接种量、灭菌条件、发酵温度和加糖量对kefir风味的影响,最终确定最佳工艺条件为:XPL-1接种量为0.0400g/L,SCY1接种量为103个/mL;灭菌条件为80~85℃下灭菌10min;发酵温度为32℃;加糖量为4%。通过此工艺条件制备的kefir,具有独特的风味和较高的营养价值。     

Sequence comparison of the ARG4 chromosomal regions from the two related yeasts,Saccharomyces cerevisiae and Saccharomyces douglasii

A 3·6 kb DNA fragment from Saccharomyces douglasii, containing the ARG4 gene, has been cloned, sequenced and compared to the corresponding region from Saccharomyces cerevisiae. The organization of this region is identical in both yeasts. It contains besides the ARG4 gene, another complete open reading frame (ORF) (YSD83) and a third incomplete one (DED81). The ARG4 and the YSD83 coding regions differ from their S. cerevisiae homologs by 8.1% and 12·5%, respectively, of base substitutions. The encoded proteins have evolved differently: amino acid replacements are significantly less frequent in Arg4 (2·8%) than in Ysc83 (12·4%) and most of the changes in Arg4 are conservative, which is not the case for Ysc83. The non-coding regions are less conserved, with small AT-rich insertions/deletions and 20% base substitutions. However, the level of divergence is smaller in the aligned sequences of these regions than in silent sites of the ORFs, probably revealing a higher degree of constraints. The Gcn4 binding site and the region where meiotic double-strand breaks occur, are fully conserved. The data confirm that these two yeasts are evolutionarily closely related and that comparisons of their sequences might reveal conserved protein and DNA domains not expected to be found in sequence comparisons between more diverged organisms.     

Glycosylation of human α1-antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts

Human α₁-antitrypsin (α₁-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α₁-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α₁-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α₁-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α₁-AT. The human α₁-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α₁-AT in the methylotrophic yeasts appeared to be relatively shorter than those of α₁-AT in S. cerevisiae. The α₁-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α₁-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein. © 1998 John Wiley & Sons, Ltd.     

Low-Affinity glucose carrier gene LGT1 of Saccharomyces cerevisiae,a homologue of the Kluyveromyces lactis RAG1 gene

A low-affinity glucose transporter gene of Saccharomyces cerevisiae was cloned by complementation of the rag1 mutation in a strain of Kluyveromyces lactis defective in low-affinity glucose transport. Gene sequence and effects of null mutation in S. cerevisiae were described. Data indicated that there are multiple genes for low-affinity glucose transport.     

GUT2, a gene for mitochondrial glycerol 3-phosphate dehydrogenase of Saccharomyces cerevisiae

A gut2 mutant of Saccharomyces cerevisiae is deficient in the mitochondrial glycerol 3-phosphate dehydrogenase and hence cannot utilize glycerol. Upon transformation of a gut2 mutant strain with a low-copy yeast genomic library, hybrid plasmids were isolated which complemented the gut2 mutation. The nucleotide sequence of a 3·2 kb PstI-XhoI fragment complementing a gut2 mutant strain is presented. The fragment reveals an open reading frame (ORF) encoding a polypeptide with a predicted molecular weight of 68·8 kDa. Disruption of the ORF leads to a glycerol non-utilizing phenotype. A putative flavin-binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins. The cloned gene has been mapped both physically and genetically to the left arm of chromosome IX, where the original gut2 mutation also maps. We conclude that the presented ORF is the GUT2 gene and propose that it is the structural gene for the mitochondrial glycerol 3-phosphate dehydrogenase.     

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.     

Mechanisms of Salt Tolerance Conferred by Overexpression of the HAL1 Gene in Saccharomyces cerevisiae

Overexpression of the HAL1 gene improves the tolerance of Saccharomyces cerevisiae to NaCl by increasing intracellular K⁺ and decreasing intracellular Na⁺. The effect of HAL1 on intracellular Na⁺ was mediated by the PMR2/ENA1 gene, corresponding to a major Na⁺ efflux system. The expression level of ENA1 was dependent on the gene dosage of HAL1 and overexpression of HAL1 suppressed the salt sensitivity of null mutants in calcineurin and Hal3p, other known regulators of ENA1 expression. The effect of HAL1 on intracellular K⁺ was independent of the TRK1 and TOK1 genes, corresponding to a major K⁺ uptake system and to a K⁺ efflux system activated by depolarization, respectively. Overexpression of HAL1 reduces K⁺ loss from the cells upon salt stress, a phenomenon mediated by an unidentified K⁺ efflux system. Overexpression of HAL1 did not increase NaCl tolerance in galactose medium. NaCl poses two types of stress, osmotic and ionic, counteracted by glycerol synthesis and sodium extrusion, respectively. As compared to glucose, with galactose as carbon source glycerol synthesis is reduced and the expression of ENA1 is increased. As a consequence, osmotic adjustment through glycerolsynthesis, a process not affected by HAL1, is the limiting factor for growth on galactose under NaCl stress. © 1997 John Wiley & Sons, Ltd.     

