A novel yeast secretion signal isolated from 28K killer precursor protein encoded on the linear DNA plasmid pGKL1.

Saccharomyces cerevisiae harboring linear dsDNA plasmids, pGKL1 and pGKL2, secretes a killer toxin consisting of 97, 31 and 28 kilodalton subunits (Nucleic Acids Res., 15, 1031-1046, 1987). We isolated the DNA encoding the N-terminal pre-sequence of the 28K precursor protein and constructed a new secretion vector in S. cerevisiae. Mouse alpha-amylase fused to the 28K signal sequence was secreted into the culture medium with a high efficiency similar to those fused to the mating factor alpha and 97K-31K killer signal sequences. This data clearly indicates that 28K presequence functions as a secretion signal. Glycosylated and nonglycosylated alpha-amylase molecules were detected in the culture medium. The secretion of alpha-amylase was blocked by sec18-1 mutation. The secreted alpha-amylase recovered from the medium was found to migrate faster in SDS-polyacrylamide gel than the precursor form of alpha-amylase synthesized in vitro. These lines of evidence suggest that mouse alpha-amylase fused to 28K killer signal sequence was processed, glycosylated and secreted through the normal secretion pathway of the yeast.     

Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae

By the kar1-mediated cytoduction, linear double-stranded DNA plasmids pGKL1 and pGKL2, encoding killer toxin complex, have been successfully transferred to the recipient strains with about 30% frequency. The killer toxin was found to be secreted through the normal yeast secretory pathway by introducing pGKL plasmids into the several Saccharomyces cerevisiae sec mutants and examining the secretion of killer toxin. S. cerevisiae cells, harboring newly isolated deletion plasmid pGKL1D, expressed only the 28K protein among three killer subunits, and secreted the 28K subunit at a level of zero to 20% efficiency of the cells containing intact pGKL1 plasmid. These data indicated that subunit interaction (cosecretion) of killer proteins is required for the efficient secretion of 28K subunit. The 28K precursor protein was found to translocate across the canine pancreatic endoplasmic reticulum membrane under the direction of its own signal peptide in vitro without any other subunits. From kex2 mutant cells harboring pGKL1 plasmid, the 97K subunit, and its precursor 128K protein were not secreted, however, the 28K subunit was secreted in the same amount as that secreted from KEX2 cells. These lines of evidence suggest that the final assembly of killer toxin complex after KEX2 site of Golgi apparatus is not essential for the secretion of 28K subunit, and therefore, that putative interaction between 128K protein and 28K subunit for the transport between endoplasmic reticulum and Golgi apparatus may be required for the efficient secretion of 28K subunit.     

A novel yeast secretion vector utilizing secretion signal of killer toxin encoded on the yeast linear DNA plasmid pGKL1

The NH2-terminal signal region comprising of approximately 70% length of the prepro-sequence of the pGKL killer precursor protein was found to direct an efficient secretion of the mouse alpha-amylase into the culture medium of Saccharomyces cerevisiae. The alpha-amylase molecule secreted into the culture medium was identified by both immuno-blotting and assay of the enzyme activity. The amount of alpha-amylase secreted via the killer toxin signal was comparable to that directed by the leader sequence of mating factor alpha. The secretion of alpha-amylase using the killer toxin signal was blocked at 37C but not at 25C in sec18-1 host, indicating that alpha-amylase is exported through the normal secretion pathway of S. cerevisiae.     

Cloning and nucleotide sequences of the linear DNA killer plasmids from yeast.

The linear DNA killer plasmids (pGKL1 and pGKL2) isolated from a Kluyveromyces lactis killer strain are also maintained and expressed its killer character in Saccharomyces cerevisiae. After these killer plasmid DNAs isolated from S. cerevisiae were treated with alkali, four terminal fragments from each plasmid DNAs were cloned separately. Using these and other cloned DNA fragments, the terminal nucleotide sequences of pGKL2 and the complete nucleotide sequence of pGKL1 were determined. The inverted terminal repetitions of 202 bp and 182 bp were found in pGKL1 and pGKL2, respectively. The pGKL1 sequence showed an extremely high A + T content of 73.2% and it contained five large open reading frames. The largest of these open reading frame was suggested to code for a membrane-bound precursor of glycoprotein subunit of the killer toxin.     

