Overexpression of Sbe2p, a Golgi Protein, Results in Resistance to Caspofungin in Saccharomyces cerevisiae

ABSTRACT Caspofungin inhibits the synthesis of 1, 3-β-d-glucan, an essential cell wall target in fungi. Genetic studies in the model yeast Saccharomyces cerevisiae have shown that mutations in FKS1 and FKS2 genes result in caspofungin resistance. However, direct demonstration of the role of gene overexpression in caspofungin resistance has been lacking. We transformed wild-type S. cerevisiae with an S. cerevisiae URA3-based GAL1 cDNA library and selected transformants in glucose synthetic complete plates lacking uracil (glucose SC minus uracil plates). We then moved the transformants to galactose SC minus uracil plates containing caspofungin (1 μg/ml) and looked for caspofungin-resistant colonies. We retested the candidates (true positives were sensitive on glucose caspofungin and resistant on galactose caspofungin media, respectively). We identified 16 caspofungin-resistant candidates. Restriction analysis and hybridization confirmed that 15 of the 16 clones were identical. We sequenced one of the cDNA clones and found that it contained the cDNA for SBE2. SBE2 has been described in S. cerevisiae to encode a Golgi protein involved in the transport of cell wall components (B. Santos and M. Snyder, Mol. Biol. Cell, 11:435-452, 2000). The SBE2 cDNA plasmid conferred again galactose-dependent caspofungin resistance when transformed back into the wild-type S. cerevisiae. Finally, the SBE2 deletion mutant was hypersensitive to caspofungin. In conclusion, overexpression of Sbe2p under the regulated control of the GAL1 promoter results in caspofungin resistance in S. cerevisiae. This transport pathway may provide insight into the tolerance or lack of sensitivity to caspofungin of some pathogenic fungi.

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Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells.

The Journal of Cell Biology ◽

10.1083/jcb.110.1.105 ◽

1990 ◽

Vol 110 (1) ◽

pp. 105-114 ◽

Cited By ~ 153

Author(s):

B K Haarer ◽

S H Lillie ◽

A E Adams ◽

V Magdolen ◽

W Bandlow ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Actin Polymerization ◽

Yeast Cells ◽

Predicted Amino Acid Sequence ◽

Temperature Sensitive ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Ellipsoidal Shape ◽

Hydrolysis Of

We have isolated profilin from yeast (Saccharomyces cerevisiae) and have microsequenced a portion of the protein to confirm its identity; the region microsequenced agrees with the predicted amino acid sequence from a profilin gene recently isolated from S. cerevisiae (Magdolen, V., U. Oechsner, G. Müller, and W. Bandlow. 1988. Mol. Cell. Biol. 8:5108-5115). Yeast profilin resembles profilins from other organisms in molecular mass and in the ability to bind to polyproline, retard the rate of actin polymerization, and inhibit hydrolysis of ATP by monomeric actin. Using strains that carry disruptions or deletions of the profilin gene, we have found that, under appropriate conditions, cells can survive without detectable profilin. Such cells grow slowly, are temperature sensitive, lose the normal ellipsoidal shape of yeast cells, often become multinucleate, and generally grow much larger than wild-type cells. In addition, these cells exhibit delocalized deposition of cell wall chitin and have dramatically altered actin distributions.

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Use of Saccharomyces cerevisiae expressing beta-galactosidase to screen for antimycotic agents directed against yeast cell wall biosynthesis and possible application to pathogenic fungi.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.34.4.660 ◽

1990 ◽

Vol 34 (4) ◽

pp. 660-662 ◽

Cited By ~ 7

Author(s):

P G Zaworski ◽

G S Gill

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Yeast Cell ◽

Pathogenic Fungi ◽

Yeast Cell Wall ◽

Cell Wall Biosynthesis ◽

Beta Galactosidase

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Deletion of YLR358C Contributes to Reduce Cell Wall Integrity in Saccharomyces Cerevisiae

10.21203/rs.3.rs-1235133/v1 ◽

2022 ◽

Author(s):

Yu Zhang ◽

Mengyan Li ◽

Hanying Wang ◽

Juqing Deng ◽

Jianxing Liu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Sodium Dodecyl ◽

Wall Stress ◽

Pathogenic Fungi ◽

Cell Wall Integrity ◽

Calcofluor White ◽

Fungal Cell ◽

Reading Frame ◽

Stress Signals

Abstract The mechanism of fungal cell wall synthesis and assembly is still unclear. Saccharomyces cerevisiae (S. cerevisiae) and pathogenic fungi are conserved in cell wall construction and response to stress signals, and often respond to cell wall stress through activated cell wall integrity (CWI) pathways. Whether the YLR358C open reading frame regulates CWI remains unclear. This study found that the growth of S. cerevisiae with YLR358C knockout was significantly inhibited on the medium containing different concentrations of cell wall interfering agents Calcofluor White (CFW), Congo Red (CR) and sodium dodecyl sulfate (SDS). CFW staining showed that the cell wall chitin was down-regulated, and transmission electron microscopy also observed a decrease in cell wall thickness. Transcriptome sequencing and analysis showed that YLR358C gene may be involved in the regulation of CWI signaling pathway. It was found by qRT-PCR that WSC3, SWI4 and HSP12 were differentially expressed after YLR358C was knocked out. The above results suggest that YLR358C may regulate the integrity of the yeast cell walls and has some potential for application in fermentation.

