Mutations in SNF1 complex genes affect yeast cell wall strength

The trimeric SNF1 complex from Saccharomyces cerevisiae, a homolog of mammalian AMP-activated kinase, has been primarily implicated in signaling for the utilization of alternative carbon sources to glucose. We here find that snf1 deletion mutants are hypersensitive to different cell wall stresses, such as the presence of Calcofluor white, Congo red, Zymolyase or the glucan synthase inhibitor Caspofungin in the growth medium. They also have a thinner cell wall. Caspofungin treatment triggers the phosphorylation of the catalytic Snf1 kinase subunit at Thr210 and removal of this phosphorylation site by mutagenesis (Snf1-T210A) abolishes the function of Snf1 in cell wall integrity. Deletion of the PFK1 gene encoding the α-subunit of the heterooctameric yeast phosphofructokinase suppresses the cell wall phenotypes of a snf1 deletion, which suggests a compensatory effect of central carbohydrate metabolism. Epistasis analyses with mutants in cell wall integrity (CWI) signaling confirm that the SNF1 complex and the CWI pathway independently affect yeast cell integrity.     

The cell wall of the human pathogen Candida glabrata: differential incorporation of novel adhesin-like wall proteins

The cell wall of the human pathogen Candida glabrata governs initial host-pathogen interactions that underlie the establishment of fungal infections. With the aim of identifying species-specific features that may directly relate to its virulence, we have investigated the cell wall of C. glabrata using a multidisciplinary approach that combines microscopy imaging, biochemical studies, bioinformatics, and tandem mass spectrometry. Electron microscopy revealed a bilayered wall structure in which the outer layer is packed with mannoproteins. Biochemical studies showed that C. glabrata walls incorporate 50% more protein than Saccharomyces cerevisiae walls and, consistent with this, have a higher mannose/glucose ratio. Evidence is presented that C. glabrata walls contain glycosylphosphatidylinositol (GPI) proteins, covalently bound to the wall 1,6-β-glucan, as well as proteins linked through a mild-alkali-sensitive linkage to 1,3-β-glucan. A comprehensive genome-wide in silico inspection showed that in comparison to other fungi, C. glabrata contains an exceptionally large number, 67, of genes encoding adhesin-like GPI proteins. Phylogenetically these adhesin-like proteins form different clusters, one of which is the lectin-like EPA family. Mass spectrometric analysis identified 23 cell wall proteins, including 4 novel adhesin-like proteins, Awp1/2/3/4, and Epa6, which is involved in adherence to human epithelia and biofilm formation. Importantly, the presence of adhesin-like proteins in the wall depended on the growth stage and on the genetic background used, and this was reflected in alterations in adhesion capacity and cell surface hydrophobicity. We propose that the large repertoire of adhesin(-like) genes of C. glabrata contributes to its adaptability and virulence.     

Cu,Zn-superoxide dismutase is required for cell wall structure and for tolerance to cell wall-perturbing agents in Saccharomyces cerevisiae

Here we report that deletion of SOD1, the Cu,Zn-superoxide dismutase in Saccharomyces cerevisiae is sensitive to cell wall-perturbing agents, such as Calcofluor white and Congo red. The sensitivity was restored by retransformation with wild type SOD1 or the addition of N-acetylcysteine or reduced glutathione to the medium. Additionally, the accumulation of reactive oxygen species was observed in sod1Δ mutant in the presence of Calcofluor white or Congo red. Cell wall analysis indicated an increase of cell wall chitin and cell wall thickness in sod1Δ mutant compared to wild type. These results indicate a novel direct connection between antioxidative functions and cell wall homeostasis.     

A novel yeast gene, RHK1, is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits β-1,3-glucan synthesis

The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall β-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast. A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin. These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups. A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated. DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids. Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S. cerevisiae cells with complete killer resistance. A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of β-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin.     

Cooperation between ER stress and calcineurin signaling contributes to the maintenance of cell wall integrity in Candida glabrata

Candida glabrata is the second most common source of Candida infections in humans. In this pathogen, the maintenance of cell wall integrity (CWI) frequently precludes effective pharmacological treatment by antifungal agents. In numerous fungi, cell wall modulation is reported to be controlled by endoplasmic reticulum (ER) stress, but how the latter affects CWI maintenance in C. glabrata is not clearly understood. Here, we characterized a C. glabrata strain harboring a mutation in the CNE1 gene, which encodes a molecular chaperone associated with nascent glycoprotein maturation in the ER. Disruption of cne1 induced ER stress and caused changes in the normal cell wall structure, specifically a reduction in the β-1,6-glucan content and accumulation of chitin. Conversely, a treatment with the typical ER stress inducer tunicamycin up-regulated the production of cell wall chitin but did not affect β-1,6-glucan content. Our results also indicated that C. glabrata features a uniquely evolved ER stress-mediated CWI pathway, which differs from that in the closely related species Saccharomyces cerevisiae. Furthermore, we demonstrated that ER stress-mediated CWI pathway in C. glabrata is also induced by the disruption of other genes encoding proteins that function in a correlated manner in the quality control of N-linked glycoproteins in the ER. These results suggest that calcineurin and ER quality control system act as a platform for maintaining CWI in C. glabrata.     

