Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin.

Strains of Saccharomyces cerevisiae harboring M1-dsRNA, the determinant of type 1 killer and immunity phenotypes, secrete a dimeric 19-kd toxin that kills sensitive yeast cells by the production of cation-permeable pores in the cytoplasmic membrane. The preprotoxin, an intracellular precursor to toxin, has the domain sequence delta-alpha-gamma-beta where alpha and beta are the 9.5-and 9.0-kd subunits of secreted toxin. Plasmids containing a partial cDNA copy of M1, in which alpha, gamma, and beta are fused to the PH05 promoter and signal peptide, have previously been shown to express phosphate-repressible toxin production and immunity. Here the construction of a complete DNA copy of the preprotoxin gene and its mutagenesis are described. Analysis of the expression of these mutants from the PH05 promoter elucidates the functions of the preprotoxin domains. delta acts as a leader peptide and efficiently mediates the secretion, glycosylation and maturation of killer toxin. Mutations within the beta subunit indicate it to be essential for binding of toxin to and killing of whole cells but unnecessary for the killing of spheroplasts. Mutations within the putative active site of alpha prevent killing of both cells and spheroplasts. The probable role of beta is therefore recognition and binding to the cell wall receptor whereas alpha is the active ionophore. Mutations within alpha causing loss of toxicity also cause loss of immunity, while the mutants described within gamma and beta retain partial or complete immunity. Expression of gamma without alpha or beta confers no phenotype. The immunity determinant may minimally consist of the alpha domain and the N-terminal portion of gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Zygosaccharomyces bailii antifungal virus toxin zygocin: cloning and expression in a heterologous fungal host

Zygocin, a monomeric protein toxin secreted by a virus-infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double-stranded (ds)RNA killer virus (ZbV-M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N-terminal amino acid sequence was determined, and a full-length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238-amino-acid preprotoxin precursor (pptox) consisting of a hydrophobic N-terminal secretion signal, followed by a potentially N-glycosylated pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N-terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix-assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post-translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin-sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.     

Synthesis and processing of Semliki forest virus polyprotein in Saccharomyces cerevisiae: a yeast type glycosylation of E1 envelope protein

A cDNA coding for the structural proteins of Semliki Forest virus (SFV) was ligated between the ADC1 promoter and terminator in a yeast expression vector, pAAH5. Synthesis of the SFV-specific proteins in Saccharomyces cerevisiae transformed with this vector was shown by immunoblotting and immunoprecipitation. Detection of the N-terminal and the C-terminal components of the viral polyprotein, capsid protein and E1 envelope protein, respectively, suggested that the entire polyprotein was translated in yeast. The capsid protein was effectively released from the polyprotein as a normal size polypeptide, but the following protein, p62 (E3, E2 precursor) was not detected, suggesting that it was rapidly degraded. Electrophoretic analyses indicated that the final protein, E1, entered the secretory pathway, the signal sequence was cleaved off and the protein became extensively and heterogeneously glycosylated. These data suggest that E1 was transported to the Golgi complex and that yeast-characteristic outer-chain glycans were added to the protein.     

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.     

Expression of high levels of human tissue plasminogen activator in yeast under the control of an inducible GAL promoter

Summary The human tissue plasminogen activator (h-tPA) cDNA was fused either with the leader sequence of the killer toxin of Kluyveromyces lactis or with the Saccharomyces diastaticus glucoamylase leader peptide and cloned in the yeast expression vector under the control of the inducible UAS _gal/CYC1 promoter. The recombinant tPA is produced in yeast as a single-chain glycosylated polypeptide of 66–72 kDa, which accumulates intracellularly associated with a membrane fraction. Using two-step fed-batch fermentation, a productivity up to 100 mg/l of active intracellular tPA was obtained. Correspondence to: E. Martegani     

Cloning and sequencing of the preprotoxin-coding region of the yeast M1 double-stranded RNA.

Complementary DNA (cDNA) copies of the M1-1, toxin-coding region of the yeast M1 double-stranded RNA (dsRNA) have been cloned and sequenced. These sequences, in combination with the known terminal sequence of M1-1 dsRNA, identify a translation reading frame for a 316 amino acid protein of 34.7 kd, similar in size to the preprotoxin produced from M1 dsRNA by in vitro translation. Potential glycosylation sites in the preprotoxin peptide are identified. Based on its methionine content the extracellular yeast toxin appears to be contained within the C-terminal region of the precursor.     