Structure of yeast pGKL 128-kDa killer-toxin secretion signal sequence. Processing of the 128-kDa killer-toxin-secretion-signal-alpha-amylase fusion protein.

The linear double-stranded DNA plasmid pGKL1 in yeast encodes a killer toxin consisting of 97-kDa, 31-kDa and 28-kDa subunits. A 128-kDa protein precursor of the 97-kDa and 31-kDa subunits, was first synthesized with a 29-amino-acid extension at its NH2-terminus as a secretion signal sequence. In the present study, the property of this signal sequence was studied by the analysis of a fusion protein with mouse alpha-amylase. Using the secretion signal sequence of the killer protein, the mouse alpha-amylase was successfully secreted into the culture medium. An intracellular precursor form of alpha-amylase was identified and purified. Analysis of the NH2-terminal sequence of this precursor molecule indicated that it corresponded to the secretory intermediate (pro form) of alpha-amylase with the removal of the hydrophobic segment (Met1-Gly16) of the secretion signal. Both the secretion of alpha-amylase into the culture medium and the detection of the pro-alpha-amylase species in the cells were prohibited by a sec 11 mutation, or by the conversion of Gly to Val at the 16th position of the secretion signal. These results strongly suggest that the cleavage occurs between Gly16 and Leu17 by a signal peptidase, and that this cleavage is required for the secretion of alpha-amylase into the medium. Based on the data from the NH2-terminal amino acid sequences of secreted alpha-amylases, we conclude that the 29-amino-acid secretion signal present in the 128-kDa killer toxin precursor protein is a prepro structure.     

Novel yeast killer toxins provoke S-phase arrest and DNA damage checkpoint activation

Certain strains of Pichia acaciae and Wingea robertsiae (synonym Debaryomyces robertsiae) harbour extranuclear genetic elements that confer a killer phenotype to their host. Such killer plasmids (pPac1-2 of P. acaciae and pWR1A of W. robertsiae) were sequenced and compared with the zymocin encoding pGKL1 of Kluyveromyces lactis. Both new elements were found to be closely related to each other, but they are only partly similar to pGKL1. As for the latter, they encode functions mediating binding of the toxin to the target cell's chitin and a hydrophobic region potentially involved in uptake of a toxin subunit by target cells. Consistently, mutations affecting the target cell's major chitin synthase (Chs3) protect it from toxin action. Heterologous intracellular expression of respective open reading frames identified cell cycle-arresting toxin subunits deviating structurally from the likewise imported gamma-subunit of the K. lactis zymocin. Accordingly, toxicity of both P. acaciae and Wingea toxins was shown to be independent of RNA polymerase II Elongator, which is indispensable for zymocin action. Thus, P. acaciae and Wingea toxins differ in their mode of action from the G1-arresting zymocin. Fluorescence-activated cell sorting analysis and determination of budding indices have proved that such novel toxins mediate cell cycle arrest post-G1 during the S phase. Concomitantly, the DNA damage checkpoint kinase Rad53 is phosphorylated. As a mutant carrying the checkpoint-deficient allele rad53-11 displays toxin hypersensitivity, damage checkpoint activation apparently contributes to coping with toxin stress, rather than being functionally implemented in toxin action.     

Isolation, purification, and characterization of a killer protein from Schwanniomyces occidentalis

The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. The protein was stable between pH 2.0 and 5.0 and inactivated at temperatures above 40 degrees C. The killer protein was chromosomally encoded. Mannan, but not beta-glucan or laminarin, prevented sensitive yeast cells from being killed by the killer protein, suggesting that mannan may bind to the killer protein. Identification and characterization of a killer strain of S. occidentalis may help reduce the risk of contamination by undesirable yeast strains during commercial fermentations.     

