Isolation of a gene encoding a G protein α subunit involved in the regulation of cAMP levels in the yeast Kluyveromyces lactis

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0·5 kb (P-1) and 0·8 kb (P-2). Sequencing showed that these two fragments share high homology with genes coding for the Gα subunits from different sources. Using the P-1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein α subunits from Saccharomyces cerevisiae. KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this Gα subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian Gαs family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence. The sequence reported in this paper has been deposited in the GenBank data base (Accession No. L45105).     

The KlTrk1 gene encodes a low affinity transporter of the K+ uptake system in the budding yeast Kluyveromyces lactis

Potassium uptake in Saccharomyces cerevisiae is mediated by at least two proteins, known as Trk1p and Trk2p. Direct involvement in cation movements has been demonstrated for Trk1p, which is the high affinity transporter. S. cerevisiae cells also show low affinity potassium uptake, perhaps mediated by Trk2p. Mutants lacking Trk1p, lose high affinity system, but when grown with moderate potassium concentrations, Trk2p seems to replace it. Mutants lacking both proteins are viable but require at least 10 mM K(+) in the medium to sustain growth. Here we report the cloning and characterization of a gene from Kluyveromyces lactis encoding a homologue of these two proteins. KlTrkp is a 1070 amino acid peptide that shows, overall, higher homology with Trk2p than with Trk1p, and its disruption gives rise to cells with deficient potassium transport and with an increased K(+) requirement for normal growth. Determination of kinetic parameters in the K. lactis wild-type and Kltrk1Delta strains, as well as in Sctrk1Delta Sctrk2Delta S. cerevisiae cells expressing KlTrk1, indicated that this is a low affinity component of a major potassium uptake system in K. lactis.     

Cloning and sequencing of the gene for apocytochrome b of the yeast Kluyveromyces lactis strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140)

The apocytochrome b genes from two strains of the yeast Kluyveromyces lactis, have been isolated and sequenced. The coding sequences in strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140) were identical but the upstream noncoding regions were slightly different. The sequences demonstrated the presence of a continuous open reading frame with no introns. The amino acid sequence, derived from the coding strand, showed 82% homology to the apocytochrome b of Saccharomyces cerevisiae strain D273-10B and only 58% homology to the protein from Schizosaccharomyces pombe strain 50. CUN and CGN codon families were absent from the K. lactis gene. Codon usage was very similar to that of other mitochondrial genomes with mostly U or A in the third position. There were two unusual features. All threonines were coded by ACA(U) and all arginines by AGA.     

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.     

Cloning,sequencing and disruption of the ARG8 gene encoding acetylornithine aminotransferase in the petite-negative yeast Kluyveromyces lactis

A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68·1% and 35·0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine. The nucleotide sequence of KlARG8 has been submitted to GenBank under Accession Number U93209.     

Separate roles for N- and C-termini of the STE4 (β) subunit of the Saccharomyces cerevisiae G protein in the mediation of the growth arrest. Lack of growth-arresting activity of mammalian βγ complexes

Mating pheromone signal transduction in Saccharomyces cerevisiae involves a G protein composed to Scg1p (Gpa1p), Ste4p and Ste18p subunits, homologous to the α, β and γ subunits of mammalian G proteins. Growth arrest in G1 phase is activated by the Ste4p/Ste18p complex via a downstream pathway and it is negatively controlled by the Scg1p subunit. Here we explored whether mammalian β or γ subunits could functionally substitute for their yeast homologues. While no evidence was obtained for functional replacement of Ste4p and Ste18p, we found that overexpression of Ste18p potentiated the effect of hybrid proteins in which the N terminus of the Ste4p subunit was replaced by that of the mammalian β, ste4 mutants having deletions in the N terminus showed a decreased activity in signalling to the downstream effector of the pheromone response. This defect was totally cured by overexpression of Ste18p, indicating that the truncated forms of Ste4p have retained their ability to form an active complex with Ste18p. Removal of six amino acids from the C terminus of Ste4p rendered a completely inactive subunit and this defect persisted in hybrids where the C terminus was placed by that of the β subunit, indicating that the C terminus of Ste4p is essential to trigger the effector of the yeast pheromone response pathway.     