Cohesin dysfunction results in cell wall defects in budding yeast

Cohesin is a conserved chromatin-binding multisubunit protein complex involved in diverse chromosomal transactions such as sister-chromatid cohesion, chromosome condensation, regulation of gene expression, DNA replication, and repair. While working with a budding yeast temperature-sensitive mutant, mcd1-1, defective in a cohesin subunit, we observed that it was resistant to zymolyase, indicating an altered cell wall organization. The budding yeast cell wall is a strong but elastic structure essential for maintenance of cell shape and protection from extreme environmental challenges. Here, we show that the cohesin complex plays an important role in cell wall maintenance. Cohesin mutants showed high chitin content in the cell wall and sensitivity to multiple cell wall stress-inducing agents. Interestingly, temperature-dependent lethality of cohesin mutants was osmoremedial, in a HOG1-MAPK pathway-dependent manner, suggesting that the temperature sensitivity of these mutants may arise partially from cell wall defects. Moreover, Mpk1 hyper-phosphorylation indicated activation of the cell wall integrity (CWI) signaling pathway in cohesin mutants. Genetic interaction analysis revealed that the CWI pathway is essential for survival of mcd1-1 upon additional cell wall stress. The cell wall defect was independent of the cohesion function and accompanied by misregulation of expression of several genes having cell wall-related functions. Our findings reveal a requirement of cohesin in maintenance of CWI that is independent of the CWI pathway, and that may arise from cohesin’s role in regulating the expression of multiple genes encoding proteins involved in cell wall organization and biosynthesis.     

Roles of septins in prospore membrane morphogenesis and spore wall assembly in Saccharomyces cerevisiae

The highly conserved family of septin proteins has important functions in cytokinesis in mitotically proliferating cells. A different form of cytokinesis occurs during gametogenesis in Saccharomyces cerevisiae, in which four haploid meiotic products become encased by prospore membrane (PSMs) and specialized, stress-resistant spore walls. Septins are known to localize in a series of structures near the growing PSM, but previous studies noted only mild sporulation defects upon septin mutation. We report that directed PSM extension fails in many septin-mutant cells, and, for those that do succeed, walls are abnormal, leading to increased susceptibility to heating, freezing, and digestion by the Drosophila gut. Septin mutants mislocalize the leading-edge protein (LEP) complex required for normal PSM and wall biogenesis, and ectopic expression of the LEP protein Ssp1 perturbs mitotic septin localization and function, suggesting a functional interaction. Strikingly, extra copies of septin CDC10 rescue sporulation and LEP localization in cells lacking Sma1, a phospholipase D–associated protein dispensable for initiation of PSM assembly and PSM curvature but required for PSM extension. These findings point to key septin functions in directing efficient membrane and cell wall synthesis during budding yeast gametogenesis.     

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.     

The Golgi GDPase of the fungal pathogen Candida albicans affects morphogenesis,glycosylation, and cell wall properties

Cell wall mannoproteins are largely responsible for the adhesive properties and immunomodulation ability of the fungal pathogen Candida albicans. The outer chain extension of yeast mannoproteins occurs in the lumen of the Golgi apparatus. GDP-mannose must first be transported from the cytosol into the Golgi lumen, where mannose is transferred to mannans. GDP is hydrolyzed by a GDPase, encoded by GDA1, to GMP, which then exits the Golgi lumen in a coupled, equimolar exchange with cytosolic GDP-mannose. We isolated and disrupted the C. albicans homologue of the Saccharomyces cerevisiae GDA1 gene in order to investigate its role in protein mannosylation and pathogenesis. CaGda1p shares four apyrase conserved regions with other nucleoside diphosphatases. Membranes prepared from the C. albicans disrupted gda1/gda1 strain had a 90% decrease in the ability to hydrolyze GDP compared to wild type. The gda1/gda1 mutants showed a severe defect in O-mannosylation and reduced cell wall phosphate content. Other cell wall-related phenotypes are present, such as elevated chitin levels and increased susceptibility to attack by β-1,3-glucanases. Our results show that the C. albicans organism contains β-mannose at their nonreducing end, differing from S. cerevisiae, which has only α-linked mannose residues in its O-glycans. Mutants lacking both alleles of GDA1 grow at the same rate as the wild type but are partially blocked in hyphal formation in Lee solid medium and during induction in liquid by changes in temperature and pH. However, the mutants still form normal hyphae in the presence of serum and N-acetylglucosamine and do not change their adherence to HeLa cells. Taken together, our data are in agreement with the hypothesis that several pathways regulate the yeast-hypha transition. Gda1/gda1 cells offer a model for discriminating among them.     