Expression in yeast of a cDNA copy of the K2 killer toxin gene

Summary A cDNA copy of the M2 dsRNA encoding the K2 killer toxin ofSaccharomyces cerevisiae was expressed in yeast using the yeastADH1 promoter. This construct produced K2-specific killing and immunity functions. Efficient K2-specific killing was dependent on the action of the KEX2 endopeptidase and the KEX1 carboxypeptidase, while K2-specific immunity was independent of these proteases. Comparison of the K2 toxin sequence with that of the K1 toxin sequence shows that although they share a common processing pathway and are both encoded by cytoplasmic dsRNAs of similar basic structure, the two toxins are very different at the primary sequence level. Site-specific mutagenesis of the cDNA gene establishes that one of the two potential KEX2 cleavage sites is critical for toxin action but not for immunity. Immunity was reduced by an insertion of two amino acids in the hydrophobic amino-terminal region which left toxin activity intact, indicating an independence of toxin action and immunity.     

Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae.

A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated. It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated. The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon. The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was purified and subsequently characterized. It has slightly slower mobility on SDS/polyacrylamide-gel electrophoresis than standard pea vicilin and forms a mixture of multimers, some of which resemble the native protein.     

Determination of the carboxyl termini of the alpha and beta subunits of yeast K1 killer toxin. Requirement of a carboxypeptidase B-like activity for maturation

The carboxyl-terminal sequences of the two polypeptide chains of the Saccharomyces cerevisiae K1 killer toxin were determined by protein sequencing and amino acid analysis of peptide fragments generated from the mature, secreted toxin. The COOH-terminal amino acid of the beta chain is histidine 316, the final residue encoded by the precursor gene. The COOH terminus of the alpha chain is at alanine 147 of the preprotoxin. Amino acid composition data for the purified toxin are consistent with that predicted from the gene sequence of the preprotoxin where the alpha and beta subunits consist of amino acid residues 45-147 and 234-316, respectively. The molecular weight of the mature alpha beta dimer is about 20,658. The COOH-terminal sequence determination completes the location of the toxin subunits in the precursor, and its configuration may be represented as prepropeptide-Pro-Arg-alpha-Arg-Arg-gamma-Lys-Arg-beta, where gamma represents the interstitial glycosylated peptide. The COOH terminal side of the paired basic residues (Arg-148 Arg-149 and Lys-232 Arg-233 of preprotoxin) are endoproteolytic processing sites for the product of the KEX2 gene (Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984) Cell 37, 1075-1089), and thus maturation of the alpha subunit of killer toxin apparently requires a carboxypeptidase B-like activity. A possible candidate for this activity is the product of the KEX1 gene (Dmochowska, A., Dignard, D., Henning, D., Thomas, D.Y., and Bussey, H. (1987) Cell, in press).     

In vivo evidence for posttranslational translocation and signal cleavage of the killer preprotoxin of Saccharomyces cerevisiae.

A full-length cDNA of the M1 double-stranded RNA killer preprotoxin coding region successfully directed the synthesis of secreted K1 toxin when expressed in Saccharomyces cerevisiae from a plasmid vector. Three protein species immunoreactive with antitoxin antiserum were detected intracellularly in transformants harboring this killer cDNA plasmid. These toxin precursor species were characterized by using secretory-defective hosts, by comparative electrophoretic mobilities, and by tunicamycin susceptibility. Such studies indicate that these three protein species represent intermediates generated by signal cleavage of the preprotoxin and its subsequent glycosylation and provide evidence that these events occur posttranslationally.     

Expression, purification and characterization of recombinant murine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast

Expression and secretion of two lymphokines, murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and bovine interleukin-2 (BoIL-2), to levels of 50-60 mg per liter were achieved by placing these cDNAs in a Saccharomyces cerevisiae expression vector that utilized the yeast alcohol dehydrogenase-2 promoter and alpha-factor leader peptide. These lymphokines were purified to homogeneity by direct application of the crude yeast medium to reversed-phase high-performance liquid chromatography. Despite the fact that both lymphokines contain at least one N-glycosylation site and have identical N-terminal residues (Ala-Pro-Thr), recombinant (R) GM-CSF was found to be heterogeneously glycosylated by yeast while RBoIL-2 was secreted without glycosylation. Additionally, approximately 40% of the RGM-CSF was found to be proteolytically cleaved after the second amino acid residue, while RBoIL-2 was found to be intact.     

Yeast killer toxin: site-directed mutations implicate the precursor protein as the immunity component

Yeast killer toxin and a component giving immunity to it are both encoded by a gene specifying a single 35 kd precursor polypeptide. This precursor is composed of a leader peptide, the alpha and beta subunits of the secreted toxin, and a glycosylated gamma peptide separating the latter. The toxin subunits are proteolytically processed from the precursor during toxin secretion. Using site-directed mutagenesis, we have identified a region of the precursor gene necessary for expression of the immunity phenotype. This immunity-coding region extends through the C-terminal half of the alpha subunit into the N-terminal part of the gamma glycopeptide. Mutations in other parts of the gene allow full immunity but produce precursors that fail to be processed. The precursor can therefore confer immunity, and we propose that it does so in the wild type by competing with mature toxin for binding to a membrane receptor.     