The KlSTE2 and KlSTE3 genes encode MATalpha- and MATa-specific G-protein-coupled receptors, respectively, which are required for mating of Kluyveromyces lactis haploid cells

Mating in yeast is initiated by binding of pheromone to G-protein-coupled receptors expressed in haploid cells. We analysed the role of KlSte2p and KlSte3p receptors in the Kluyveromyces lactis mating pathway. By sequence analysis, KlSte2p and KlSte3p are the homologues of the Saccharomyces cerevisiae alpha-pheromone and a-pheromone receptors, respectively. However, by expression experiments, we determined that KlSTE2 gene is expressed in the cells typified as MATalpha and therefore is the receptor for the K. lactis a-pheromone and KlSTE3 gene is expressed in the MATa cells and binds the alpha-pheromone. The KlSTE2 gene is silent in MATa cells, while it is highly expressed in MATalpha cells, and conversely the KlSTE3 gene is expressed in MATa cells and repressed in MATalpha cells. Disruption mutants of both genes were found to be sterile, and this defect is reversed by plasmidic copies of each gene. The cytoplasmic C-terminus of KlSte3p interacts strongly with the KlGpa1p (Galpha) subunit, which is involved in the transduction of the pheromone stimulus to induce mating. Remarkably, this same domain does not interact with a constitutive active allele of the Galpha subunit, indicating that the C-terminus is able to discriminate between the active (GTP-bound) and inactive (GDP-bound) forms of the Galpha subunit.     

The lysine-rich C-terminal repeats of the centromere-binding factor 5 (Cbf5) of Kluyveromyces lactis are not essential for function

The gene coding for the centromere-binding factor 5 (CBF5) of Kluyveromyces lactis has been isolated by hybridization of a Saccharomyces cerevisiae CBF5 DNA probe to a K. lactis library. The amino acid sequence of KlCbf5 is highly homologous, 88% identity, to ScCbf5, but also to the rat protein Nap57 (64% identity). The main difference between both yeast proteins and the rat protein is the presence of a lysine-rich domain with KKE/D repeats in the C-terminal part of the protein. These repeats are thought to be involved in binding of the protein to microtubules. Deletion of the KKE/D domain in KlCbf5 however, has no discernible effect on growth on rich medium, sensitivity to the microtubule-destabilizing drug benomyl or segregation of a reporter plasmid. On the other hand, insertion of two leucine residues adjacent to the KKE domain increases the loss rate of a reporter plasmid. In both yeasts complementation of a lethal CBF5 disruption with the heterologous gene results in a slight increase in benomyl sensitivity. A possible role of CBF5 in chromosome segregation will be discussed. © 1998 John Wiley & Sons, Ltd.     

An analysis of inter- and intraspecific genetic variabilities in the Kluyveromyces marxianus group of yeast species for the reconsideration of the K. lactis taxon

In the present work, we analyse the sequences of the 5.8S rRNA gene and the two internal transcribed spacers 1 and 2 (5.8S-ITS region), obtained from 39 strains belonging to the species Kluyveromyces aestuarii, K. dobzhanskii, K. lactis and K. marxianus, K. nonfermentans and K. wickerhamii, to solve the phylogenetic relationships among these species and also to determine the possible genetic basis for the delimitation of the two currently accepted K. lactis varieties: lactis, including lactose-positive strains isolated from dairy products, and drosophilarum, comprising lactose-negative strains isolated from insects and plant exudates. The determination of the phylogenetic relationships within the species K. lactis, together with the examination of the electrophoretic karyotypes and phenotypic characterization of strains representatives of K. lactis var. lactis and var. drosophilarum, allowed differentiation of two groups of strains. The first, and ancestral, group comprises lactose-negative strains isolated from natural habitats in North America. The second, and derived, group includes both lactose-negative strains isolated from natural habitats in Europe and wine fermentation in South Africa, and lactose-positive strains associated with dairy products. These results suggest that the present taxon K. lactis is a complex of different species, subspecies or, at least, genetically structured populations.     

