scholarly journals A mutational analysis of killer toxin resistance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1-->6)-beta-glucan synthesis.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 837-849 ◽  
Author(s):  
J L Brown ◽  
Z Kossaczka ◽  
B Jiang ◽  
H Bussey
Keyword(s):  
Cell Wall ◽  
Mutational Analysis ◽  
Killer Toxin ◽  
Similar Proportion ◽  
Wild Type ◽  
Beta Glucan ◽  
Toxin Resistance ◽  
Recessive Mutations ◽  
New Genes ◽  
Glucan Synthesis

Abstract Recessive mutations leading to killer resistance identify the KRE9, KRE10 and KRE11 genes. Mutations in both the KRE9 and KRE11 genes lead to reduced levels of (1-->6)-beta-glucan in the yeast cell wall. The KRE11 gene encodes a putative 63-kD cytoplasmic protein, and disruption of the KRE11 locus leads to a 50% reduced level of cell wall (1-->6)-glucan. Structural analysis of the (1-->6)-beta-glucan remaining in a kre11 mutant indicates a polymer smaller in size than wild type, but containing a similar proportion of (1-->6)- and (1-->3)-linkages. Genetic interactions among cells harboring mutations at the KRE11, KRE6 and KRE1 loci indicate lethality of kre11 kre6 double mutants and that kre11 is epistatic to kre1, with both gene products required to produce the mature glucan polymer at wild-type levels. Analysis of these KRE genes should extend knowledge of the beta-glucan biosynthetic pathway, and of cell wall synthesis in yeast.

Mutational analysis of the functional domains of yeast K1 killer toxin

1991 ◽  
Vol 11 (1) ◽  
pp. 175-181
Author(s):  
H Zhu ◽  
H Bussey
Keyword(s):  
Cell Wall ◽  
Receptor Binding ◽  
Mutational Analysis ◽  
Killer Toxin ◽  
Alpha Subunit ◽  
Functional Domains ◽  
Beta Subunits ◽  
Channel Formation ◽  
Beta Glucan ◽  
Beta Toxin

To determine the functional domains of K1 killer toxin, we analyzed the phenotypes of a set of mutations throughout regions encoding the alpha- and beta-toxin subunits that allow secretion of mutant toxins. A range of techniques have been used to examine the ability of mutant toxins to bind to beta-glucan cell wall receptor and to form lethal ion channels. Our results indicate that both the alpha and beta subunits are involved in beta-glucan receptor binding. Defects in ion channel formation and toxin immunity are confined to the hydrophobic alpha subunit of the toxin.

SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis

1993 ◽  
Vol 13 (7) ◽  
pp. 4039-4048
Author(s):  
T Roemer ◽  
S Delaney ◽  
H Bussey
Keyword(s):  
Genomic Library ◽  
Killer Toxin ◽  
Resistant Mutant ◽  
Null Alleles ◽  
Dependent Manner ◽  
Wild Type ◽  
Beta Glucan ◽  
Glucan Synthesis ◽  
Dose Dependent

KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T. Roemer and H. Bussey, Proc. Natl. Acad. Sci. USA 88:11295-11299, 1991). The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant. To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p. SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels. skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant. Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure. Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis.

Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis

1994 ◽  
Vol 14 (2) ◽  
pp. 1017-1025
Author(s):  
Z Hong ◽  
P Mann ◽  
N H Brown ◽  
L E Tran ◽  
K J Shaw ◽  
...  
Keyword(s):  
Cell Wall ◽  
Killer Toxin ◽  
Structural Defects ◽  
Beta Glucan ◽  
Calculated Molecular Mass ◽  
Beta Glucanase ◽  
Reading Frame ◽  
Glucan Synthesis ◽  
The Right

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

scholarly journals SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis.

1993 ◽  
Vol 13 (7) ◽  
pp. 4039-4048 ◽  
Author(s):  
T Roemer ◽  
S Delaney ◽  
H Bussey
Keyword(s):  
Genomic Library ◽  
Killer Toxin ◽  
Resistant Mutant ◽  
Null Alleles ◽  
Dependent Manner ◽  
Wild Type ◽  
Beta Glucan ◽  
Glucan Synthesis ◽  
Dose Dependent

KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T. Roemer and H. Bussey, Proc. Natl. Acad. Sci. USA 88:11295-11299, 1991). The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant. To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p. SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels. skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant. Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure. Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis.

scholarly journals Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis.

1994 ◽  
Vol 14 (2) ◽  
pp. 1017-1025 ◽  
Author(s):  
Z Hong ◽  
P Mann ◽  
N H Brown ◽  
L E Tran ◽  
K J Shaw ◽  
...  
Keyword(s):  
Cell Wall ◽  
Killer Toxin ◽  
Structural Defects ◽  
Beta Glucan ◽  
Calculated Molecular Mass ◽  
Beta Glucanase ◽  
Reading Frame ◽  
Glucan Synthesis ◽  
The Right

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

scholarly journals Mutational analysis of the functional domains of yeast K1 killer toxin.