Expression of Torpedo nicotinic acetylcholine receptor subunits in yeast is enhanced by use of yeast signal sequences

We have produced the four subunits of the nicotinic acetylcholine receptor of Torpedo californica, an integral membrane protein, in the yeast Saccharomyces cerevisiae. Two of the subunits (alpha and delta) were readily produced from their cDNAs after simply subcloning them into a yeast shuttle vector adjacent to a yeast promoter. The other two protein subunits (beta and gamma) were not produced by this strategy, although the amounts of mRNA produced from these expression constructs are similar to those for alpha and delta. Replacing the DNA coding for the normal N-terminal signal sequences for the beta and gamma subunits with DNA coding for the signal sequence of yeast invertase results in successful protein synthesis. The yeast signal sequence allows these subunits to be translocated across the membrane of the endoplasmic reticulum and to be glycosylated. The appropriate final size of the subunit proteins suggests that the yeast signal sequence has been properly cleaved after translocation.     

Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.     

Cloning of the cDNA and genomic clones for glutathione synthetase fromArabidopsis thaliana and complementation of agsh2 mutant in fission yeast

Glutathione is essential for protecting plants from a range of environmental stresses, including heavy metals where it acts as a precursor for the synthesis of phytochelatins. A 1658 bp cDNA clone for glutathione synthetase (gsh2) was isolated fromArabidopsis thaliana plants that were actively synthesizing glutathione upon exposure to cadmium. The sequence of the clone revealed a protein with an estimated molecular mass of 53858 Da that was very similar to the protein from higher eukaryotes, was less similar to the gene from the fission yeast,Schizosaccharomyces pombe, and shared only a small region of similarity with theEscherichia coli protein. A 4.3 kbSstI fragment containing the genomic clone for glutathione synthetase was also isolated and sequenced. A comparison of the cDNA and genomic sequences revealed that the gene was composed of twelve exons.When theArabidopsis cDNA cloned in a special shuttle vector was expressed in aS. pombe mutant deficient in glutathione synthetase activity, the plant cDNA was able to complement the yeast mutation. Glutathione synthetase activity was measurable in wild-type yeast cells, below detectable levels in thegsh2 ^- mutant, and restored to substantial levels by the expression of theArabidopsis cDNA. TheS. pombe mutant expressing the plant cDNA had near wild type levels of total cellular thiols,¹⁰⁹Cd²⁺ binding activity, and cadmium resistance. Since theArabidopsis cDNA was under control of a thiamine-repressible promoter, growth of the transformed yeast on thiamine-free medium increased expression of the cDNA resulting in increases in cadmium resistance.     

The conformation of mature human alpha-amylase conditions its secretion from yeast

The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary alpha-amylase (Amy) under control of the yeast PHO5 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.     

Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant

Sulfur plays an important role in plants, being used for the biosynthesis of amino acids, sulfolipids and secondary metabolites. After uptake sulfate is activated and subsequently reduced to sulfide or serves as donor for sulfurylation reactions. The first step in the activation of sulfate in all cases studied so far is catalyzed by the enzyme ATP-sulfurylase (E.C. 2.7.7.4.) which catalyzes the formation of adenosine-5′-phosphosulfate (APS). Two cDNA clones from potato encoding ATP-sulfurylases were identified following transformation of a Saccharomyces cerevisiae mutant deficient in ATP-sulfurylase activity with a cDNA library from potato source leaf poly(A)⁺ RNA cloned in a yeast expression vector. Several transformants were able to grow on a medium with sulfate as the only sulfur source, this ability being strictly linked to the presence of two classes of cDNAs. The clones StMet3-1 and StMet3-2 were further analyzed. DNA analysis revealed an open reading frame encoding a protein with a molecular mass of 48 kDa in the case of StMet3-1 and 52 kDa for StMet3-2. The deduced polypeptides are 88% identical at the amino acid level. The clone StMet3-2 has a 48 amino acid N-terminal extension which shows common features of a chloroplast transit peptide. Sequence comparison of the ATP-sulfurylase Met3 from Saccharomyces cerevisiae with the cDNA StMet3-1 (StMet3-2) reveals 31% (30%) identity at the amino acid level. Protein extracts from the yeast mutant transformed with the clone StMet3-1 displayed ATP-sulfurylase activity. RNA blot analysis demonstrated the expression of both genes in potato leaves, root and stem, but not in tubers. To the best of the authors' knowledge this is the first cloning and identification of genes involved in the reductive sulfate assimilation pathway from higher plants.     

The core region of human glutaminyl-tRNA synthetase homologies with the Escherichia coli and yeast enzymes.

We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases. A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E. coli and yeast glutaminyl (Gln)-tRNA synthetase. The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase. Thus, the human enzyme is about three times larger than the E. coli and two times larger than the yeast Gln-tRNA synthetase. The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core.