Nutritional and toxicological evaluation of yeast (Saccharomyces cerevisiae) biomass and a yeast protein concentrate

Brewer's yeast was prepared by alkaline treatment for debittering, cell wall rupture and dehydration by spray drying. Yeast protein concentrate was prepared by centrifugation of the ruptured cell suspension, treatment of the supernatant with sodium perchlorate, precipitation of the protein at isoelectric pH (4.2) and neutralisation of the isoelectric protein to pH 6.5 with sodium hydroxide, prior to lyophylisation. Chemical characterisation was performed on the biomass and protein concentrate. Amino acid scores were 98.1 and 87.2% for the whole biomass and protein concentrate respectively, based on available lysine and compared with the FAO/WHO/UNU reference standard. The growth‐promoting property of the yeast biomass protein was roughly 85% of casein and was significantly better than for the yeast protein concentrate. No difference in growth was found between 15 and 30% dietary protein for all three sources, ie casein, whole yeast biomass and yeast protein concentrate. When tested for subchronical toxicity at 15 and 30% protein concentration, no evidence of toxicity was found for the whole yeast biomass, compared with casein, after 45 and 90 days of feeding. Retarded growth and discrete liver steatosis were observed in rats fed the yeast protein concentrate at both dietary levels. © 2000 Society of Chemical Industry     

Activation of trehalase during growth induction by nitrogen sources in the yeast Saccharomyces cerevisiae depends on the free catalytic subunits of camp-dependent protein kinase,but not on functional ras proteins

Addition of a nitrogen-source to glucose-repressed, nitrogen-starved G0 cells of the yeast Saccharomyces cerevisiae in the presence of a fermentable carbon source induces growth and causes within a few minutes a five-fold, protein-synthesis-independent increase in the activity of trehalase. Nitrogen-activated trehalase could be deactivated in vitro by alkaline phosphatase treatment, supporting the idea that the activation is triggered by phosphorylation. Yeast strains containing only one of the three TPK genes (which encode the catalytic subunit of cAMP-dependent protein kinase) showed different degrees of nitrogen-induced trehalase activation. The order of effectiveness was different from that previously reported for glucose-induced activation of trehalase in glucose-derepressed yeast cells. Further reduction of TPK-encoded catalytic subunit activity by partially inactivating point mutations in the remaining TPK gene further diminished nitrogen-induced trehalase activation, while deletion of the BCY1 gene (which encodes the regulatory subunit) in the same strains resulted in an increase in the extent of activation. Deletion of the RAS genes in such a tpk^w1 bcy1 strain had no effect. These results are consistent with mediation of nitrogen-induced trehalase activation by the free catalytic subunits alone. They support our previous conclusion that cAMP does not act as second messenger in this nitrogen-induced activation process and our suggestion that a novel nitrogen-induced signaling pathway integrates with the cAMP pathway at the level of the free catalytic subunits of protein kinase A. Western blot experiments showed that the differences in the extent of trehalase activation were not due to differences in trehalase expression. On the other hand, we cannot completely exclude that protein kinase A influences the nitrogen-induced activation mechanism itself rather than acting directly on trehalase. However, any such alternative explanation requires the existence of an additional, yet unknown, mechanism for activation of trehalase besides the well-established regulation by protein kinase A.     

Viability of probiotic micro-organisms (Lactobacillus casei Shirota and Bifidobacterium animalis subspp. lactis) in a milk-based dessert with cranberry sauce

The objective of this study was to evaluate the survival of two probiotic micro-organisms, Lactobacillus casei Shirota and Bifidobacterium animalis subspp . lactis , in a milk-based dessert (2.7% fat) with cranberry sauce added. B. lactis had a final concentration of 1.99 × 10⁶ cfu/g after 21 days of study, with a logarithmic decrease of 8.87%. On other hand, L. casei Shirota had a final concentration of 2.05 × 10⁷ cfu/g at the end of the same period, a logarithmic decrease of 8.41%. Statistical analysis showed that significant differences existed between both micro-organisms and over various storage times, the more viable micro-organism being L. casei Shirota , which decreased less than one logarithmic cycle after 21 days.     

Deletion of the N-terminal domain of the yeast vacuolar (Na+,K+)/H+ antiporter Vnx1p improves salt tolerance in yeast and transgenic Arabidopsis

Cation/proton antiporters play a major role in the control of cytosolic ion concentrations in prokaryotes and eukaryotes organisms. In yeast, we previously demonstrated that Vnx1p is a vacuolar monovalent cation/H⁺ exchanger showing Na⁺/H⁺ and K⁺/H⁺ antiporter activity. We have also shown that disruption of VNX1 results in an almost complete abolishment of vacuolar Na⁺/H⁺ exchange, but yeast cells overexpressing the complete protein do not show improved salinity tolerance. In this study, we have identified an autoinhibitory N-terminal domain and have engineered a constitutively activated version of Vnx1p, by removing this domain. Contrary to the wild type protein, the activated protein has a pronounced effect on yeast salt tolerance and vacuolar pH. Expression of this truncated VNX1 gene also improves Arabidopsis salt tolerance and increases Na⁺ and K⁺ accumulation of salt grown plants thus suggesting a biotechnological potential of activated Vnx1p to improve salt tolerance of crop plants.     

