Identification and kinetic analysis of a functional homolog of elongation factor 3, YEF3 in Saccharomyces cerevisiae

Yeast and other fungi contain a soluble elongation factor 3 (EF-3) which is required for growth and protein synthesis. EF-3 contains two ABC cassettes, and binds and hydrolyses ATP. We identified a homolog of the YEF3 gene in the Saccharomyces cerevisiae genome database. This gene, designated YEF3B, is 84% identical in protein sequence to YEF3, which we will now refer to as YEF3A. YEF3B is not expressed during growth under laboratory conditions, and thus cannot rescue growth of YEF3A deletion strains. However, YEF3B can take the place of YEF3A in vivo when expressed from the YEF3A or ADH1 promoters. The products of the YEF3A and YEF3B genes, EF-3A and EF-3B, respectively, were expressed from the ADH1 promoter and purified. Both factors possessed basal and ribosomal-stimulated ATPase activity, and had similar affinity for yeast ribosomes (103 to 113 nm). K_m values for ATP were similar, but the K_cat values differed significantly. Ribosome-dependent ATPase activity of EF-3A was more efficient than EF-3B, since the K_cat and K_cat/K_m values for EF-3A were about two-fold higher; however, the difference in K_cat/K_m values between the two factors was small for basal ATPase activity. © 1998 John Wiley & Sons, Ltd.     

Hut1 proteins identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe are functional homologues involved in the protein-folding process at the endoplasmic reticulum

The Saccharomyces cerevisiae HUT1 gene (scHUT1) and the Schizosaccharomyces pombe hut1(+) gene (sphut1(+)) encode hydrophobic proteins with approximately 30% identity to a human UDP-galactose transporter-related gene (UGTrel1) product. These proteins show a significant similarity to the nucleotide sugar transporter and are conserved in many eukaryotic species, but their physiological functions are not known. Both scHUT1 and sphut1(+) genes are non-essential for cell growth under normal conditions, and their disruptants show no defects in the modification of O- and N-linked oligosaccharides, but are sensitive to a membrane-permeable reducing agent, dithiothreitol (DTT). Consistent with this phenotype, scHUT1 has genetic interaction with ERO1, which plays an essential role in the oxidation of secretory proteins at the endoplasmic reticulum (ER). Overexpression of the MPD1 or MPD2 genes, which were isolated as multicopy suppressors of protein disulphide isomerase (PDI) depletion, could not replace the essential function of PDI in Delta hut1 S. cerevisiae cells. Our results indicate that scHut1p and spHut1p are functional homologues, and their physiological function is to maintain the optimal environment for the folding of secretory pathway proteins in the ER.     

The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae

The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19‐1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19‐1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes. The GenBank Accession Nos of the sequences are AF255332 (K.lactis GDI1) and AY007574 (P. pastoris GDI1). Copyright © 2001 John Wiley & Sons, Ltd.     

Isolation of GCR1, a major transcription factor of glycolytic genes in Saccharomyces cerevisiae, from Kluyveromyces lactis

To study the function of GCR1, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, a Kluyveromyces lactis gene that complements the growth defect of a S. cerevisiae Deltagcr1 mutant was isolated. Introduction of this gene into the Deltagcr1 mutant also restored the activities of glycolytic enzymes. DNA sequencing of KlGCR1 predicted an open reading frame of a 767 amino acid protein with an overall identity of 33% and similarity of 48% to Gcr1p from S. cerevisiae. Its DDBJ/EMBL/GenBank Accession No. is AB046391.     

酿酒酵母与粟酒裂殖酵母融合子检出方法的研究

为了快速检出酿酒酵母与粟酒裂殖酵母的融合子,在双亲原生质体融合处理后,利用两亲本再生营养条件的差异及氧化邻苯二胺能力的不同,通过设计选择性底层培养基和鉴定性上层培养基,进行了融合子检出方法的研究,并确定了3株融合子。该方法简单易行,可用于酿酒酵母与粟酒裂殖酵母原生质体融合育种的研究。     

Isolation of Delta12 and omega3-fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and alpha-linolenic acids in Saccharomyces cerevisiae