Intergeneric transfer of deoxyribonucleic acid killer plasmids, pGKl1 and pGKl2, from Kluyveromyces lactis into Saccharomyces cerevisiae by cell fusion

Two novel linear deoxyribonucleic acid plasmids, pGKl1 and pGKl2, were isolated from the yeast Kluyveromyces lactis. K. lactis strains harboring the pGK1 plasmids killed a certain group of yeasts, including Saccharomyces cerevisiae, Saccharomyces italicus, Saccharomyces rouxii, K. lactis, Kluyveromyces thermotolerans, Kluyvermyces vanudenii, Torulopsis glabrata, Candida utilis, and Candida intermedia. In this experiment, the pGKl1 and pGKl2 plasmids were intergenerically transferred from a K. lactis killer strain into a non-killer (killer-sensitive) strain of S. cerevisiae by the use of a protoplast fusion technique. Both of the pGKl plasmids replicated autonomously and stably in the new host cells of S. cerevisiae and could coexist with the resident 2-micrometers deoxyribonucleic acid plasmid. The S. cerevisiae cells which accepted the pGKl plasmids expressed the same killer phenotype as that of the donor K. lactis killer and became resistant to the K. lactis killer. The pGKl plasmids existing in the S. cerevisiae cells were cured by treatment with ethidium bromide, and the killer and resistance characters were simultaneously lost. From there results, it was concluded that both the killer and the resistance genes are located on the pGKl plasmids.     

Transmission of killer activity into laboratory and industrial strains of Saccharomyces cerevisiae by electroinjection

The killer character was electrically introduced into protoplasts of three yeast strains. These were the killer-negative variant of the K1 killer strain Saccharomyces cerevisiae T 158 C (his-); the killer-sensitive laboratory strain S. cerevisiae AH 215 (leu-, his-); and the killer-sensitive industrial strain S. cerevisiae AS 4/H2 (rho-). The killer dsRNA used for electroinjection was isolated from the super-killer strain S. cerevisiae T 158 C. Optimum numbers of transformed cells were obtained after regeneration and selection in appropriate media if the protoplasts were exposed to three exponentially decaying field pulses of 18.2 kV/cm strength and 40 microseconds duration at 4 degrees C. In the case of the killer-negative variant of S. cerevisiae T 158 C the majority of the protoplasts were transformed, whereas in the case of the two other strains the yield of transformed clones was much less. This latter result is expected if the expression of the electroinjected dsRNA was diminished in these two strains. Gel electrophoresis of the dsRNA of the clones of the three strains supported the conclusion that the transformed clones exhibited killer activity. The transformed clones of all three species were stable.     

Incompatibility of linear DNA killer plasmids pGKL1 and pGKL2 from Kluyveromyces lactis with mitochondrial DNA from Saccharomyces cerevisiae.

Two linear killer plasmids (pGKL1 and pGKL2) from Kluyveromyces lactis stably replicated and expressed the killer phenotype in a neutral petite mutant [( rho0]) of Saccharomyces cerevisiae. However, when cytoplasmic components were introduced by cytoduction from a wild-type [( rho+]) strain of S. cerevisiae, the linear plasmids became unstable and were frequently lost from the cytoductant cells during mitosis, giving rise to nonkiller clones. The phenomenon was ascribed to the incompatibility with the introduced S. cerevisiae mitochondrial DNA (mtDNA), because the plasmid stability was restored by [rho0] mutations in the cytoductant cells. Incompatibility with mtDNA was also apparent for the transmission of plasmids into diploid progeny in crosses between killer cells carrying the pGKL plasmids and [rho+] nonkiller cells lacking the plasmids. High-frequency transmission of the plasmids was observed in crosses lacking mtDNA [( rho0] by [rho0] crosses) and in crosses involving mutated mtDNA with large deletions of various regions of mitochondrial genome. In contrast, mutated mtDNA from various mit- mutations also exerted the incompatibility effect on the transmission of plasmids. Double-stranded RNA killer plasmids were stably maintained and transmitted in the presence of wild-type mtDNA and stably coexisted with pGKL killer plasmids in [rho0] cells of S. cerevisiae.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Susceptibility of individual cells of Saccharomyces cerevisiae to the killer toxin K1