The DNA sequence analysis of the HAP4–LAP4 region on chromosome XI of Saccharomyces cerevisiae suggests the presence of a second aspartate aminotransferase gene in yeast

The nucleotide sequence of a 19 000 base pair region from the left arm of chromosome XI of Saccharomyces cerevisiae has been determined and analysed. It covers the HAP4–GFA1–LAP4 loci already described. As expected HAP4, GFA1 and LAP4 genes have been found and six new open reading frames (ORFs) with a coding capacity of more than 100 amino acid residues have been identified. One of them (YKL461) shows a high degree of identity with an aspartate aminotransferase gene. This raises the question of a second aspartate aminotransferase gene in yeast. A second ORF (YKL462) shows features compatible with a membranous localization. The other ORFs do not show a similarity with any known gene. A member of the highly repetitive ‘CAT’ DNA sequence is present.     

Identification and characterization of the type 2C protein phosphatase Ptc4p in the human fungal pathogen Candida albicans

Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ions. There are seven PP2C genes, named from PTC1 to PTC7, in Saccharomyces cerevisiae. In the current study we identified the CaPTC4 gene in Candida albicans and demonstrated that the CaPtc4p protein is a typical PP2C enzyme, which is highly conserved in fungal species. Deletion of CaPTC4 renders Candida cells sensitive to sodium and potassium ions as well as to antifungal azole drugs. In addition, we have shown that CaPtc4p is localized in the mitochondrion, suggesting that CaPtc4p is likely to be involved in the regulation of a mitochondrial function related to ion homeostasis. Copyright © 2009 John Wiley & Sons, Ltd.     

Cloning and characterization of WMSU1, a Williopsis saturnus var. mrakii gene encoding a new yeast SUN protein involved in the cell wall structure

SUN proteins of Saccharomyces cerevisiae have been defined on the basis of high homologies in their C-terminal domain. Recently, two of these four proteins were shown to be involved in cell wall morphogenesis (Mouassite et al., 2000a). In the present study, we have isolated WMSU1 (Accession No. AF418983), a new SUN-related gene, from W. saturnus var. mrakii MUCL 41968. Sequencing of the gene revealed an open reading frame coding for 402 amino acids. The predicted amino acid sequence of WMSU1 is closely related to the S. cerevisiae SUN proteins and to other yeast proteins involved in cell wall metabolism. WMSU1 is proposed to encode a cell wall protein since its predicted product contains a signal sequence, a Kex2p cleavage site and a serine/threonine-rich N-terminal domain. Southern blot analysis of the W. saturnus var. mrakii MUCL 41968 genome using the highly conserved domain of WMSU1 as a probe suggested that the isolated gene belongs to a multigenic family. Expression of WMSU1 in E. coli led to a 45 kDa protein, which appeared to be toxic to this host. Scanning electron microscopy analysis of a recombinant S. cerevisiae producing Wmsu1p showed that this strain exhibited an altered cell wall, thus pointing to a probable role of this protein in the cell wall structure.     

The Genes Encoding the Transcription Factor yTAFII60, the G4p1 Protein and a Putative Glucose Transporter are Contained in a 12·3 kb DNA Fragment on the Left Arm of Saccharomyces cerevisiae Chromosome VII

We report the nucleotide sequence of a DNA fragment of 12 325 base pairs from the left arm of the Saccharomyces cerevisiae chromosome VII. Inspection of the coding capacity revealed 11 open reading frames (ORFs) longer than 100 amino acids. Five ORFs are significantly homologous to known proteins. The region encoding ORF G2985 corresponds (100%) to the gene encoding the yeast TATA binding protein-associated factor TAF_II60. The G3075 ORF is 47·8% identical to the hypothetical yeast protein YB88. G3080 shows 36·7% identity to the eel calmodulin. G3085 shows 94·9% identity with the published sequence of the quadruplex DNA binding protein G4p1. G3090 reveals 46·7% identity with the probable glucose transport protein yBR1625. The DNA sequence has been submitted to the EMBL data library under Accession Number X97644. © 1997 by John Wiley & Sons, Ltd.