The Sur7 protein regulates plasma membrane organization and prevents intracellular cell wall growth in Candida albicans

The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Δ mutant are similar to the effects of inhibiting β-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains.     

Construction of a Library of Human Glycosyltransferases Immobilized in the Cell Wall of Saccharomyces cerevisiae

Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pir-glycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides.     

Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae

Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal sequence for secretion and a serine- or threonine-rich sequence. In all, 53 ORFs met these three criteria and 38 were further analyzed as follows. The sequence encoding the 40 C-terminal amino acids of each was fused with the structural gene for a reporter protein consisting of a secretion signal, α-galactosidase and a hemagglutinin (HA) epitope, and examined for the ability to become incorporated into the cell wall. On this basis, 14 of fusion proteins were classified as GPI-dependent cell wall proteins because cells expressing these fusion proteins: (i) had high levels of α-galactosidase activity on their surface; (ii) released significant amounts of the fusion proteins from the membrane on treatment with phosphatidylinositol-specific phospholipase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase. Of the 14 identified putative GPI-dependent cell wall proteins, 12 had novel ORFs adjacent to their GPI-attachment signal sequence. Amino acid sequence alignment of the C-terminal sequences of the 12 ORFs, together with those of known cell wall proteins, reveals some sequence similarities among them.     

Transposon-Induced Mutations in Two Loci of Listeria monocytogenes Serotype 1/2a Result in Phage Resistance and Lack of N-Acetylglucosamine in the Teichoic Acid of the Cell Wall

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916ΔE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.     

Cell wall residues in yeast protoplast preparations

Protoplast preparations made from Saccharomyces cerevisiae by prolonged treatment with snail digestive juice contained fibrils and chitinous bud-scar residues from the original cell wall.     

Secretory pattern of a major integral mannoprotein of the yeast cell wall

A major integral mannoprotein of the Saccharomyces cerevisiae cell wall with an apparent size of 33 kDa has been purified and polyclonal antibodies have been raised against its enzymatically deglycosylated protein moiety. These have been employed to immunodetect the molecular forms of this species accumulated in yeast sec mutants. Microsomal preparations from those mutants blocked at the initial steps of the secretory route (sec53 and sec59) contained the 30.5 kDa non-glycosylated form, although minor amounts of partially glycosylated forms and the 33 kDa one were also immunodetected in sec53 membranes. Mutants blocked at later steps (sec18, sec7 and sec1) accumulated only the glyciosylated form. Criteria such as sensitivity to pronase and solubilization by Triton-urea in mild conditions have been employed to study the topology of the 30.5 and 33 kDa forms in intact microsomal vesicles. In sec53 and sec59 cells, the non-glycosylated form accumulates at the cytoplasmic face of the endoplasmic reticulum, although in the case of the sec59 mutant the molecules appear to be tightly stuck to the membrane. In sec1 and sec18 cells, the 33 kDa mannoprotein is apparently localized at the vesicular lumen as a peripheral-membrane form or as an entirely soluble one.     

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2 (PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.Abbreviations Pi inorganic phosphate - PiBP2 phosphate binding protein 2 obtained from Candida tropicalis - Tris Tris(hydroxymethyl)-aminoethane - MES [2-(N-Morpholino)] ethanesulfonic acid - EDTA ethylene diamine tetraacetic acid, disoldium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

A Screen for Spore Wall Permeability Mutants Identifies a Secreted Protease Required for Proper Spore Wall Assembly

The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat.     

The Small GTP-Binding Proteins AgRho2 and AgRho5 Regulate Tip-Branching,Maintenance of the Growth Axis and Actin-Ring-Integrity in the Filamentous Fungus Ashbya gossypii

GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from Saccharomyces cerevisiae and of their homologs in other species is poorly understood. Here, we report on the analysis of the AgRho2 and AgRho5 proteins of the filamentous fungus Ashbya gossypii. In contrast to S. cerevisiae mutants of both encoding genes displayed a strong morphological phenotype. The Agrho2 mutants showed defects in tip-branching, while Agrho5 mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the Agrho5 mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the Agrho5 mutant, suggesting that both proteins act in the same pathway, but the AgRho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.