1991 ◽  
Vol 11 (1) ◽  
pp. 175-181 ◽  
Author(s):  
H Zhu ◽  
H Bussey
Keyword(s):  
Cell Wall ◽  
Receptor Binding ◽  
Mutational Analysis ◽  
Killer Toxin ◽  
Alpha Subunit ◽  
Functional Domains ◽  
Beta Subunits ◽  
Channel Formation ◽  
Beta Glucan ◽  
Beta Toxin

To determine the functional domains of K1 killer toxin, we analyzed the phenotypes of a set of mutations throughout regions encoding the alpha- and beta-toxin subunits that allow secretion of mutant toxins. A range of techniques have been used to examine the ability of mutant toxins to bind to beta-glucan cell wall receptor and to form lethal ion channels. Our results indicate that both the alpha and beta subunits are involved in beta-glucan receptor binding. Defects in ion channel formation and toxin immunity are confined to the hydrophobic alpha subunit of the toxin.

scholarly journals Genomic Approach to Identification of Mutations Affecting Caspofungin Susceptibility in Saccharomyces cerevisiae

2004 ◽  
Vol 48 (10) ◽  
pp. 3871-3876 ◽  
Author(s):  
Sarit Markovich ◽  
Aya Yekutiel ◽  
Itamar Shalit ◽  
Yona Shadkchan ◽  
Nir Osherov
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Ergosterol Biosynthesis ◽  
Membrane Function ◽  
Minimum Effective Concentration ◽  
Fourfold Increase ◽  
Microdilution Method ◽  
New Genes ◽  
Broth Microdilution Method ◽  
Glucan Synthesis

ABSTRACT The antifungal agent caspofungin (CAS) specifically interferes with glucan synthesis and cell wall formation. To further study the cellular processes affected by CAS, we analyzed a Saccharomyces cerevisiae mutant collection (4,787 individual knockout mutations) to identify new genes affecting susceptibility to the drug. This collection was screened for increased CAS sensitivity (CAS-IS) or increased CAS resistance (CAS-IR). MICs were determined by the broth microdilution method. Disruption of 20 genes led to CAS-IS (four- to eightfold reductions in the MIC). Eleven of the 20 genes are involved in cell wall and membrane function, notably in the protein kinase C (PKC) integrity pathway (MID2, FKS1, SMI1, and BCK1), chitin and mannan biosynthesis (CHS3, CHS4, CHS7, and MNN10), and ergosterol biosynthesis (ERG5 and ERG6). Four of the 20 genes (TPO1, VPS65, VPS25, and CHC1) are involved in vacuole and transport functions, 3 of the 20 genes (CCR4, POP2, and NPL3) are involved in the control of transcription, and 2 of the 20 genes are of unknown function. Disruption of nine additional genes led to CAS-IR (a fourfold increase of MIC). Five of these nine genes (SLG1, ERG3, VRP1, CSG2, and CKA2) are involved in cell wall function and signal transduction, and two of the nine genes (VPS67 and SAC2) are involved in vacuole function. To assess the specificity of susceptibility to CAS, the MICs of amphotericin B, fluconazole, flucytosine, and calcofluor for the strains were tested. Seven of 20 CAS-IS strains (with disruption of FKS1, SMI1, BCK1, CHS4, ERG5, TPO1, and ILM1) and 1 of 9 CAS-IR strains (with disruption of SLG1) demonstrated selective susceptibility to CAS. To further explore the importance of PKC in CAS susceptibility, the activity of the PKC inhibitor staurosporine in combination with CAS was tested against eight Aspergillus clinical isolates by the microdilution assay. Synergistic or synergistic-to-additive activities were found against all eight isolates by use of both MIC and minimum effective concentration endpoints.

The yeast KRE9 gene encodes an O glycoprotein involved in cell surface beta-glucan assembly

1993 ◽  
Vol 13 (10) ◽  
pp. 6346-6356
Author(s):  
J L Brown ◽  
H Bussey
Keyword(s):  
Cell Wall ◽  
Secretory Pathway ◽  
Null Mutant ◽  
Wild Type ◽  
Beta Glucan ◽  
Extracellular Medium ◽  
Average Molecular Mass ◽  
Growth Impairment ◽  
Additional Cell ◽  
Altered Cell

The yeast KRE9 gene encodes a 30-kDa secretory pathway protein involved in the synthesis of cell wall (1-->6)-beta-glucan. Disruption of KRE9 leads to serious growth impairment and an altered cell wall containing less than 20% of the wild-type amount of (1-->6)-beta-glucan. Analysis of the glucan material remaining in a kre9 delta null mutant indicated a polymer with a reduced average molecular mass. kre9 delta null mutants also displayed several additional cell-wall-related phenotypes, including an aberrant multiply budded morphology, a mating defect, and a failure to form projections in the presence of alpha-factor. Double mutants were generated by crossing kre9 delta strains with strains harboring a null mutation in the KRE1, KRE6, or KRE11 gene, and each of these double mutants was found to be inviable in the SEY6210 background. Similar crosses with null mutations in the KRE5 and SKN1 genes indicated that these double mutants were no more severely affected than kre5 delta or kre9 delta single mutants alone. Antibodies were generated against Kre9p and detected an O glycoprotein of approximately 55 to 60 kDa found in the extracellular medium of a strain overproducing Kre9p.

scholarly journals Characterization of the yeast (1-->6)-beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly.

1994 ◽  
Vol 127 (2) ◽  
pp. 567-579 ◽  
Author(s):  
T Roemer ◽  
G Paravicini ◽  
M A Payton ◽  
H Bussey
Keyword(s):  
Cell Wall ◽  
Beta Glucan ◽  
Homologous Proteins ◽  
Synthetic Lethal ◽  
Cell Wall Assembly ◽  
Cell Biol ◽  
Synthetic Lethal Interactions ◽  
Glucan Synthesis ◽  
Wall Assembly

A characterization of the S. cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H. Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment. Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229). Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect. When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants. These suppression and synthetic lethal interactions, as well as reduced beta-glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly. PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.

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