The susceptibility of sensitive yeast to killer toxins is known to depend on various factors, such as the selected killer toxin, the exposed yeast strain, its growth phase and the state of culture under given experimental conditions. The aim of this paper was to find whether individual cells from one culture are equally susceptible to the impact of the killer toxin. For this purpose the rhodamine B assay in a modified form was used. In order to observe the fate of individual cell the method of fluorescence video microscopy with a digital picture analysis was applied. Four selected groups of specific cells (with no, small, medium, and large bud, respectively) were investigated. Different sensitivity of Saccharomyces cerevisiae cells to the killer toxin K1 was observed in these cell groups. The most susceptible appeared to be the cells which were in S-phase (cells with the small buds); the least susceptible were the M-phase cells with large buds. The enhanced susceptibility in S-phase results probably from coincidence in higher porosity of the cell wall, accumulation of surface receptors, and enlarged growth activity at the surface cell structures.     

The ecological role of killer yeasts in natural communities of yeasts

The killer phenomenon of yeasts was investigated in naturally occurring yeast communities. Yeast species from communities associated with the decaying stems and fruits of cactus and the slime fluxes of trees were studied for production of killer toxins and sensitivity to killer toxins produced by other yeasts. Yeasts found in decaying fruits showed the highest incidence of killing activity (30/112), while yeasts isolated from cactus necroses and tree fluxes showed lower activity (70/699 and 11/140, respectively). Cross-reaction studies indicated that few killer-sensitive interactions occur within the same habitat at a particular time and locality, but that killer-sensitive reactions occur more frequently among yeasts from different localities and habitats. The conditions that should be optimal for killer activity were found in fruits and young rots of Opuntia cladodes where the pH is low. The fruit habitat appears to favor the establishment of killer species. Killer toxin may affect the natural distribution of the killer yeast Pichia kluyveri and the sensitive yeast Cryptococcus cereanus. Their distributions indicate that the toxin produced by P. kluyveri limits the occurrence of Cr. cereanus in fruit and Opuntia pads. In general most communities have only one killer species. Sensitive strains are more widespread than killer strains and few species appear to be immune to all toxins. Genetic study of the killer yeast P. kluyveri indicates that the mode of inheritance of killer toxin production is nuclear and not cytoplasmic as is found in Saccharomyces cerevisiae and Kluyveromyces lactis.     

Molecular structure of the cell wall receptor for killer toxin KT28 in Saccharomyces cerevisiae.

The adsorption of the yeast killer toxin KT28 to susceptible cells of Saccharomyces cerevisiae was prevented by concanavalin A, which blocks the mannoprotein receptor. Certain mannoprotein mutants of S. cerevisiae that lack definite structures in the mannan of their cell walls were found to be resistant to KT28, whereas the wild-type yeast from which the mutants were derived was susceptible. Isolated mannoprotein from a resistant mutant was unable to adsorb killer toxin. By comparing the resistances of different mannoprotein mutants, information about the molecular structure of the receptor was obtained. At least two mannose residues have to be present in the side chains of the outer chain of the cell wall mannan, whereas the phosphodiester-linked mannose group is not essential for binding and the subsequent action of killer toxin KT28.     

