Sensitive Yeast Cells Research Articles

Saccharomyces cerevisiae RNA Polymerase II Is Affected by Kluyveromyces lactis Zymocin

The G(1) arrest imposed by Kluyveromyces lactis zymocin on Saccharomyces cerevisiae cells requires a functional RNA polymerase II (pol II) Elongator complex. In studying a link between zymocin and pol II, progressively truncating the carboxyl-terminal domain (CTD) of pol II was found to result in zymocin hypersensitivity as did mutations in four different CTD kinase genes. Consistent with the notion that Elongator preferentially associates with hyperphosphorylated (II0) rather than hypophosphorylated (IIA) pol II, the II0/IIA ratio was imbalanced toward II0 on zymocin treatment and suggests zymocin affects pol II function, presumably in an Elongator-dependent manner. As judged from chromatin immunoprecipitations, zymocin-arrested cells were affected with regards to pol II binding to the ADH1 promotor and pol II transcription of the ADH1 gene. Thus, zymocin may interfere with pol II recycling, a scenario assumed to lead to down-regulation of pol II transcription and eventually causing the observed G(1) arrest.

Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor.

The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G(1) phase of the cell cycle. Despite its heterotrimeric (alpha beta gamma) structure, intracellular expression of its smallest subunit, the gamma-toxin, is alone responsible for the G(1) arrest. The alpha subunit, however, has a chitinase activity that is essential for holozymocin action from the cell exterior. Here we show that sensitive yeast cells can be rescued from zymocin treatment by exogenously applying crude chitin preparations, supporting the idea that chitin polymers can compete for binding to zymocin with chitin present on the surface of sensitive yeast cells. Consistent with this, holozymocin can be purified by way of affinity chromatography using an immobilized chitin matrix. PCR-mediated deletions of chitin synthesis (CHS) genes show that most, if not all, genetic scenarios that lead to complete loss (chs3 Delta), blocked export (chs7 Delta) or reduced activation (chs4 Delta), combined with mislocalization (chs4 Delta chs5 Delta; chs4 Delta chs6 Delta; chs4 Delta chs5 Delta chs6 Delta) of chitin synthase III activity (CSIII), render cells refractory to the inhibitory effects of exozymocin. In contrast, deletions in CHS1 and CHS2, which code for CSI and CSII, respectively, have no effect on zymocin sensitivity. Thus, CSIII-polymerized chitin, which amounts to almost 90% of the cell's chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells.

Expression, processing and high level secretion of a virus toxin in fission yeast.

The virally encoded K28 toxin of Saccharomyces cerevisiae kills sensitive yeast cells in a multi-step receptor-mediated fashion by cell cycle arrest and inhibition of DNA synthesis. In vivo, the toxin is translated as a 38 kDa preprotoxin (pptox) which is enzymatically processed to the biologically active alpha/beta heterodimer during passage through the yeast secretory pathway. Here, we demonstrate that Schizosaccharomyces pombe, a yeast from which no natural toxin-secreting killer strains are known, is perfectly capable of expressing a killer phenotype. Episomal as well as integrating K28 pptox gene cassettes were constructed that allowed a tightly thiamine-regulated killer phenotype expression under transcriptional control of the Sch. pombe nmt1 promotor. Northern analysis of the toxin-coding transcript as well as Western analysis of the secreted toxin indicated that fission yeast is capable of expressing a correctly processed and fully functional virus toxin. Moreover, toxin secretion in recombinant Sch. pombe was at least ten-fold higher than in any natural and/or recombinant Sac. cerevisiae killer strain, indicating that pptox-derived vectors might be attractive in the fast growing field of heterologous protein expression and secretion in yeast.

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.

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.

Commercial baker's yeast stability as affected by intracellular content of trehalose, dehydration procedure and the physical properties of external matrices.

The effects of vacuum-drying and freeze-drying on the cell viability of a commercial baker's yeast, Saccharomyces cerevisiae, strain with different endogenous contents of trehalose were analyzed. An osmotolerant Zygosaccharomyces rouxii strain was used for comparative purposes. Higher viability values were observed in cells after vacuum-drying than after freeze-drying. Internal concentrations of trehalose in the range 10-20% protected cells in both dehydration processes. Endogenous trehalose concentrations did not affect the water sorption isotherm nor the Tg values. The effect of external matrices of trehalose and maltodextrin was also studied. The addition of external trehalose improved the survival of S. cerevisiae cells containing 5% internal trehalose during dehydration. Maltodextrin (1.8 kDa) failed to protect vacuum-dried samples at 40 degrees C. The major reduction in the viability during the freeze-drying process of the sensitive yeast cells studied was attributed to the freezing step. The suggested protective mechanisms for each particular system are vitrification and the specific interactions of trehalose with membranes and/or proteins. The failure of maltodextrins to protect cells was attributed to the fact that none of the suggested mechanisms of protection could operate in these systems.

