Cwp2p, the plasma membrane receptor for Pichia membranifaciens killer toxin

PMKT is a channel-forming killer toxin secreted by Pichia membranifaciens. To identify novel genes that mediate cellular resistance to PMKT we screened a collection of 288 deletion mutants. We found 29 open reading frames (ORFs) that, when deleted, confer resistance to PMKT. In addition, the deletion of 15 ORFs was observed to increase protoplast resistance, in agreement with the initial assumption that a plasma membrane receptor for PMKT exists. Whole cells and protoplasts of a cwp2Delta mutant were found to be completely resistant to PMKT and were unable to bind PMKT, indicating that Cwp2p interacts with it. A protein with a molecular mass of 11.7 kDa was purified from PMKT-affinity columns. This protein was sequenced and identified as Cwp2p. Glycosylphosphatidylinositol (GPI) anchoring-defective mutants were much less sensitive to PMKT, as were wild-type protoplasts pretreated with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins, indicating that the GPI-anchored precursor of Cwp2p is also necessary for PMKT activity. Carboxyfluorescein-entrapped liposomes containing a purified GFP-Cwp2p fusion protein in their membranes were much more sensitive to PMKT than protein-free liposomes. Cwp2p and its GPI-anchored precursor are proposed for the first time to be involved as PMKT secondary receptors.     

Physiological conditions affecting the sensitivity of Saccharomyces cerevisiae to a Pichia kluyveri killer toxin and energy requirement for toxin action

The interaction between the killer toxin of Pichia kluyveri 1002 and cells of Saccharomyces cerevisiae SCF 1717 is strongly affected by the physiological state of sensitive cells. The killing effect is maximal for cells in the lag and early exponential phase of growth, whereas stationary cells are completely resistant. Furthermore, sensitivity is markedly enhanced by a rise of the pH (from 3.2 to 6.8) at which cells are cultured.Three successive stages can be distinguished in the killing process: (I) binding of the toxin to the primary binding site; (II) transmission of the toxin to its reactive site in the plasma membrane; (III) occurrence of functional damage (K⁺-leakage; decrease of intracellular pH). The transition from stage I to II is prevented in the absence of metabolic energy or at low temperature (below 10°C). Sensitive cells in stage I can be rescued from toxin-induced killing by a short incubation at pH 7.0, which treatment is not effective for cells in stage II. Cells in stage II are able to resume growth when plated in a rich medium containing suitable concentrations of potassium and hydrogen ions. Rescue was not observed for cells in stage III of the killing process.     

(1-->6)-beta-D-glucan as cell wall receptor for Pichia membranifaciens killer toxin

The killer toxin from Pichia membranifaciens CYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1-->6)-beta-D-glucan of the cell wall of a sensitive yeast (Candida boidinii IGC 3430). The (1-->6)-beta-D-glucan was purified from cell walls of C. boidinii by alkali and hot-acetic acid extraction, a procedure which solubilizes glucans. The major fraction of receptor activity remained with the alkali-insoluble (1-->6)-beta- and (1-->3)-beta-D-glucans. The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and (1)H nuclear magnetic resonance) analyses of the fractions obtained showed that (1-->6)-beta-D-glucan was a receptor. Adsorption of most of the killer toxin to the (1-->6)-beta-D-glucan was complete within 2 min. Killer toxin adsorption to the linear (1-->6)-beta-D-glucan, pustulan, and a glucan from Penicillium allahabadense was observed. Other polysaccharides with different linkages failed to bind the killer toxin. The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines. This toxin shows its maximum killer activity in the presence of NaCl. This report is the first to identify the (1-->6)-beta-D-glucan as a receptor for this novel toxin.     

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.     

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.     

Yeast killer toxin: purification and characterisation of the protein toxin from Saccharomyces cerevisiae.

Killer toxin from killer strains of Saccharomyces cerevisiae was isolated from concentrates of extracellular medium by precipitation in poly(ethylene glycol) and chromatography through glyceryl-controlled-pore glass. The toxin migrated as a single protein band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A molecular weight of 11470 was determined for the toxin protein from its electrophoretic mobility and amino acid composition. Gel filtration of the active toxin indicated that the 11,470-Mr monomer was the active unit. Electrophoretic comparison of extracellular concentrates from a killer strain and an isogenic non-killer showed the presence of the toxin protein only in the killer-derived material. The activity of the toxin was most stable between pH 4.2 and 4.6. At 30 degrees C toxin from a superkiller strain was more stable than that from a normal killer.     

Binding of yeast killer toxin to a cell wall receptor on sensitive Saccharomyces cerevisiae.