Enzymic activity of the K5-type yeast killer toxin and its characterization

K5-type yeast killer toxin secreted by P. anomala NCYC 434 cells has a broad killing spectrum. Competitive inhibiton of killer activity showed that glucans, mainly the beta-1,3 glucan, represent the primary toxin binding site within the cell wall of sensitive cells. Its hydrolytic activity on laminarin in an exo-like fashion revealed that the toxin exerts its killing effect by exo-beta-1,3-glucanase activity. Its specific activity on laminarin was 120 U/mg, and the Michaelis constants K(m) and V(max) for laminarin hydrolysis were 0.25 mg/ml and 370 micromol/min/mg. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Production of the toxin by the cells was induced only when they were grown in culture media rich in beta-glucan sources, and the addition of glucose increased the specific production rate. The enzymic activity of the toxin was fully inhibited by Hg(+2), but increased with some other metal ions, most of all by Pb(+2).     

Immunity to K1 killer toxin: internal TOK1 blockade

K1 killer strains of Saccharomyces cerevisiae harbor RNA viruses that mediate secretion of K1, a protein toxin that kills virus-free cells. Recently, external K1 toxin was shown to directly activate TOK1 channels in the plasma membranes of sensitive yeast cells, leading to excess potassium flux and cell death. Here, a mechanism by which killer cells resist their own toxin is shown: internal toxin inhibits TOK1 channels and suppresses activation by external toxin.     

Mannoprotein of the yeast cell wall as primary receptor for the killer toxin of Saccharomyces cerevisiae strain 28

The killer toxin KT 28 of Saccharomyces cerevisiae strain 28 is primarily bound to the mannoprotein of the cell wall of sensitive yeasts. The mannoprotein of S. cerevisiae X 2180 was purified; gel filtration and SDS-PAGE indicated an estimated Mr of 185,000. The ability to bind killer toxin KT 28 increased during purification of the mannoprotein. Removing the protein part of the mannoprotein by enzymic digestion or removing the alkali-labile oligosaccharide chains by beta-elimination did not destroy the ability to bind killer toxin KT 28. However, binding activity was lost when the 1,6-alpha-linkages of the outer carbohydrate backbone were hydrolysed by acetolysis. The separated oligomannosides of the side chains also failed to bind toxin, indicating that the main mannoside chains were essential for the receptor activity. The reversible adsorption of killer toxin to mannoprotein was demonstrated by linking it covalently to Sepharose and using this material for affinity chromatography. A 90-fold increase in the specific activity of a preparation of killer toxin KT 28 was achieved in this way.     

Characterization of a novel killer toxin encoded by a double-stranded linear DNA plasmid of Kluyveromyces lactis

A novel killer toxin, encoded by a double-stranded linear DNA plasmid pGK l-1 (5.4 MDa) in Kluyveromyces lactis IFO 1267 was purified 320 000-fold from the culture broth of yeast. The toxin was obtained in an electrophoretically homogeneous state with a yield of 24% by hydroxyapatite column chromatography, chromatofocusing and polyacrylamide gel electrophoresis. The purified toxin was dissociated into two subunits with molecular masses of 27 kDa and above 80 kDa, as estimated by Laemmli's sodium dodecylsulfate gel electrophoresis; the exact composition ratio of the two subunits remains unestablished. The isoelectric point was between 4.4 and 4.8. As compared with the reported narrow pH range of action and instability of k1 killer toxin encoded by a double-stranded RNA plasmid of Saccharomyces cerevisiae, the K. Lactis toxin was effective with sensitive strains of S. cerevisiae in a relatively wider pH range between 4 and 8; it was stable for several months at pH 6.0 when stored below -20 degrees C. In contrast to the simple protein nature of the k1 killer toxin with a molecular mass of 11.47 kDa, the K. lactis toxin maintained a mannoprotein nature, as it was absorbed by a ConA-Sepharose column and eluted by methyl alpha-D-mannoside. The growth inhibitory activity of K. lactis toxin was enhanced 2-35-fold by the presence of 4-60% glycerol.