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.

Action properties of HYI killer toxin from Williopsis saturnus var. saturnus, and antibiotics, aculeacin A and papulacandin B.

The mechanism of the killing and cytocidal effects produced by HYI toxin from Williopsis saturnus var. saturnus, and by the amphiphilic antibiotics aculeacin A and papulacandin B on yeast Saccharomyces bayanus cells was studied. When the yeast cells were treated with these molecules, a discharge of cell materials at the budding position was observed by phase-contrast and scanning electron microscopy. The cytocidal effect of these molecules was most pronounced when the cells were in the logarithmic growth phase. Washing the HYI toxin incubation mixture completely eliminated the killing activity, but in the case of the antibiotics, it only partially reduced the cytocidal activity. Full recovery of the killing activity in the supernatant of the washing solution was observed after HYI toxin incubation, but in the case of the antibiotics, the recovery of cytocidal activity was time-dependent. The activity of membrane beta-1,3-glucan synthase was potently inhibited by HYI toxin, and the concentration of this enzyme in the budding tip was observed. These results suggest that HYI toxin exerts a cytocidal effect on the budding of sensitive yeast cells by inhibiting cell wall synthesis. This mechanism is similar to that of the HM-1 toxin of W. saturnus var. mrakii, and to aculeacin A and papulacandin B, although there are some differences from that of HYI toxin.

Killer-toxin-resistant kre12 mutants of Saccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor.

The Saccharomyces cerevisiae killer toxin K1 is a secreted alpha/beta-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to beta-1,6-D-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of the KRE genes whose products are involved in synthesis and/or assembly of cell wall beta-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 strains indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), rendering kre12 mutants incapable of binding significant amounts of toxin to the membrane. Since kre12 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from a kre12 mutant and from an isogenic Kre12(+) 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 the kre12 mutant. A model for K1 killer toxin action is presented in which the gene product of KRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.

Significance of the lag phase in K1 killer toxin action on sensitive yeast cells.

The minimum period between the addition of killer toxin K1 to sensitive yeast cells and the appearance of first cells stained with bromocresol purple indicating membrane damage, is approximately 20 min. The length of this lag phase depends strongly on toxin concentration, extending sharply at toxin levels lower than 60 lethal units (LU) per cell (about one-tenth of the toxin concentration necessary for saturating all surface receptors). As the binding of the toxin to the cell is virtually complete within 1 min, the rest of the lag phase reflects processes different from actual binding, e.g. combination of several toxin molecules to form a membrane ion channel or pore.

Two Saccharomyces cerevisiae genes which control sensitivity to G1 arrest induced by Kluyveromyces lactis toxin.

The Kluyveromyces lactis toxin causes an arrest of sensitive yeast cells in the G1 phase of the cell division cycle. Two complementary genetic approaches have been undertaken in the yeast Saccharomyces cerevisiae to understand the mode of action of this toxin. First, two sequences conferring toxin resistance specifically in high copy number have been isolated and shown to encode a tRNA(Glu3) and a novel polypeptide. Disruption of the latter sequence in the yeast genome conferred toxin resistance and revealed that it was nonessential, while the effect of the tRNA(Glu)3 was highly specific and mediated resistance by affecting the toxin's target. An alpha-specific, copy number-independent suppressor of toxin sensitivity was also isolated and identified as MATa, consistent with the observation that diploid cells are partially resistant to the toxin. Second, in a comprehensive screen for toxin-resistant mutants, representatives of 13 complementation groups have been obtained and characterized to determine whether they are altered in the toxin's intracellular target. Of 10 genes found to affect the target process, one (KTI12) was found to encode the novel polypeptide previously identified as a multicopy resistance determinant. Thus, both loss of KTI12 function and elevated KTI12 copy number can cause resistance to the K. lactis toxin.

Two Saccharomyces cerevisiae genes which control sensitivity to G1 arrest induced by Kluyveromyces lactis toxin.

The Kluyveromyces lactis toxin causes an arrest of sensitive yeast cells in the G1 phase of the cell division cycle. Two complementary genetic approaches have been undertaken in the yeast Saccharomyces cerevisiae to understand the mode of action of this toxin. First, two sequences conferring toxin resistance specifically in high copy number have been isolated and shown to encode a tRNA(Glu3) and a novel polypeptide. Disruption of the latter sequence in the yeast genome conferred toxin resistance and revealed that it was nonessential, while the effect of the tRNA(Glu)3 was highly specific and mediated resistance by affecting the toxin's target. An alpha-specific, copy number-independent suppressor of toxin sensitivity was also isolated and identified as MATa, consistent with the observation that diploid cells are partially resistant to the toxin. Second, in a comprehensive screen for toxin-resistant mutants, representatives of 13 complementation groups have been obtained and characterized to determine whether they are altered in the toxin's intracellular target. Of 10 genes found to affect the target process, one (KTI12) was found to encode the novel polypeptide previously identified as a multicopy resistance determinant. Thus, both loss of KTI12 function and elevated KTI12 copy number can cause resistance to the K. lactis toxin.

