Cloning and characterization of GNS1: a Saccharomyces cerevisiae gene involved in synthesis of 1,3-beta-glucan in vitro.

The GNS1 gene product is required for the synthesis of 1,3-beta-glucan in vitro, since mutations in this gene result in exhibit an 80 to 90% reduction in 1,3-beta-glucan synthase specific activity. gns1 mutant strains display a pleiotropic phenotype including resistance to a pneumocandin B0 analog (L-733,560), slow growth, and mating and sporulation defects. The gns1-1 mutation was genetically mapped to within 1.35 centimorgans from the MAT locus on chromosome III. The wild-type GNS1 gene was isolated by complementing the pneumocandin resistance phenotype of the gns1-1 mutation and by hybridization with a chromosome III-derived sequence being used as a probe. The nucleotide sequence of GNS1 was determined and compared with the homologous region of the chromosome. The genetic and nucleotide sequence analyzes revealed that GNS1 and the open reading frame, YCR34 [S. Oliver, Q. van der Aart, M. Agostoni-Carbone, and the Chromosome III Sequencing Group, Nature (London) 357:38-46, 1992], represent identical loci in the genome. Cells deleted for GNS1 are viable but exhibit slow growth as well as the pleiotropic phenotype of the gns1 mutants. The putative protein product is predicted to be an integral membrane protein with five transmembrane helices displaying an exoplasmic orientation for the N terminus and a cytoplasmic orientation for the C terminus. This protein may be a subunit of 1,3-beta-glucan synthase.     

Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae

The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Delta2Delta mutant and the quintuple yapsin mutant (5ypsDelta) undergo osmoremedial cell lysis at 37 degrees C. The cell walls of both 5ypsDelta and yps1Delta2Delta mutants have decreased amounts of 1,3- and 1,6-beta-glucan. Although there is decreased incorporation of both 1,3- and 1,6-beta-glucan in the 5ypsDelta mutant in vivo, in vitro specific activity of both 1,3- and 1,6-beta-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsDelta. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Delta mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.     

The CRH family coding for cell wall glycosylphosphatidylinositol proteins with a predicted transglycosidase domain affects cell wall organization and virulence of Candida albicans

In Candida albicans UTR2 (CSF4), CRH11, and CRH12 are members of a gene family (the CRH family) that encode glycosylphosphatidylinositol-dependent cell wall proteins with putative transglycosidase activity. Deletion of genes of this family resulted in additive sensitivity to compounds interfering with normal cell wall formation (Congo red, calcofluor white, SDS, and high Ca(2+) concentrations), suggesting that these genes contribute to cell wall organization. A triple mutant lacking UTR2, CRH11, and CRH12 produced a defective cell wall, as inferred from increased sensitivity to cell wall-degrading enzymes, decreased ability of protoplasts to regenerate a new wall, constitutive activation of Mkc1p, the mitogen-activated protein kinase of the cell wall integrity pathway, and an increased chitin content of the cell wall. Importantly, this was accompanied by a decrease in alkali-insoluble 1,3-beta-glucan but not total glucan content, suggesting that formation of the linkage between 1,3-beta-glucan and chitin might be affected. In support of this idea, localization of a Utr2p-GFP fusion protein largely coincided with areas of chitin incorporation in C. albicans.As UTR2 and CRH11 expression is regulated by calcineurin, a serine/threonine protein phosphatase involved in tolerance to antifungal drugs, cell wall morphogenesis, and virulence, this points to a possible relationship between calcineurin and the CRH family. Deletion of UTR2, CRH11, and CRH12 resulted in only a partial overlap with calcineurin-dependent phenotypes, suggesting that calcineurin has additional targets. Interestingly, cells deleted for UTR2, CRH11, and CRH12 were, like a calcineurin mutant, avirulent in a mouse model of systemic infection but retained the capacity to colonize target organs (kidneys) as the wild type. In conclusion, this work establishes the role of UTR2, CRH11, and CRH12 in cell wall organization and integrity.     

Isolation from Candida albicans of a functional homolog of the Saccharomyces cerevisiae KRE1 gene, which is involved in cell wall beta-glucan synthesis.

