Isolation and characterization of Saccharomyces cerevisiae mutants with a different degree of resistance to killer toxins K1 and K2

Killer toxin K1 of Saccharomyces cerevisiae kills sensitive cells of the same species by disturbing the ion gradient across the plasma membrane after binding to the receptor at cell wall beta-1,6-glucan. Killer protein K2 is assumed to act by a similar mechanism. To identify the putative plasma membrane receptors for both toxins we mutagenized three sensitive S. cerevisiae strains and searched for clones with killer-resistant spheroplasts. The well diffusion assay identified three phenotypically different groups of clones: clones resistant simultaneously to both toxins, clones with lowered sensitivity to only K1 toxin and those with strongly lowered sensitivity to K2 and partially lowered sensitivity to K1 toxin. These phenotypes are controlled by recessive mutations that belong to at least four different complementation groups. This indicates certain differences at the level of interaction of K1 and K2 toxin with sensitive cells.     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Immunity to killer toxin K1 is connected with the golgi-to-vacuole protein degradation pathway

Killer strains of Saccharomyces cerevisiae producing killer toxin K1 kill sensitive cells but are resistant to their own toxin. It is assumed that in the producer, an effective interaction between the external toxin and its plasma membrane receptor or the final effector is not possible on the grounds of a conformation change of the receptor or its absence in a membrane. Therefore, it is possible that some mutants with defects in intracellular protein transport and degradation can show a suicidal phenotype during K1 toxin production. We have examined these mutants in a collection of S. cerevisiae strains with deletions in various genes transformed by the pYX213+M1 vector carrying cDNA coding for the K1 toxin under the control of the GAL1 promoter. Determination of the quantity of dead cells in colony population showed that (1) the toxin production from the vector did not support full immunity of producing cells, (2) the suicidal phenotype was not connected with a defect in endocytosis or autophagy, (3) deletants in genes VPS1, VPS23, VPS51 and VAC8 required for the protein degradation pathway between the Golgi body and the vacuole exhibited the highest mortality. These results suggest that interacting molecule(s) on the plasma membrane in the producer might be diverted from the secretion pathway to degradation in the vacuole.     

Mutual antagonism among killer yeasts: competition between K1 and K2 killers and a novel cDNA-based K1-K2 killer strain of Saccharomyces cerevisiae

Mutually antagonistic K1 and K2 killer strains compete when mixed and serially subcultured. At pH 4.6, where the K1 killer toxin is more stable in vitro, the K1 strain outcompeted the K2 strains at both 18 and 30 degrees C. At pH 4.0, closer to the in vitro pH optimum of the K2 killer toxin, the K1 strain again predominated at 18 degrees C, but at 30 degrees C the K2 strains became the sole cell type on subculture. To show more clearly that these results were dependent upon the respective killer toxins, control experiments were conducted with isogenic, nonkiller strains cured of the dsRNA-based killer virions. Such nonkiller strains were unable to compete with antagonistic killers under conditions where their isogenic killer parents could, strongly suggesting that the killer phenotype was important in these competitions. Double K1-K2 killer strains cannot stably exist, as their dsRNA genomes compete at a replicative level. Using recombinant DNA methodology, a stable K1-K2 killer strain was constructed. This strain outcompeted both K1 and K2 killers when serially subcultured under conditions where either the K1 or the K2 strains would normally predominate in mixed cultures. Such a double killer may be useful in commercial fermentations, where there is a risk of contamination by killer yeasts.     

Isolation from Candida albicans of a functional homolog of the Saccharomyces cerevisiae KRE1 gene, which is involved in cell wall beta-glucan synthesis.

The KRE1 gene of Saccharomyces cerevisiae, sacKRE1, appears to be involved in the synthesis of cell wall beta-glucan. S. cerevisiae strains with mutations in the KRE1 gene produce a structurally altered cell wall (1----6)-beta-glucan, which results in resistance to K1 killer toxin. We isolated the canKRE1 gene from Candida albicans by its ability to complement a kre1 mutation in S. cerevisiae and confer sensitivity to killer toxin. Sequence analysis revealed that the predicted protein encoded by canKRE1 shares an overall structural similarity with that encoded by sacKRE1. The canKRE1 protein is composed of an N-terminal signal sequence, a central domain of 46% identity with the sacKRE1 protein, and a C-terminal hydrophobic tract. These structural and functional similarities imply that the canKRE1 gene carries out a function in C. albicans cell wall assembly similar to that observed for sacKRE1 in S. cerevisiae.     

Changes in plasma membrane fluidity lower the sensitivity of<Emphasis Type="Italic">S. cerevisiae</Emphasis> to killer toxin K1

The possible correlation between plasma membrane fluidity changes induced by modified cultivation conditions and cell sensitivity to the killer toxin K1 of Saccharomyces cerevisiae were investigated. Cells grown under standard conditions exhibited high toxin sensitivity. Both a membrane fluidity drop and fluidity rise brought about markedly reduced sensitivity to the toxin. These results do not fit the hypothesis of physiological relevance of direct toxin-lipid interaction, suggesting that the essential event in killer toxin action is interaction with membrane protein(s) that can be negatively influenced by any changes of membrane fluidity.     