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Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.3.5.922-930.1983 ◽

1983 ◽

Vol 3 (5) ◽

pp. 922-930

Author(s):

R L Roberts ◽

B Bowers ◽

M L Slater ◽

E Cabib

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Cell Wall ◽

Permissive Temperature ◽

Wild Type ◽

Chitin Synthesis ◽

Specific Staining ◽

Septal Region ◽

Wall Components ◽

Generalized Distribution

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.

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Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways

Microbiology ◽

10.1099/mic.0.26110-0 ◽

2003 ◽

Vol 149 (5) ◽

pp. 1193-1204 ◽

Cited By ~ 73

Author(s):

Iwona Wojda ◽

Rebeca Alonso-Monge ◽

Jan-Paul Bebelman ◽

Willem H. Mager ◽

Marco Siderius

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Osmotic Stress ◽

Map Kinase ◽

Growth Temperature ◽

Wild Type ◽

Hog Pathway ◽

Map Kinase Pathway ◽

Kinase Pathway ◽

Intracellular Glycerol

In the yeast Saccharomyces cerevisiae, response to an increase in external osmolarity is mediated by the HOG (high osmolarity glycerol) MAP kinase pathway. HOG pathway mutant strains display osmosensitive phenotypes. Recently evidence has been obtained that the osmosensitivity of HOG pathway mutants is reduced during growth at elevated temperature (37 °C). A notable exception is the ste11ssk2ssk22 mutant, which displays hypersensitivity to osmotic stress at 37 °C. This paper reports that overexpression of FPS1 or GPD1 (encoding the glycerol transport facilitator and glycerol-3-phosphate dehydrogenase, respectively, and both affecting intracellular glycerol levels) reduces the hypersensitivity to osmotic stress of ste11ssk2ssk22 at 37 °C. Although in this particular HOG pathway mutant a correlation between suppression of the phenotype and glycerol content could be demonstrated, the absolute level of intracellular glycerol per se does not determine whether a strain is osmosensitive or not. Rather, evidence was obtained that the glycerol level may have an indirect effect, viz. by influencing signalling through the PKC (protein kinase C) MAP kinase pathway, which plays an important role in maintenance of cellular integrity. In order to validate the data obtained with a HOG pathway mutant strain for wild-type yeast cells, MAP kinase signalling under different growth conditions was examined in wild-type strains. PKC pathway signalling, which is manifest at elevated growth temperature by phosphorylation of MAP kinase Mpk1p, is rapidly lost when cells are shifted to high external osmolarity conditions. Expression of bck1-20 or overexpression of WSC3 in wild-type cells resulted in restoration of PKC signalling. Both PKC and HOG signalling, cell wall phenotypes and high osmotic stress responses in wild-type cells were found to be influenced by the growth temperature. The data taken together indicate the intricate interdependence of growth temperature, intracellular glycerol, cell wall structure and MAP kinase signalling in the hyperosmotic stress response of yeast.

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Evidence for a Role for the Plasma Membrane in the Nanomechanical Properties of the Cell Wall as Revealed by an Atomic Force Microscopy Study of the Response of Saccharomyces cerevisiae to Ethanol Stress

Applied and Environmental Microbiology ◽

10.1128/aem.01213-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4789-4801 ◽

Cited By ~ 21

Author(s):

Marion Schiavone ◽

Cécile Formosa-Dague ◽

Carolina Elsztein ◽

Marie-Ange Teste ◽

Helene Martin-Yken ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Atomic Force Microscopy ◽