The KRE1 gene of Saccharomyces cerevisiae, sacKRE1, appears to be involved in the synthesis of cell wall beta-glucan. S. cerevisiae strains with mutations in the KRE1 gene produce a structurally altered cell wall (1----6)-beta-glucan, which results in resistance to K1 killer toxin. We isolated the canKRE1 gene from Candida albicans by its ability to complement a kre1 mutation in S. cerevisiae and confer sensitivity to killer toxin. Sequence analysis revealed that the predicted protein encoded by canKRE1 shares an overall structural similarity with that encoded by sacKRE1. The canKRE1 protein is composed of an N-terminal signal sequence, a central domain of 46% identity with the sacKRE1 protein, and a C-terminal hydrophobic tract. These structural and functional similarities imply that the canKRE1 gene carries out a function in C. albicans cell wall assembly similar to that observed for sacKRE1 in S. cerevisiae.     

Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall

A novel 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus was recently characterized. This enzyme splits internally a 1,3-beta-glucan molecule and transfers the newly generated reducing end to the non-reducing end of another 1, 3-beta-glucan molecule forming a 1,3-beta linkage, resulting in the elongation of 1,3-beta-glucan chains. The GEL1 gene encoding this enzyme was cloned and sequenced. The predicted amino acid sequence of Gel1p was homologous to several yeast protein families encoded by GAS of Saccharomyces cerevisiae, PHR of Candida albicans, and EPD of Candida maltosa. Although the expression of these genes is required for correct morphogenesis in yeast, the biochemical function of the encoded proteins was unknown. The biochemical assays performed on purified recombinant Gas1p, Phr1p, and Phr2p showed that these proteins have a 1,3-beta-glucanosyltransferase activity similar to that of Gel1p. Biochemical data and sequence analysis have shown that Gel1p is attached to the membrane through a glycosylphosphatidylinositol in a similar manner as the yeast homologous proteins. The activity has been also detected in membrane preparations, showing that this 1,3-beta-glucanosyltransferase is indeed active in vivo. Our results show that transglycosidases anchored to the plasma membrane via glycosylphosphatidylinositols can play an active role in fungal cell wall synthesis.     

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

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.     

Yeast KRE genes provide evidence for a pathway of cell wall beta-glucan assembly

The Saccharomyces cerevisiae KRE1 gene encodes a Ser/Thr-rich protein, that is directed into the yeast secretory pathway, where it is highly modified, probably through addition of O-linked mannose residues. Gene disruption of the KRE1 locus leads to a 40% reduced level of cell wall (1----6)-beta-glucan. Structural analysis of the (1----6)-beta-glucan fraction, isolated from a strain with a krel disruption mutation, showed that it had an altered structure with a smaller average polymer size. Mutations in two other loci, KRE5 and KRE6 also lead to a defect in cell wall (1----6)-beta-glucan production and appear to be epistatic to KRE1. These findings outline a possible pathway of assembly of yeast cell wall (1----6)-beta-glucan.     

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

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.     

Cell wall composition and structure of Yarrowia lipolytica transposon mutants affected in calcofluor sensitivity

A collection of transposon-mutagenized strains of Yarrowia lipolytica was screened for wall defects by determination of their sensitivity to calcofluor white. A number of strains were hypersensitive, whereas others were resistant. Different non-allelic mutants displayed increased sensitivity to autolysis and lytic enzymes, independently of whether they were sensitive or resistant to calcofluor white. A thorough analysis of their cell walls revealed minor quantitative alterations, and no significant changes in chitin content. Electrophoretic analysis of wall-bound and excreted proteins proved to be a sensitive method that revealed defects in the cell wall structure of the mutants. Important alterations in the patterns of the wall proteins extracted by SDS or by enzymatic treatments were noticed for the mutants, as compared to the parental strain. Mutants released to the growth medium a larger number of protein species than the parental strain, suggesting impairment in wall assembly of certain polypeptides. Patterns of wall-bound and excreted proteins, as well as alterations in wall chemical composition were not diagnostic of calcofluor white sensitivity or resistance, but were specific for each mutant. Our data show that an increase in either sensitivity or resistance of Y. lipolytica to certain levels of calcofluor is equally indicative of alterations in cell wall structure, independent of chitin levels. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Analysis of beta-1,3-glucan assembly in Saccharomyces cerevisiae using a synthetic interaction network and altered sensitivity to caspofungin

