A putative serine/threonine protein kinase gene on chromosome III of Saccharomyces cerevisiae

We have sequenced a gene on chromosome III of Saccharomyces cerevisiae which codes for a putative serine/threonine protein kinase of 726 amino acids (calculated molecular weight 82 kDa). We have called this gene KIN82. The amino acid sequence of KIN82 is most similar to the cyclic nucleotide-dependent protein kinase subfamily and the protein kinase C subfamily. Gene disruption of KIN82 did not produce any phenotype when tested under a variety of conditons. Reduced stringency hybridizations revealed the presence of another genomic sequence with high homology to the carboxy-terminal catalytic domain of KIN82.     

Characterization of the KIN2 gene product in Saccharomyces cerevisiae and comparison between the kinase activities of p145KIN1 and p145KIN2

We have isolated two yeast genes, KIN1 and KIN2, by their homology to the protein kinase family of viral oncogenes. Previous studies have identified the yeast KIN1 gene product (pp145^KIN1) as a 145 kilodalton (kDa) phosphoprotein with serine/threonine-specific protein kinase activity. To identify and biochemically characterize the KIN2 gene product, antibodies were raised against a bacterial β-galactosidase/KIN2 fusion polypeptide. In vivo, the KIN2 gene product is a 145 kDa phosphoprotein, pp145^KIN2. In immune complexes, pp145^KIN2 demonstrates serine/threonine protein kinase activity, transferring phosphate from [γ-³²P]ATP to either itself or the exogenously added substrates α-casein, acid-denatured enolase, or phosvitin. In vitro, kinase activity is dependent on either Mn²⁺ or Mg²⁺ ions. Both enzymes, pp145^KIN1 and pp145^KIN2, prefer ATP over GTP as their phosphoryl donor. Since a new class of yeast protein kinases has been identified which are serine/tyrosine-specific, we analysed a wide range of substrates to see if any could be phosphorylated by pp145^KIN1 or pp145^KIN2 on tyrosine residues. Both enzymes phosphorylate α-casein, acid-denatured enolase, and phosvitin on serine and threonine residues. Neither enzyme could phosphorylate tyrosine residues even though good substrates for tyrosine-specific kinases such as enolase, angiotensin II, and the synthetic polymer GLU⁸⁰TYR²⁰ were used. The biochemical analysis of KIN2 kinase activity shows remarkable similarity to that of its most closely related yeast kinase, KIN1. It remains to be seen if these two yeast protein kinases share any functional relationships or substrates in vivo.     

Sequence and analysis of a 26·9 kb fragment from chromosome XV of the yeast Saccharomyces cerevisiae

We have determined the nucleotide sequence of a fragment of chromosome XV of Saccharomyces cerevisiae cloned into cosmid pEOA048. The analysis of the 26 857 bp sequence reveals the presence of 19 open reading frames (ORFs), and of one RNA-coding gene (SNR17A). Six ORFs correspond to previously known genes (MKK1/SSP32, YGE1/GRPE/MGE1, KIN4/KIN31/KIN3, RPL37B, DFR1 and HES1, respectively), all others were discovered in this work. Only five of the new ORFs have significant homologs in public databases, the remaining eight correspond to orphans (two of them are questionable). O5248 is a probable folylpolyglutamate synthetase, having two structural homologs already sequenced in the yeast genome. O5273 shows homology with a yeast protein required for vanadate resistance. O5268 shows homology with putative oxidoreductases of different organisms. O5257 shows homology with the SAS2 protein and another hypothetical protein from yeast. The last one, O5245, shows homology with a putative protein of Caenorhabditis elegans of unknown function. The present sequence corresponds to coordinates 772 331 to 799 187 of the entire chromosome XV sequence which can be retrieved by anonymous ftp (ftp. mips. embnet. org).     

DNA sequence analysis of a 17 kb fragment of yeast chromosome XI physically localizes the MRB1 gene and reveals eight new open reading frames,including a homologue of the KIN1/KIN2 and SNF1 protein kinases

We report in this paper the sequence of a part of chromosome XI of Saccharomyces cerevisiae. This 17 kbp nucleotide sequence represents the right half of cosmid pUKG151 and contains nine open reading frames, YKL453, 450, 449, 448, 445, 443, 442, 441 and the 5′ part of YKL440. YKL440 was previously identified as the MBR1 gene and plays a role in mitochondrial biogenesis. YKL443 is a homologue of the yeast serine-rich protein (SRP1), while YKL453 presents strong homologies with the KIN1/KIN2/SNF1 kinase family. It must be pointed out that the size of this gene is well above average for yeast.     

The product of the KIN1 locus in Saccharomyces cerevisiae is a serine/threonine-specific protein kinase.