Cell Wall ◽

Cell Membrane ◽

Yeast Cells ◽

Ethanol Stress ◽

Wild Type ◽

Force Microscopy ◽

Nanomechanical Properties ◽

Atomic Force

ABSTRACTA wealth of biochemical and molecular data have been reported regarding ethanol toxicity in the yeastSaccharomyces cerevisiae. However, direct physical data on the effects of ethanol stress on yeast cells are almost nonexistent. This lack of information can now be addressed by using atomic force microscopy (AFM) technology. In this report, we show that the stiffness of glucose-grown yeast cells challenged with 9% (vol/vol) ethanol for 5 h was dramatically reduced, as shown by a 5-fold drop of Young's modulus. Quite unexpectedly, a mutant deficient in the Msn2/Msn4 transcription factor, which is known to mediate the ethanol stress response, exhibited a low level of stiffness similar to that of ethanol-treated wild-type cells. Reciprocally, the stiffness of yeast cells overexpressingMSN2was about 35% higher than that of the wild type but was nevertheless reduced 3- to 4-fold upon exposure to ethanol. Based on these and other data presented herein, we postulated that the effect of ethanol on cell stiffness may not be mediated through Msn2/Msn4, even though this transcription factor appears to be a determinant in the nanomechanical properties of the cell wall. On the other hand, we found that as with ethanol, the treatment of yeast with the antifungal amphotericin B caused a significant reduction of cell wall stiffness. Since both this drug and ethanol are known to alter, albeit by different means, the fluidity and structure of the plasma membrane, these data led to the proposition that the cell membrane contributes to the biophysical properties of yeast cells.IMPORTANCEEthanol is the main product of yeast fermentation but is also a toxic compound for this process. Understanding the mechanism of this toxicity is of great importance for industrial applications. While most research has focused on genomic studies of ethanol tolerance, we investigated the effects of ethanol at the biophysical level and found that ethanol causes a strong reduction of the cell wall rigidity (or stiffness). We ascribed this effect to the action of ethanol perturbing the cell membrane integrity and hence proposed that the cell membrane contributes to the cell wall nanomechanical properties.

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The genetic basis of aneuploidy tolerance in wild yeast

eLife ◽

10.7554/elife.52063 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 13

Author(s):

James Hose ◽

Leah E Escalante ◽

Katie J Clowers ◽

H Auguste Dutcher ◽

DeElegant Robinson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Drug Treatment ◽

Genetic Basis ◽

Rna Binding ◽

Laboratory Strain ◽

Pathogenic Fungi ◽

Wild Type ◽

Wild Yeast ◽

Mechanistic Understanding

Aneuploidy is highly detrimental during development yet common in cancers and pathogenic fungi – what gives rise to differences in aneuploidy tolerance remains unclear. We previously showed that wild isolates of Saccharomyces cerevisiae tolerate chromosome amplification while laboratory strains used as a model for aneuploid syndromes do not. Here, we mapped the genetic basis to Ssd1, an RNA-binding translational regulator that is functional in wild aneuploids but defective in laboratory strain W303. Loss of SSD1 recapitulates myriad aneuploidy signatures previously taken as eukaryotic responses. We show that aneuploidy tolerance is enabled via a role for Ssd1 in mitochondrial physiology, including binding and regulating nuclear-encoded mitochondrial mRNAs, coupled with a role in mitigating proteostasis stress. Recapitulating ssd1Δ defects with combinatorial drug treatment selectively blocked proliferation of wild-type aneuploids compared to euploids. Our work adds to elegant studies in the sensitized laboratory strain to present a mechanistic understanding of eukaryotic aneuploidy tolerance.

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Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in Schizosaccharomyces pombe

Journal of Bacteriology ◽

10.1128/jb.181.4.1356-1359.1999 ◽

1999 ◽

Vol 181 (4) ◽

pp. 1356-1359 ◽

Cited By ~ 25

Author(s):

Naotaka Tanaka ◽

Atsuro Awai ◽

M. Shah Alam Bhuiyan ◽

Kiyotaka Fujita ◽

Hiroshi Fukui ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Cell Surface ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Wild Type ◽

Liquid Media ◽

Calcium Dependent ◽

Yeast Mutants

ABSTRACT We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, thegsf1 mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike Saccharomyces cerevisiae FLO mutants. The gsf1 mutant coflocculated withSchizosaccharomyces pombe wild-type cells, while no coflocculation was found with galactose-deficient (gms1Δ) cells. Moreover, flocculation of the gsf1 mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from gms1Δ cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors ofgsf1-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in S. pombe.

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Mitochondrial Function in Cell Wall Glycoprotein Synthesis in Saccharomyces cerevisiae NCYC 625 (Wild Type) and [rho0] Mutants

Applied and Environmental Microbiology ◽

10.1128/aem.65.12.5398-5402.1999 ◽

1999 ◽

Vol 65 (12) ◽

pp. 5398-5402 ◽

Cited By ~ 11

Author(s):

Annie Rakotoarivony Iung ◽

Joël Coulon ◽

Ferenc Kiss ◽

Jacques Ngondi Ekome ◽

Judit Vallner ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Mitochondrial Function ◽

Type Strain ◽

Wild Type Strain ◽

Extracellular Enzymes ◽

Biologically Active ◽

Cell Recognition ◽

Wild Type ◽

Glycoprotein Synthesis

ABSTRACT We studied phosphopeptidomannans (PPMs) of two Saccharomyces cerevisiae NCYC 625 strains (S. diastaticus): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a [rho 0] mutant grown aerobically and anaerobically. The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent. Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type. The [rho 0] mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities. PPMs extracted from different cells differ in the protein but not in the glycosidic moiety. The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis. The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a [rho 0] mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon. These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs.

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