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

Flegelová, H.; Novotná, Drahomíra; Vojtikova, K; Janderová, B.

doi:10.1016/s1567-1356(01)00063-0

Cited by 7 publications

(13 citation statements)

References 14 publications

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“…The following S. cerevisiae strains were used: (a) wild‐type GRF18 (α his3‐11,15 leu2‐3,112 ) from the DMUP yeast collection, Charles University, Faculty of Science, Prague. Its isogenic mutant GRF18/a was isolated previously [24]; (b) all single deletion strains used were haploid derivatives of the reference strains BY4741 ( MATa; his3 Δ 1; leu2 Δ 0; met15 Δ 0; ura3 Δ 0 ) or BY4742 ( MAT α ; his3 Δ 1; leu2 Δ 0; lys2 Δ 0; ura3 Δ 0 ); (c) kre5 , kre9 , kre29 , kre30 , kre33 and kre34 were diploid strains with heterozygous deletions derived from the reference strain BY4743 ( MATa/MAT α ; his3 Δ 1/his3 Δ 1; leu2 Δ 0/leu2 Δ 0; met15 Δ 0/MET15; LYS2/lys2 Δ 0; ura3 Δ 0/ura3 Δ 0 ). These strains from the Euroscarf collection (http://www.uni-frankfurt.de/fb15/mikro/euroscarf/; [25]) are listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

“…The cells were grown at 28 °C with shaking in YEPG medium [24] until they reached an exponential growth phase (cell counts 5 × 10 6 ‐3 × 10 7 ml −1 ). Solid YEPGA medium (YEPG with 20 g l −1 agar) was used for mating, solid OYEPGA medium (YEPGA supplemented with 1 M sorbitol) was used for plating the spheroplasts.…”

Section: Methodsmentioning

confidence: 99%

“…Spheroplasts were embedded into OSA (3% agar containing only 1 M sorbitol). Solid medium OWA (osmotically stabilised wort agar) [24], buffered by McIlvaine citrate phosphate buffer to pH 4.7 or 3.8 for K1 or K2 killer toxins, respectively, and supplemented with 1 M sorbitol, was used for the well assays. For replica‐plating of transformants it was supplemented with 1% methylene blue (OWAB).…”

Section: Methodsmentioning

confidence: 99%

“…For replica‐plating of transformants it was supplemented with 1% methylene blue (OWAB). JG medium buffered to pH 4.7 or 3.8 similarly as for OWA [24] was used for the cultivation of killer toxin‐producing strains S. cerevisiae X3 and V10‐25‐16 and for the cell survival assay. JG medium supplemented with sorbitol to 1 M final concentration (OJG medium) was used for the spheroplast survival assays.…”

Section: Methodsmentioning

confidence: 99%

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Different action of killer toxins K1 and K2 on the plasma membrane and the cell wall of

Novotná

Flegelová

Janderová

2004

FEMS Yeast Research

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Study of Saccharomyces cerevisiae killer toxin-sensitive strains with the deltakre2 phenotype (resistant to toxin K1, sensitive to toxin K2) showed that the phenotype is complemented by the KRE2 gene not only in intact cells but also in spheroplasts, and resistance to K1 thus resides very probably in the plasma membrane. deltakre1 deletant displays a faulty interaction with both K1 and K2 toxin. Hence, Kre1p probably serves as plasma membrane receptor for both toxins. Deletants in seven other genes (GDA1, SAC1, LUV1, KRE23, SAC2, KRE21, ERG4) exhibit different degrees of the deltakre2-like resistance pattern, but the phenotype in deltagda1 and deltasac1 is not connected with a defect in K1 toxin interaction with the plasma membrane, similarly as in deltakre6 and deltakre11 strains with a higher resistance to K2 toxin. Differences between the K1 and K2 killer toxin thus occur on the level of both the plasma membrane and the cell wall.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Different action of killer toxins K1 and K2 on the plasma membrane and the cell wall of

Novotná

Flegelová

Janderová

2004

FEMS Yeast Research

Self Cite

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“…Secondly, K1 and K2 toxins are biochemically distinct: they differ in primary sequence, preprotoxin organization, molecular weight, isoelectric point, and killing pH optimum [19], [21], [22]. Thirdly, numerous budding yeast mutants have distinctive patterns of sensitivity/resistance towards K1 or K2 [23]. For example, Δ kre2 cells lacking an α-1,2-mannosyltransferase are sensitive to K2 but are resistant to K1 [24], [25].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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BackgroundUnderstanding 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 FindingsWe 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.SignificanceOur 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.

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

et al. 2006

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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.

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

Cited by 7 publications

References 14 publications

Different action of killer toxins K1 and K2 on the plasma membrane and the cell wall of

Different action of killer toxins K1 and K2 on the plasma membrane and the cell wall of

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

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

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