Two clones with homology to known fatty acid desaturase genes were isolated from the yeast Kluyveromyces lactis. The first gene, which we designate KlFAD2, consists of 411 amino acids with an overall identity of 73.0% to FAD2 from Saccharomyces kluyveri. It exhibited Delta12 fatty acid desaturase activity when expressed in S. cerevisiae under the control of ADH1 promoter and produced endogenous linoleic acid. The second clone, which we designate KlFAD3, consists of 415 amino acids with an overall identity of 79.3% to FAD3 from S. kluyveri. It exhibited omega3 fatty acid desaturase activity in S. cerevisiae when expressed under the control of ADH1 promoter in the presence of the exogenous substrate linoleic acid and produced alpha-linolenic acid. Co-expression of KlFAD2 and KlFAD3 resulted in the endogenous production of both linoleic and alpha-linolenic acids. The yield of alpha-linolenic acid reached 0.8% of total fatty acids and its production was not increased by adding exogenous oleic acid; alpha-linolenic acid reached 8.7% when exogenous linoleic acid was available.     

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated,sorted and matured in the fission yeast Schizosaccharomyces pombe

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPY^sc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPY^sc produced in the fission yeast has a higher molecular mass than mature CPY^sc produced by the budding yeast. CPY^sc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPY^sc produced by S. cerevisiae. CPY^sc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPY^sc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.     

Review: The Cct eukaryotic chaperonin subunits of Saccharomyces cerevisiae and other yeasts

All eight of the CCT1-CCT8 genes encoding the subunits of the Cct chaperonin complex in Saccharomyces cerevisiae have been identified, including three that were uncovered by the systematic sequencing of the yeast genome. Although most of the properties of the eukaryotic Cct chaperonin have been elucidated with mammalian systems in vitro, studies with S. cerevisiae conditional mutants revealed that Cct is required for assembly of microtubules and actin in vivo. Cct subunits from the other yeasts, Candida albicans and Schizosaccharomyces pombe, also have been identified from partial and complete DNA sequencing of genes. Cct8p from C. albicans, the only other completely sequenced Cct protein from a fungal species other than S. cerevisiae, is 72% and 61% similar to the S. cerevisiae and mouse Cct8 proteins, respectively. The C. albicans CCT8 sequence has been assigned the Accession Number U37371 in the GenBank/EMBL database.     

KlSEC53 is an essential Kluyveromyces lactis gene and is homologous with the SEC53 gene of Saccharomyces cerevisiae

Phosphomannomutase (PMM) is a key enzyme, which catalyses one of the first steps in the glycosylation pathway, the conversion of D-mannose-6-phosphate to D-mannose-1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for the glycosylation reactions in eukaryotic cells. In the yeast Saccharomyces cerevisiae PMM is encoded by the gene SEC53 (ScSEC53) and the deficiency of PMM activity leads to severe defects in both protein glycosylation and secretion. We report here on the isolation of the Kluyveromyces lactis SEC53 (KlSEC53) gene from a genomic library by virtue of its ability to complement a Saccharomyces cerevisiae sec53 mutation. The sequenced DNA fragment contained an open reading frame of 765 bp, coding for a predicted polypeptide, KlSec53p, of 254 amino acids. The KlSec53p displays a high degree of homology with phosphomannomutases from other yeast species, protozoans, plants and humans. Our results have demonstrated that KlSEC53 is the functional homologue of the ScSEC53 gene. Like ScSEC53, the KlSEC53 gene is essential for K. lactis cell viability. Phenotypic analysis of a K. lactis strain overexpressing the KlSEC53 gene revealed defects expected for impaired cell wall integrity.     

Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac⁺ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac⁺ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac⁺ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield. © 1998 John Wiley & Sons, Ltd.     