Factors affecting the susceptibility of sensitive yeast cells to killer toxin K1.

Optimum conditions for action of the killer toxin K1 on sensitive strain S. cerevisiae S6 were established. Maximum killing was reached in a very narrow pH range of 4.5-4.6. Maximum susceptibility to toxin was displayed by highly energized fresh cells from the early exponential phase in the presence of an external energy source (at least 200 mmol/L glucose). Further, maintenance of maximum membrane potential was necessary for killer action, as documented by decreasing toxin activity in the presence of increasing concentrations of KCl. The killing was strongly stimulated in the presence of millimolar concentrations of Ca2+ and Mg2+.

K1 killer toxin, a pore‐forming protein from yeast

K1 killer toxin is a secreted, pore-forming protein that kills sensitive yeast cells. The heterodimeric toxin is processed from a precursor in the Golgi, and has allowed identification of the KEX2- and KEX1-encoded proteases. The toxin binds to a glucan receptor on the cell wall of target yeast, and mutational analysis implicates both the alpha- and beta-toxin subunits in receptor binding. Toxin-resistant mutants with altered cell-wall glucans have helped to outline a pathway of assembly of these polysaccharides. Patch-clamp technology has demonstrated the nature of the lethal channel in toxin-treated plasma membranes. The hydrophobic alpha-subunit-encoding region is the site of all mutations affecting channel formation. Immunity to the toxin is conferred by the toxin precursor, and immunity mutations map to the region encoding the alpha subunit. The precursor probably competes with the toxin to prevent channel formation in toxin-producing cells, but the basis of this remains unknown. This toxin/immunity system has a domain structure that differs from that of other characterized toxins and has no known homologues.

Intracellular expression of Kluyveromyces lactis toxin γ subunit mimics treatment with exogenous toxin and distinguishes two classes of toxin‐resistant mutant

The Kluyveromyces lactis toxin is a heterotrimeric protein which irreversibly arrests proliferation of sensitive Saccharomyces cerevisiae cells in the G1 phase of the cell cycle. By expressing the gamma subunit of the toxin in sensitive yeast cells from a conditional promoter, it was previously demonstrated that it alone is required for inhibition (Tokunaga et al. (1989). Nucleic Acids Res. 17, 3435-3446). Here we show that, like native exogenous toxin, intracellular gamma subunit expression promotes a striking arrest of sensitive cells in G1. However, unlike the G1 arrest caused by native toxin, that induced by the gamma subunit alone does not result in reduced cellular viability and is fully and rapidly reversible, suggesting that the G1 arrest and the irreversibility of action may reflect different aspects of the toxin's interaction with sensitive cells. We have selected a large number of S. cerevisiae mutants which are highly resistant to the toxin in order to study its mode of action in more detail. Complementation analysis demonstrated that all but one of the mutants were recessive and these defined four separate genes. Members of two complementation groups concurrently acquired resistance to intracellular gamma subunit expression, suggesting that they contain a modified toxin target site. The other two genes appear to be required for entry of the gamma subunit into the sensitive cells since these mutants, while refractory to exogenous toxin, were fully sensitive to intracellular gamma subunit expression.

Kluyveromyces lactis toxin has an essential chitinase activity

The Kluyveromyces lactis toxin is a protein containing three subunits (alpha, beta and gamma) which causes sensitive yeast cells to arrest proliferation in the G1 phase of the cell cycle. Despite the toxin's complex structure, the gamma subunit appears to be the only component required for it to arrest proliferation since intracellular expression of the gamma polypeptide alone in a sensitive yeast strain mimics the effect of the exogenous native toxin. The toxin alpha subunit shows sequence similarity to a variety of chitinases and here we report that the toxin is a potent exochitinase. The exochitinase activity is absolutely required for its biological activity against sensitive Saccharomyces cerevisiae cells and allosamidin, a specific inhibitor of chitinases, abolishes the biological activity of the toxin. However, since the alpha subunit is not required for the G1 arrest induced by the toxin, the chitinase activity of the toxin cannot be directly responsible for the ultimate effect of the toxin and most likely plays a role in the initial interaction of the toxin with sensitive cells.

K28, a unique double-stranded RNA killer virus of Saccharomyces cerevisiae.