The catalytic domain (30 kDa) of all protein kinases can be aligned for maximum homology, thereby revealing both invariant and highly conserved residues. The KIN1 locus from Saccharomyces cerevisiae was isolated by hybridization to a degenerate oligonucleotide encoding the conserved protein kinase domain, DVWSFG. The predicted amino acid sequence revealed significant homology to the catalytic domain of protein kinases. Using antibodies raised against a bacterial LacZ/KIN1 fusion protein, we have identified by immunoprecipitation the yeast KIN1 gene product as a 145,000 dalton protein (p145KIN1). In exponentially growing yeast cells, the KIN1 protein is phosphorylated primarily on serine residues. The gene product of KIN1 was shown to be a serine/threonine-specific protein kinase in immune complexes, as determined by the transfer of label from [gamma-32P]ATP to either pp145KIN1 or to an exogenously added substrate, alpha-casein. The optimal metal ion concentration in this assay was 20 mM-MnCl2. Subsequent phosphoamino acid analysis of the radiolabelled product, pp145KIN1, demonstrated that this autophosphorylation was specific for serine/threonine residues. There is no apparent difference between wild-type cells and cells containing a disrupted KIN1 gene. The biochemical characterization of protein kinases in simple eukaryotes such as yeast will aid us in determining the role of phosphorylation in cellular growth and physiology.     

IV. XV. Yeast mapping reports. RPL44 and RPL44′, encoding acidic ribosomal phosphoproteins YP2α(l44) and YP1β(l44′), are adjacent to rig and STF1 on Saccharomyces cerevisiae chromosomes XV and IV respectively

The RPL44′ gene from Saccharomyces cerevisiae encoding the ribosomal protein YP1β(L44′) has been found to be linked to the STF1 gene, encoding a stabilizing factor of the F₁F₀-ATPase inhibitor protein from mitochondria. Evidence of this linkage comes from results obtained from Northern hybridization using a DNA probe that contains a complementary region to the 5′ end of the mRNA of RPL44′. Similarly, a data bank search has shown that RPL44, encoding ribosomal protein YP2α(L44) is linked to the rig gene that encodes ribosomal protein S21.     

IX. Yeast sequencing reports. Cloning and sequence of REV7, a gene whose function is required for DNA damage-induced mutagenesis in Saccharomyces cerevisiae

The function of the REV7 gene is required for DNA damage-induced mutagenesis in budding yeast, Saccharomyces cerevisiae, and is therefore thought to promote replication past sites of mutagen damage in the DNA template. We have cloned this gene by complementation of the rev7-2 mutant defect, and determined its sequence. REV7 encodes a predicted protein of M_r 28 759 which is unlike any other protein in the NCBI non-redundant protein sequence data base, and which is inessential for viability. The sequence of the 3·88 kb yeast genomic fragment containing REV7 has been deposited in Genbank accession number U07228.     

Identification of peroxisomal membrane ghosts with an epitope-tagged integral membrane protein in yeast mutants lacking peroxisomes

Many yeast peroxisome biogenesis mutants have been isolate in which peroxisomes appear to be completely absent. Introduction of a wild-type copy of the defective gene causes the reappearance of peroxisomes, despite the fact that new peroxisomes are thought to form only from pre-existing peroxisomes. This apparent paradox has been explained for similar human mutant cell lines (from patients with Zellweger syndrome) by the discovery of peroxisomal membrane ghosts in the mutant cells (Santos, M. J., T. Imanaka, H. Shio, G. M. Small and P. B. Lazarow. 1988. Science 239 , 1536–1538). Introduction of a wild-type gene is thought to restore to the ghosts the ability to import matrix proteins, and thus lead to the refilling of the peroxisomes. It is vitally important to our understanding of peroxisome biogenesis to determine whether the yeast mutants contain ghosts. We have solved this problem by introducing an epitope-tagged version of Pas3p, a peroxisome integral membrane protein (that is essential for peroxisome biogenesis). Nucleotides encoding a nine amino acid HA epitope were added to the PAS3 gene immediately before the stop codon. The tagged gene (PAS3_HA) was inserted in the genome, replacing the wild-type gene at its normal locus. It was fully functional (the cells assembled peroxisomes normally and grew on oleic acid) but the expression level was too low to detect the protein with monoclonal antibody 12CA5. PAS3_HA was expressed in greater quantity from an episomal plasmid with the CUP1 promoter. The gene product, Pas3p_HA, was detected by immunogold labelling on the membranes of individual and clustered peroxisomes; the clusters appeared as large spots in immunofluorescence. PAS3_HA was similarly expressed in peroxisome biogenesis mutants peb2 and peb4, which lack morphologically recognizable peroxisomes. Gold-labelled membranes were clearly visible in both mutants: in peb2 the labelled membrane vesicles were generally much smaller than those in peb4, which resembled normal peroxisomes in size.     