Killer systems in Saccharomyces cerevisiae: three distinct modes of exclusion of M2 double-stranded RNA by three species of double-stranded RNA, M1, L-A-E, and L-A-HN.

M1 and M2 double-stranded RNAs (dsRNAs) code for the K1R1 and K2R2 killer toxin and resistance functions, respectively. Natural variants of a larger dsRNA (L-A) carry various combinations of the [EXL], [HOK], and [NEX] genes, which affect the K1 and K2 killer systems. Other dsRNAs, the same size as L-A, called L-B and L-C, are often present with L-A. We show that K1 killer strains have [HOK] and [NEX] but not [EXL] on their L-A (in disagreement with Field et al., Cell 31:193-200, 1982). These strains also carry other L-size molecules detectable after heat-curing has eliminated L-A. The exclusion of M2 dsRNA observed on mating K2 strains with K1 strains is due to the M1 dsRNA (not the L-A dsRNA as claimed by Field et al.) in the K1 strains. Four independent mutants of a [KIL-k2] [NEX-o] [HOK-o] strain were selected for resistance to [EXL] exclusion of M2 ([EXLR] phenotype). The [EXLR] phenotype showed non-Mendelian inheritance in each case, and these mutants had simultaneously each acquired [HOK]. The mutations were located on L-A and not on M2, and did not confer resistance to M1 exclusion of M2.     

Kre1p, the plasma membrane receptor for the yeast K1 viral toxin

Saccharomyces cerevisiae K1 killer strains are infected by the M1 double-stranded RNA virus encoding a secreted protein toxin that kills sensitive cells by disrupting cytoplasmic membrane function. Toxin binding to spheroplasts is mediated by Kre1p, a cell wall protein initially attached to the plasma membrane by its C-terminal GPI anchor. Kre1p binds toxin directly. Both cells and spheroplasts of Deltakre1 mutants are completely toxin resistant; binding to cell walls and spheroplasts is reduced to 10% and < 0.5%, respectively. Expression of K28-Kre1p, an inactive C-terminal fragment of Kre1p retaining its toxin affinity and membrane anchor, fully restored toxin binding and sensitivity to spheroplasts, while intact cells remained resistant. Kre1p is apparently the toxin membrane receptor required for subsequent lethal ion channel formation.     

Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

Background

Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts.

Principal Findings

We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility.

Significance

Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.     

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.     

Construction and properties of K1 type killer wine yeasts

Summary With the use of a protoplast fusion technique the killer character of K1 type was transferred into four industrial Saccharomyces wine yeasts. The prototrophic yeast strains active against standard sensitive and K2 killer Saccharomyces strains, resistant to K1 killer toxin were constructed with no changes in technological properties.     

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.     

Isolation of Candida glabrata Homologs of the Saccharomyces cerevisiae KRE9 and KNH1 Genes and Their Involvement in Cell Wall β-1,6-Glucan Synthesis

The Candida glabrata KRE9 (CgKRE9) and KNH1 (CgKNH1) genes have been isolated as multicopy suppressors of the tetracycline-sensitive growth of a Saccharomyces cerevisiae mutant with the disrupted KNH1 locus and the KRE9 gene placed under the control of a tetracycline-responsive promoter. Although a cgknh1Δ mutant showed no phenotype beyond slightly increased sensitivity to the K1 killer toxin, disruption of CgKRE9 resulted in several phenotypes similar to those of the S. cerevisiae kre9Δ null mutant: a severe growth defect on glucose medium, resistance to the K1 killer toxin, a 50% reduction of β-1,6-glucan, and the presence of aggregates of cells with abnormal morphology on glucose medium. Replacement in C. glabrata of the cognate CgKRE9 promoter with the tetracycline-responsive promoter in a cgknh1Δ background rendered cell growth tetracycline sensitive on media containing glucose or galactose. cgkre9Δ cells were shown to be sensitive to calcofluor white specifically on glucose medium. In cgkre9 mutants grown on glucose medium, cellular chitin levels were massively increased.     

Viral induced yeast apoptosis

In an analogous system to mammals, induction of an apoptotic cell death programme (PCD) in yeast is not only restricted to various exogenous factors and stimuli, but can also be triggered by viral killer toxins and viral pathogens. In yeast, toxin secreting killer strains are frequently infected with double-stranded (ds)RNA viruses that are responsible for killer phenotype expression and toxin secretion in the infected host. In most cases, the viral toxins are either pore-forming proteins (such as K1, K2, and zygocin) that kill non-infected and sensitive yeast cells by disrupting cytoplasmic membrane function, or protein toxins (such as K28) that act in the nucleus by blocking DNA synthesis and subsequently causing a G1/S cell cycle arrest. Interestingly, while all these virus toxins cause necrotic cell death at high concentration, they trigger caspase- and ROS-mediated apoptosis at low-to-moderate concentration, indicating that even low toxin doses are deadly by triggering PCD in enemy cells. Remarkably, viral toxins are not solely responsible for cell death induction in vivo, as killer viruses themselves were shown to trigger apoptosis in non-infected yeast. Thus, as killer virus-infected and toxin secreting yeasts are effectively protected and immune to their own toxin, killer yeasts bear the intrinsic potential to dominate over time in their natural habitat.     