Characterization of the Saccharomyces cerevisiae cdc42-1ts Allele and New Temperature-Conditional-Lethal cdc42 Alleles

Cdc42p is a highly conserved GTPase involved in controlling cell polarity and polarizing the actin cytoskeleton. The CDC42 gene was first identified by the temperature-sensitive cell-division-cycle mutant cdc42-1^ts in Saccharomyces cerevisiae. We have determined the DNA and predicted amino-acid sequence of the cdc42-1^ts allele and identified multiple mutations in the coding region and 5′ promoter region, thereby limiting its usefulness in genetic screens. Therefore, we generated additional temperature-conditional-lethal alleles in highly conserved amino-acid residues of both S. cerevisiae and Schizosaccharomyces pombe Cdc42p. The cdc42^W97R temperature-sensitive allele in S. cerevisiae displayed the same cell-division-cycle arrest phenotype (large, round unbudded cells) as the cdc42-1^ts mutant. However, it exhibited a bud-site selection defect and abnormal bud morphologies at the permissive temperature of 23°C. These phenotypes suggest that Cdc42p functions in bud-site selection early in the morphogenetic process and also in polarizing growth patterns leading to proper bud morphogenesis later in the process. In S. pombe, the cdc42^W97R mutant displayed a cold-sensitive, loss-of-function phenotype when expressed from the thiamine-repressible nmt1 promoter under repressing conditions. In addition, cdc42^T58A and cdc42^S71P mutants showed a temperature-sensitive loss-of-function phenotype when expressed in S. pombe; these mutants did not display a conditional phenotype when expressed in S. cerevisiae. These new conditional-lethal cdc42 alleles will be important reagents for the further dissection of the cell polarity pathway in both yeasts. © 1997 John Wiley & Sons, Ltd.     

Plasmids with the Cre-recombinase and the dominant nat marker, suitable for use in prototrophic strains of Saccharomyces cerevisiae and Kluyveromyces lactis

Two plasmids are described which can be used to remove the "loxP-markerMX-loxP" cassettes in strains lacking the ura3 mutation. Both contain the Cre-recombinase under control of the GAL1 promoter and the natMX cassette with the dominant marker nat, which gives yeasts resistance to the antibiotic ClonNat. pNatCre contains ARSH and CEN6 for maintenance in Saccharomyces cerevisiae. pKlNatCre has a Kluyveromyces lactis replication origin and centromere in addition.     

The red ade mutants of Kluyveromyces lactis and their classification by complementation with cloned ADE1 or ADE2 genes from Saccharomyces cerevisiae

Seventy-six red adenine mutants of Kluyveromyces lactis were isolated. By complementation they could be assigned to two groups with 31 and 45 mutants. Transformation of several strains from each group with plasmids containing the Saccharomyces cerevisiae ADE1 or ADE2 gene showed that the largest group was ade2 and the other group was ade1. Several previously isolated ‘ade1’ mutants were classified to either group and given new gene and allele numbers. ADE1 was localized at chromosome III, closely linked to the mating type gene, making it a convenient marker for mating type. ADE2 was localized at chromosome V.     

Kluyveromyces lactis SSO1 and SEB1 genes are functional in Saccharomyces cerevisiae and enhance production of secreted proteins when overexpressed

The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae. Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion. Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S. cerevisiae, which suggests that they may be rate-limiting factors in this process. Here we report isolation of Kluyveromyces lactis homologues of these genes. KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature. The encoded Kluyveromyces proteins are up to 70% identical with the S. cerevisiae homologues at the amino acid level and can functionally replace them. Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S. cerevisiae when overexpressed. In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S. cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K. lactis and Pichia pastoris, the latter also in Candida utilis. The GenBank Accession Nos are AF307983 (K. lactis SSO1) and AF318314 (K. lactis SEB1).     

Isolation and study of KlLSM4, a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae

We have isolated the KlLSM4 gene as a multicopy suppressor of a Kluyveromyces lactis mutant which shows a rag(-) phenotype (resistance to antimycin A on glucose). This gene is homologous to the ScLSM4 of Saccharomyces cerevisiae, which codes for an essential 187 amino acid protein containing Sm-like domains. These motifs are present in the evolutionarily conserved family of the Sm-like proteins, which are involved in a large number of cellular processes, including pre-mRNA splicing and mRNA decapping. We demonstrated that the first 72 amino acids of KlLsm4p, which contain the Sm-like domains, can restore cell viability in both K. lactis and S. cerevisiae cells lacking the wild-type protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of cell viability in the stationary phase. The KlLSM4 sequence has been deposited in the EMBL Data library under Accession No. AJ311719.     