The double-stranded RNA (dsRNA) viruses of Saccharomyces cerevisiae consist of 4.5-kilobase-pair (kb) L species and 1.7- to 2.1-kb M species, both found in cytoplasmic viruslike particles (VLPs). The L species encode their own capsid protein, and one (LA) has been shown to encode a putative capsid-polymerase fusion protein (cap-pol) that presumably provides VLPs with their transcriptase and replicase functions. The M1 and M2 dsRNAs encode the K1 and K2 toxins and specific immunity mechanisms. Maintenance of M1 and M2 is dependent on the presence of LA, which provides capsid and cap-pol for M dsRNA maintenance. Although a number of different S. cerevisiae killers have been described, only K1 and K2 have been studied in any detail. Their secreted polypeptide toxins disrupt cytoplasmic membrane functions in sensitive yeast cells. K28, named for the wine S. cerevisiae strain 28, appears to be unique; its toxin is unusually stable and disrupts DNA synthesis in sensitive cells. We have now demonstrated that 4.5-kb L28 and 2.1-kb M28 dsRNAs can be isolated from strain 28 in typical VLPs, that these VLPs are sufficient to confer K28 toxin and immunity phenotypes on transfected spheroplasts, and that the immunity of the transfectants is distinct from that of either M1 or M2. In vitro transcripts from the M28 VLPs show no cross-hybridization to denatured M1 or M2 dsRNAs, while L28 is an LA species competent for maintenance of M1. K28, encoded by M28, is thus the third unique killer system in S. cerevisiae to be clearly defined. It is now amenable to genetic analysis in standard laboratory strains.

K28, A Unique Double-Stranded RNA Killer Virus of Saccharomyces cerevisiae

The double-stranded RNA (dsRNA) viruses of Saccharomyces cerevisiae consist of 4.5-kilobase-pair (kb) L species and 1.7- to 2.1-kb M species, both found in cytoplasmic viruslike particles (VLPs). The L species encode their own capsid protein, and one (LA) has been shown to encode a putative capsid-polymerase fusion protein (cap-pol) that presumably provides VLPs with their transcriptase and replicase functions. The M1 and M2 dsRNAs encode the K1 and K2 toxins and specific immunity mechanisms. Maintenance of M1 and M2 is dependent on the presence of LA, which provides capsid and cap-pol for M dsRNA maintenance. Although a number of different S. cerevisiae killers have been described, only K1 and K2 have been studied in any detail. Their secreted polypeptide toxins disrupt cytoplasmic membrane functions in sensitive yeast cells. K28, named for the wine S. cerevisiae strain 28, appears to be unique; its toxin is unusually stable and disrupts DNA synthesis in sensitive cells. We have now demonstrated that 4.5-kb L28 and 2.1-kb M28 dsRNAs can be isolated from strain 28 in typical VLPs, that these VLPs are sufficient to confer K28 toxin and immunity phenotypes on transfected spheroplasts, and that the immunity of the transfectants is distinct from that of either M1 or M2. In vitro transcripts from the M28 VLPs show no cross-hybridization to denatured M1 or M2 dsRNAs, while L28 is an LA species competent for maintenance of M1. K28, encoded by M28, is thus the third unique killer system in S. cerevisiae to be clearly defined. It is now amenable to genetic analysis in standard laboratory strains.

Yeast K1 killer toxin forms ion channels in sensitive yeast spheroplasts and in artificial liposomes.

The patch-clamp technique was used to examine the plasma membranes of sensitive yeast spheroplasts exposed to partially purified killer toxin preparations. Asolectin liposomes in which the toxin was incorporated were also examined. Excised inside-out patches from these preparations often revealed at 118 pS conductance appearing in pairs. The current through this conductance flickered rapidly among three states: dwelling mostly at the unit-open state, less frequently at the two-unit-open state, and more rarely at the closed state. Membrane voltages from -80 to 80 mV had little influence on the opening probability. The current reversed near the equilibrium potential of K+ in asymmetric KCl solutions and also reversed near O mV at symmetric NaCl vs. KCl solutions. The two levels of the conductance were likely due to the toxin protein, as treatment of spheroplasts or liposomes with extracellular protein preparations from isogenic yeasts deleted for the toxin gene gave no such conductance levels. These results show that in vivo the killer-toxin fraction can form a cation channel that seldom closes regardless of membrane voltage. We suggest that this channel causes the death of sensitive yeast cells.

Blockage of cell wall receptors for yeast killer toxin KT28 with antimannoprotein antibodies

Binding of yeast killer toxin KT28 to its primary cell wall receptor was specifically blocked with polyclonal antimannoprotein antibodies which masked all toxin-binding sites on the surface of sensitive yeast cells. By indirect immunofluorescence, it was shown that KT28 binds to the cell wall mannoprotein and that the toxin resistance of mannoprotein mutants (mnn) of Saccharomyces cerevisiae was due to a lack of killer toxin-binding sites within the yeast cell wall. Structural analysis of acetylated mannoprotein from KT28-resistant mutant strains identified the outer mannotriose side chains as the actual killer toxin-binding domains.