Assembly of phosphofructokinase-1 from Saccharomyces cerevisiae in extracts of single-deletion mutants

Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non-identical subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell-free extracts of single-deletion mutants. In contrast to the previously described lack of phosphofructokinase-1 activity in cell-free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption. Upon mixing of separately prepared cell-free extracts of both deletion mutants very low activity could be measured. About 40% of the wild-type activity was regained when both mutants were mixed prior to disruption. The reactivation rate could be slightly increased by addition of ATP and fructose 6-phosphate and was found to be a function of the growth state, particularly of the β-subunit-carrying cells. The individual subunits did not interact with Cibacron Blue F3G-A, a biomimetic ligand of phosphofructokinase-1. After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase-1 to the dye-ligand was observed. The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption. These changes give rise to induce an unproductive side reaction like self-aggregation of the subunits. Because reconstitution of phosphofructokinase-1 from S. cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo. © 1998 John Wiley & Sons, Ltd.     

The linkage of (1–3)-β-glucan to chitin during cell wall assembly in Saccharomyces cerevisiae

Pulse-chase experiments with [¹⁴C]glucose demonstrated that in the cell wall of wild-type Saccharomyces cerevisiae alkali-soluble (1–3)-β-glucan serves as a precursor for alkali-insoluble (1–3)-β-glucan. The following observations support the notion that the insolubilization of the glucan is caused by linkage to chitin: (i) degradation of chitin by chitinase completely dissolved the glucan, and (ii) disruption of the gene for chitin synthase 3 prevented the formation of alkali-insoluble glucan. These cells, unable to form a glucan–chitin complex, were highly vulnerable to hypo-osmotic shock indicating that the linkage of the two polymers significantly contributes to the mechanical strength of the cell wall. Conversion of alkali-soluble glucan into alkali-insoluble glucan occurred both early and late during budding and also in the ts-mutant cdc24-1 in the absence of bud formation.     

Sequence and Analysis of a 36·2 kb Fragment from the Right Arm of Yeast Chromosome XV Reveals 19 Open Reading Frames Including SNF2 (5′ end), CPA1, SLY41, a Putative Transport ATPase,a Putative Ribosomal Protein and an SNF2 Homologue

The complete sequence of a 36 196 bp DNA segment located on the right arm of chromosome XV of Saccharomyces cerevisiae has been determined and analysed. The sequence includes the 5′ coding region of the SNF2 gene, the CPA1 leader peptide sequence and 17 open reading frames (ORFs) of at least 100 amino acids. Two of these correspond to previously known genes (CPA1, SLY41), whereas 15 correspond to new genes. The putative translation products of three ORFs show significant similarity with known proteins: one is a putative transport ATPase, another appears to be a ribosomal protein, and the third is an Snf2p homologue. The sequence has been deposited in the EMBL databank under Accession Numbers: Z75198, Z75199, Z75200, Z75201, Z75202, Z75203, Z75204, Z75205, Z75206, Z75207, Z75209, Z75210, Z75211, Z75212, Z75213, Z75214, Z75215, Z75216. © 1997 John Wiley & Sons, Ltd.     

Cloning and genetic analysis of the gene encoding a new protein kinase in Saccharomyces cerevisiae

We have isolated a single gene from the yeast Saccharomyces cerevisiae encoding a potential 800 amino acid polypeptide of calculated M_r 90 098 Da. This protein consists of an N-terminal region that shares significant homology with the catalytic domains of several serine- and threonine-specific protein kinases, as well as a large, unique, C-terminal domain of unknown function. Haploid disruption mutants are viable and do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients or osmotic strength. Due to the apparent structural similarity between this kinase and the protein products of the KIN1 and KIN2 genes, we have chosen to name this new gene KIN3.