Chr4, a Schizosaccharomyces pombe homologue of the Saccharomyces cerevisiae Chs4p/Skt5p protein, is related to septum formation and is required for the proper localization of Chs2

In Saccharomyces cerevisiae, Chs4p directly interacts with chitin synthase III (Chs3p) to act as a post-translational regulator of the Chs3p complex. We identified four Chs4p homologous proteins in Schizosaccharomyces pombe which we named Chr1, Chr2, Chr3 and Chr4 (putative chitin synthase regulatory factor). We assessed the functions of these proteins and found that while overproduction of Chr1, Chr2 or Chr3 did not affect the cellular morphology of wild-type Sz. pombe cells, overproduction of Chr4 caused the cells to form multi-septa and delayed their growth. All multiple disruptants of chr1, chr2, chr3 and chr4 grew normally under a variety of growth conditions. However, while chitin synthase II (Chs2) normally localizes exclusively at the septum, in many chr4-disrupted cells it was found in the cytoplasm and the septa. Chs2 did localize at the abnormal septa caused by the overproduction of chr4+. Chr4-13Myc expression was unaffected by the different media or growth conditions in both wild-type and the chs2 disruptant. Chs2 expression was also unaltered by the absence of Chr4. Moreover, Chr4-13Myc localized mostly at the tips and the septum during vegetative growth in chs2, chr1, chr2 and chr3 disruptants as well as in wild-type. Thus, chr4+ is involved in septum formation and is required for the proper localization of Chs2 at the septum in Sz. pombe.     

Identification of novel and conserved functional and structural elements of the G1 cyclin Cln3 important for interactions with the CDK Cdc28 in Saccharomyces cerevisiae

Regions of the budding yeast G1 cyclin Cln3 were characterized using mutational analysis and viability assays to identify functionally relevant and novel mutant alleles of CLN3. Cyclin proteins are conserved, and Cln3 contains a region with homology to the cyclin box, which is thought to mediate physical interactions with the cyclin-dependent kinase. CLN3 was found to have characteristics similar to the conserved cyclin fold found in higher eukaryotic cyclin boxes, which consist of five alpha-helices. Peptide linker sequences inserted within helices 1, 2, 3 and 5 resulted in a loss of Cln3 function, showing cyclin fold structure similar to that previously observed for the G1 cyclin Cln2. A clustered-charge-to-alanine scan mutagenesis revealed two regions of Cln3 important for Cln3-dependent viability. The first region encompasses the conserved cyclin box. The second region is identified with alanine substitutions located well past the cyclin box, just prior to the C-terminal region of Cln3 important for protein stability. Cln3 with mutational changes in each of these regions are expressed at steady-state levels higher than wild-type Cln3, and show some defect in binding to Cdc28. The conserved hydrophobic patch domain (HPD) of cyclins is present within the first helix of the cyclin box. Alanine substitutions introduced into the HPD of Cln3 and Cln2 show functional defects while maintaining physical interaction with Cdc28 as measured by co-immunoprecipitation assay.     

Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1

Nicotinamidase (NAMase) from the budding yeast, Saccharomyces cerevisiae, was purified by Ni(2+) affinity chromatography and gel filtration. N-terminal microsequencing revealed sequence identity with a hypothetical polypeptide encoded by the yeast YGL037C open reading frame sharing 30% sequence identity with Escherichia coli pyrazinamidase/nicotinamidase. A yeast strain in which the NAMase gene, hereafter named PNC1, was deleted shows a decreased intracellular NAD(+) concentration, consistent with the loss of NAMase activity in the null mutant. In wild-type strains, NAMase activity is stimulated during the stationary phase of growth, by various hyperosmotic shocks or by ethanol treatment. Using a P(PNC1)::lacZ gene fusion, we have shown that this stimulation of NAMase activity results from increased levels of the protein and requires stress response elements in the 5'non-coding region of PNC1. These results suggest that NAMase helps yeast cells to adapt to various stress conditions and nutrient depletion, most likely via the activation of NAD-dependent biological processes.