35S-labeled killer toxin protein bound to cells of sensitive Saccharomyces cerevisiae S14a. Strains that were resistant to toxin through mutation in the nuclear genes kre1 kre2 bound toxin only weakly. Non-radioactive toxin competed effectively with 35S-labeled toxin for binding to S14a, but did not compete significantly in the binding to mutant kre1-1. This implied that binding to kre1-1 was nonspecific. A Scatchard analysis of the specific binding to S14a gave a linear plot, with an association constant of 2.9 x 10(6) M-1 and a receptor number of 1.1 x 10(7) per cell. Killer toxin receptors were solubilized from the cell wall by zymolyase digestion. Soluble, non-dialyzable cell wall digest from S14a competed with sensitive yeast cells for 35S-labeled toxin binding and reduced toxin-dependent killing of a sensitive strain. Wall digest from kre1-1 competed only weakly for toxin binding with sensitive cells and caused little reduction of toxin-dependent killing. Although the abundant (1.1 x 10(7) per cell) receptor appeared necessary for toxin action, as few as 2.8 x 10(4) toxin molecules were necessary to kill a sensitive cell of S14a. The kinetics killing of S14a suggested that some component was saturated with toxin at a concentration 50-fold lower than that needed to saturate the wall receptor.     

Induction of DNA damage and apoptosis in Saccharomyces cerevisiae by a yeast killer toxin

The cellular response of Saccharomyces cerevisiae to a linear plasmid encoded killer toxin from Pichia acaciae was analysed. As for the Kluyveromyces lactis zymocin, such toxin was recently shown to bind to the target cell's chitin and probably acts by facilitating the import of a toxin subunit. However, as distinct from zymocin, which arrests cells in G1, it provokes S-phase arrest and concomitant DNA damage checkpoint activation. Here, we report that such novel toxin type causes cell death in a two-step process. Within 4 h in toxin, viability of cells is immediately reduced to approximately 30%. Elevated mutation rates at the CAN1 locus prove DNA damaging mediated by the toxin. Cells arrested artificially in G1 or G2/M are very rapidly affected, while cells arrested in S loose their viability at a slower rate. S-phase arrest is, thus, a response of target cells to cope with DNA damage induced by the toxin. A second decline in viability requiring metabolically active target cells emerges upon toxin exposure over 10 h. During this phase, toxin treated cells develop abnormal nuclear morphology and react positive to terminal deoxynucleotidyl transferase-mediated nick end-labelling (TUNEL), indicative of DNA fragmentation. Furthermore, as judged from staining with fluorescein conjugated annexinV, cells expose phosphatidylserine at the outer membrane face and the formation of reactive oxygen species (ROS) is increased. ROS formation and concomitant cell death was heavily suppressed in a rho- derivative of the tester strain, while immediate reduction of viability was indistinguishable from the wild type. As a strain lacking the cellular target because of defects in the major chitinsynthase (Chs3) did not display such characteristic changes, the chitin binding and DNA-damaging P. acaciae toxin constitutes an apoptosis inducing protein. Both, DNA-damaging and apoptosis induction are unique features of this novel toxin type.     

Reclassification of Pichia membranifaciens sensu Kurtzman

Forty strains which were stocked as Pichia membranifaciens sensu Kurtzman and 9 strains stocked as Candida valida, anamorphs of P. membranifaciens, in the Institute for Fermentation, Osaka (IFO) were reclassified based on the data of base composition of nuclear DNA, DNA/DNA hybridization, coenzyme Q system, electrophoretic karyotype, and base sequence of 18S rDNA. P. membranifaciens complex was assigned into 3 groups: (I) P. membranifaciens group, including 25 strains with high DNA homologies to the type strain of P. membranifaciens (72–98%); (II) P. manshurica group, including 18 strains with high DNA homology of 79–95% to the type strain of P. manshurica; and a group including the remaining 6 strains, which had low DNA homology to the above two species. GC content was 42.9–45.3 mol% for the P. membranifaciens group, 40.0–42.0 mol% for the P. manshurica group, and 27.2–44.7 mol% for the remaining group. All three groups had ubiquinone Co Q-7. Of the 6 anomalous strains, IFO 0162 was identified as Pichia deserticola, and IFO 0839 and IFO 0840 were identified as Issatchenkia occidentalis; but IFO 0842, IFO 0843, and IFO 1788 were thought to be unknown strains.     

Mode of action of yeast toxins: energy requirement for Saccharomyces cerevisiae killer toxin.

The role of the energy status of the yeast cell in the sensitivity of cultures to two yeast toxins was examined by using 12K release from cells as a measure of toxin action. The Saccharomyces cerevisiae killer toxin bound to sensitive cells in the presence of drugs that interfered with the generation or use of energy, but it was unable to efflux 12K from the cells under these conditions. In direct contrast, the Torulopsis glabrata pool efflux-stimulating toxin induced efflux of the yeast 42K pool was insensitive to the presence of energy poisons in cultures. The results indicate that an energized state, maintained at the expense of adenosine 5'-triphosphate from either glycolytic or mitochondrial reactions, is required for the action of the killer toxin on the yeast cell.     

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.