Cloning and characterization of the Pichia pastoris PRC1 gene encoding carboxypeptidase Y

We purified a 58 kDa serine protease from culture-supernatant of Pichia pastoris and found that the NH₂-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharomyces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRC1 gene. Using the S. cerevisiae PRC1 gene as a hybridization probe, a cross-hybridizing fragment of P. pastoris genomic DNA was identified and the gene, PRC1, encoding CPY, was cloned. The open reading frame of the P. pastoris PRC1 gene consists of 1569 bp encoding a protein of 523 amino acids. The molecular mass of the protein is calculated to be 59·44 kDa without sugar chains. The protein comprises 20 amino acids of pre (signal)-peptide, 87 amino acids of pro-peptide and 416 amino acids of mature peptide, and has four N-glycosylation sites. The NH₂-terminal amino acid sequence of mature peptide is completely identical with that of the protease purified from the culture-supernatant. There is 61% identity between the amino acid sequences of P. pastoris Prc1p and S. cerevisiae Prc1p. Chromosomal disruption of the PRC1 gene resulted in the loss of CPY activity. Over-expression of the PRC1 gene under regulation of the P. pastoris AOX1 promoter resulted in accumulation of a large amount of active CPY in the intracellular fraction, and secretion of a slightly larger molecule that is thought to be pro-CPY. The nucleotide sequence data reported in this paper will appear in the EMBL Nucleotide Sequence Databases under the Accession Number X87987.     

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end‐products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast‐based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1·0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA‐1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA‐1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA‐1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1_P‐MFα1_S‐pedA‐ADH1_T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1_P) and terminator (ADH1_T). Secretion of the PED1‐encoded pediocin PA‐1 was directed by the yeast mating pheromone α‐factor's secretion signal (MFα1_S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4·6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria. Copyright © 1999 John Wiley & Sons, Ltd.     

The VPH2 gene encodes a 25 kDa protein required for activity of the yeast vacuolar H+-ATPase

Strains bearing the vph2 mutation are defective in vacuolar acidification. The VPH2 gene was isolated from a genomic DNA library by complementation of the zinc-sensitive phenotype of the mutant. Deletion analysis localized the complementing activity to a 1·2 kb DNA fragment. Sequence analysis of this fragment revealed the presence of a single open reading frame that encoded a protein of 215 amino acids. Computer analysis indicated that the protein, which has a predicted molecular mass of 25 286 Daltons, has two distinct membrane-spanning domains. Biochemical studies indicated that strains bearing the vph2 mutation have greatly reduced levels of vacuolar proton pumping and ATPase activity and that the nucleotide binding subunits of the multimeric vacuolar H⁺-ATPase failed to be correctly targeted to the vacuolar membrane. The vph2 mutant fails to grow on YEP glycerol medium and on media containing 100 mM -CaCl₂ or 4 mM -ZnCl₂ or buffered to pH 7·5, a phenotype observed in strains carrying deletions in the genes encoding several vacuolar H⁺-ATPase subunits. The VPH2 gene is identical to the VMA12 gene (T. Stevens and Y. Anraku, personal communication).     

Extracellular Expression of a Recombinant Ganoderma lucidium Immunomodulatory Protein by Food-Grade Lactococcus lactis System

Lactococcus lactis is a food-grade microorganism of major commercial importance in food industry. For commercial application of genetically modified L. lactis, a food-grade expression system is strongly recommended. In this study, two food-grade selection markers, nisin immunity gene nisI and nisin resistance gene nsr, were evaluated as dominant markers for the L. lactis food-grade expression system. By using an efficient P_SlpAl promoter fused to a signal peptide from subtilisin YaB (SP_YAB), a functional recombinant Ganoderma lucidium immunomodulatory protein rLZ8 was expressed extracellularly in L. lactis. Replacing the antibiotic marker gene into the proper food-grade selection marker nsr gene, the rLZ8 was expressed extracellularly in the food-grade L. lactis system. This study provides a rationale basis for a food-grade system to express functional peptides extracellularly as an important tool for oral administration of genetically modified L. lactis.     

Nucleotide sequence and characterization of the Saccharomyces cerevisiae RPL19A gene encoding a homolog of the mammalian ribosomal protein l19

A gene designated RPL19A has been identified in the region downstream from the 3′-end of the Saccharomyces cerevisiae MIS1 gene encoding the mitochondrial C₁-tetrahydrofolate synthase. The gene codes for the yeast ribosomal protein YL19 which exhibits 57·5% identity with the mammalian ribosomal protein L19. RPL19A is one of two functional copies of the YL19 gene located on chromosome II. The disruption of RPL19A has no effect on the growth of the yeast. The RPL19A gene contains an intron located near the 5′-end. The 5′-flanking region contains one similar and one complete UAS_rpg upstream activating sequence. RPL19A was also found to be adjacent to the chromosome II AAC3 gene, encoding the mitochondrial ADP/ATP carrier protein. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^tm/EMBL data bank with the accession number Z36751.     