Curing of the killer character of Saccharomyces cerevisiae with acridine orange

Acridine orange, an intercalating dye usually employed in the curing of bacterial plasmids, was tested for its ability to cure K1 and K2 killer strains (laboratory and wine strains). The results showed a high curing percentage of the killer character. This was demonstrated by the loss of M1 or M2 dsRNAs (responsible for toxin production and resistance to it) and because the meiotic products exhibited non-Mendelian segregation. The curing percentages varied, depending on the strain but not on the killer type, and showed similar efficiency as compared with other known curing agents.     

Linear plasmids pWR1A and pWR1B of the yeast Wingea robertsiae are associated with a killer phenotype

Wingea robertsiae CBS6693 (synonym Debaryomyces robertsiae) was previously reported to harbor two cryptic linear plasmids, designated pWR1A (8.3 kb) and pWR1B (14.6 kb). Reexamination of a putative plasmid encoded killer phenotype involved UV-curing as well as a highly sensitive toxin assay. Killer activities of concentrated culture supernatants prepared from both, a plasmid carrying and a cured plasmid-free strain, were examined in liquid media. Supernatants collected from plasmid carrying strains subjected to cultures of the plasmid-free derivative had clear concentration-dependent inhibitory effects, whereas plasmid harboring cells were not affected. Incubation at 65 degrees C for 10 min totally destroyed the toxin. Since supernatants prepared from the plasmid-free strain did not possess such killer activity and the presence of the plasmids confered resistance, toxin as well as immunity functions appear plasmid encoded. Beyond this, chitin affinity chromatography and Western blot analysis proved plasmid specific expression and secretion of a protein displaying similarities to the alpha-subunit of the Kluyveromyces lactis killer toxin. The assay applied in this study will most probably allow disclosure of other hidden killer phenomena, which may have escaped detection by conventionally applied plate assays.     

A Saccharomyces cerevisiae genome-wide mutant screen for altered sensitivity to K1 killer toxin

Using the set of Saccharomyces cerevisiae mutants individually deleted for 5718 yeast genes, we screened for altered sensitivity to the antifungal protein, K1 killer toxin, that binds to a cell wall beta-glucan receptor and subsequently forms lethal pores in the plasma membrane. Mutations in 268 genes, including 42 in genes of unknown function, had a phenotype, often mild, with 186 showing resistance and 82 hypersensitivity compared to wild type. Only 15 of these genes were previously known to cause a toxin phenotype when mutated. Mutants for 144 genes were analyzed for alkali-soluble beta-glucan levels; 63 showed alterations. Further, mutants for 118 genes with altered toxin sensitivity were screened for SDS, hygromycin B, and calcofluor white sensitivity as indicators of cell surface defects; 88 showed some additional defect. There is a markedly nonrandom functional distribution of the mutants. Many genes affect specific areas of cellular activity, including cell wall glucan and mannoprotein synthesis, secretory pathway trafficking, lipid and sterol biosynthesis, and cell surface signal transduction, and offer new insights into these processes and their integration.     

A molecular target for viral killer toxin: TOK1 potassium channels.

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes.     

The K3 type killer strains of genusSaccharomyces for wine production

Using simultaneous fusion of protoplasts of three strains the auxotrophic K3 killer designated MH1 was prepared at a very low frequency and further employed for the transfer of the K3 killer character into a commercial wine yeast. A novelSaccharomyces Bratislava 1K3 strain with desirable technological and anti-yeast killer abilities was thus constructed. Attempts to prepare double K1/K3 killers were made. The obtained fusion products contained M₁ dsRNA and were able to produce only the K1 type killer toxin.     

Killer toxin producing strains of the yeasts Hanseniaspora uvarum and Pichia kluyveri

By heat treatment killer strains of the type K1 of Saccharomyces cerevisiae that are known to harbour dsRNA plasmids were completely cured, whereas only a small fraction of the clones of the killer type K2 had lost the dsRNA dependent killer character. The K2 killers but not the strains of killer type K1 were easily cured by cycloheximide. Killer strains of Hanseniaspora uvarum were not curable by heat treatment. Curing was successfull with cycloheximide or 5-fluorouracil. Two double-stranded RNA plasmids were detected in the killer strains of H. uvarum. The smaller dsRNA plasmid was absent in the strains that were cured of their killer character by 5-fluorouracil. The killer character of H. uvarum was transferred to S. cerevisiae by spheroplast fusion. The fusion products showing the killer character contained both dsRNA plasmids, obviously the smaller plasmid (M-dsRNA) carries the genes for killer toxin formation. Killer strains of Pichia kluyveri were not curable of their killer character, in these strains no dsRNA plasmids were detected.This paper was kindly supported by a grant from the Deutsche Forschungsgemeinschaft