Expression of yeast homolog of the mammal BCRP gene coding for riboflavin efflux protein activates vitamin B2 production in the flavinogenic yeast Candida famata

Candida famata is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B₂) in response to iron limitation. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The corresponding protein and gene have not been identified in yeasts. At the same time, the corresponding gene BCRP has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in Debaryomyces hansenii. The closest homolog was expressed under the control of D. hansenii TEF1 promoter in the riboflavin overproducing strain of C. famata. Resulted transformants overexpressed the corresponding gene and produced 1.4- to 1.8-fold more riboflavin as compared with the parental strain. They also were characterized by overexpression of RIB1 and RIB6 genes of riboflavin synthesis and exhibited elevated specific activity of GTP-cyclohydrolase II. Membrane localization of the riboflavin excretase was confirmed by fluorescent microscopy.     

Sequence,map position and genome organization of the RPL17B gene,encoding ribosomal protein L17b in Saccharomyces cerevisiae

Sequence analysis of the newly defined SSU81 gene revealed an adjacent open reading frame (ORF) encoding a protein whose deduced amino acid sequence is identical to that of ribosomal protein L17. The DNA sequence of this region is different from that of the RPL17A gene and therefore represents a duplicate gene encoding L17. We have designated this gene RPL17B. The RPL17B coding region is split by an intron that occurs in the same position (codons 14/15) as the intron in RPL17A. The RPL17B promoter region includes two TATA boxes, a canonical UAS_RPG motif, and several pyrimidine-rich tracts. RPL17B was mapped by CHEF and lambda clone grid hybridization blots to the right arm of chromosome V, linked to the TRP2 and RAD51 genes. A partial ORF was identified adjacent to RPL17B and SSU81 that is homologous to an ORF (designated A509) physically linked to RPL17A. This observation, and the identical position of the introns within the RPL17 genes, suggest that one RPL17 locus arose by duplication and translocation of the other. The complete 3·8 kbp DNA sequence encompassing RPL17B has been entered in the GenBank data library under Accession Number U15653.     

The nucleotide sequence and initial characterization of pyruvate decarboxylase from the yeast Hanseniaspora uvarum

We have isolated a pyruvate decarboxylase (PDC) gene from the yeast Hanseniaspora uvarum using the Saccharomyces cerevisiae PDC1 gene as a probe. The nucleotide sequence of this gene was determined and compared to PDC genes from yeast and other organisms. The H. uvarum PDC gene is more than 70% identical to the S. cerevisiae PDC isozymes and possesses a putative thiamine diphosphate binding site. The PDC enzyme was purified and partially characterized. The H. uvarum PDC was very similar to other known PDCs; the K_m for pyruvate was 0·75 mM, and the enzyme is a homotetramer with subunits of M_r = 57 000. The sequence has been submitted to GenBank under Accession No. U13635.     

The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source

Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full‐length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l ‐cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l ‐cystine and some other cysteine conjugates, but not l ‐cystathionine or l ‐methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l ‐cysteine as a nitrogen source, and that overexpression of the gene results in increased H₂S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S‐ethyl‐l ‐cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

Purification and molecular characterization of a quinoprotein alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes IFO 14464

We have cloned and verified a gene for a novel quinoprotein alcohol dehydrogenase (ADH) from Pseudogluconobacter saccharoketogenes IFO 14464 that has the ability to oxidize -sorbose to 2-keto- -gulonic acid (2-KLGA). The enzyme was purified from the soluble fraction of the bacterium and was estimated to be a monomeric protein with a molecular weight of 65 kDa from the analyses of SDS-PAGE and gel-filtration chromatography. An open reading frame of 1824 bp for 608 amino acid residues was estimated as the gene for ADH because of the consistency of the calculated molecular mass and the elucidated partial amino acid sequences of the native enzyme. Homology search revealed that the enzyme showed close similarity to quinoprotein alcohol dehydrogenases isolated from Methylobacterium extorquens and Acetobacter aceti, particularly in the tryptophan docking motifs in the α-subunits of those dehydrogenases. The ability to convert -sorbose to 2-KLGA was found when the lysate of recombinant Escherichia coli DH10B transformed with the gene for ADH was mixed with CaCl₂ and pyrroloquinoline quinone (PQQ). These data indicate that the cloned DNA is the desired gene for the ADH in which CaCl₂ and PQQ are essential for enzymatic activity.