Large-scale screening of genetic and chemical-genetic interactions was used to examine the assembly and regulation of beta-1,3-glucan in Saccharomyces cerevisiae. Using the set of deletion mutants in approximately 4600 nonessential genes, we scored synthetic interactions with genes encoding subunits of the beta-1,3-glucan synthase (FKS1, FKS2), the glucan synthesis regulator (SMI1/KNR4), and a beta-1,3-glucanosyltransferase (GAS1). In the resulting network, FKS1, FKS2, GAS1, and SMI1 are connected to 135 genes in 195 interactions, with 26 of these genes also interacting with CHS3 encoding chitin synthase III. A network core of 51 genes is multiply connected with 112 interactions. Thirty-two of these core genes are known to be involved in cell wall assembly and polarized growth, and 8 genes of unknown function are candidates for involvement in these processes. In parallel, we screened the yeast deletion mutant collection for altered sensitivity to the glucan synthase inhibitor, caspofungin. Deletions in 52 genes led to caspofungin hypersensitivity and those in 39 genes to resistance. Integration of the glucan interaction network with the caspofungin data indicates an overlapping set of genes involved in FKS2 regulation, compensatory chitin synthesis, protein mannosylation, and the PKC1-dependent cell integrity pathway.     

Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase,in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated multicopy suppressors of the GS mutation. We demonstrate that all of the multicopy suppressors obtained (WSC1, WSC3, MTL1, ROM2, LRE1, ZDS1, and MSB1) and the constitutively active RHO1 mutations tested restore 1,3-beta-glucan synthesis in the GS mutant. A deletion of either ROM2 or WSC1 leads to a significant defect of 1,3-beta-glucan synthesis. Analyses of the degree of Mpk1p phosphorylation revealed that among the multicopy suppressors, WSC1, ROM2, LRE1, MSB1, and MTL1 act positively on the Pkc1p-MAPK pathway, another signaling pathway regulated by Rho1p, while WSC3 and ZDS1 do not. We have also found that MID2 acts positively on Pkc1p without affecting 1,3-beta-glucan synthesis. These results suggest that distinct networks regulate the two effector proteins of Rho1p, Fks1p and Pkc1p.     

Homologous subunits of 1,3-beta-glucan synthase are important for spore wall assembly in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-beta-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-beta-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-beta-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-beta-glucan synthase.     

Enhanced Extraction Efficiency of Recombinant Proteins by Use of the KNR4-Disrupted Strains of Saccharomyces Cerevisiae

A fragile cell wall mutant strain of Saccharomyces cerevisiae was prepared by a single step disruption of the KNR4 gene, which is involved in cell wall biosynthesis. The enhanced transport property of the mutant cell across the loosen cell wall was confirmed by the increased drug permeability. The KNR4-disrupted mutant strain released the ectopically expressed foreign protein more easily than the wild type by mechanical disruption of the cell walls using glass beads, demonstrating a potential utility of using the mutant strain as a host for the efficient extraction of recombinant proteins.     

Up-regulation of the cell integrity pathway in saccharomyces cerevisiae suppresses temperature sensitivity of the pgs1Delta mutant

We have previously shown that mutants in the cardiolipin (CL) pathway exhibit temperature-sensitive growth defects that are not associated with mitochondrial dysfunction. The pgs1Delta mutant, lacking the first enzyme of the CL pathway, phosphatidylglycerolphosphate synthase (Pgs1p), has a defective cell wall due to decreased beta-1,3-glucan (Zhong, Q., Gvozdenovic-Jeremic, J., Webster, P., Zhou, J., and Greenberg, M. L. (2005) Mol. Biol. Cell 16, 665-675). Disruption of KRE5, a gene involved in cell wall biogenesis, restores beta-1,3-glucan synthesis and suppresses pgs1Delta temperature sensitivity. To gain insight into the mechanisms underlying the cell wall defect in pgs1Delta, we show in the current report that pgs1Delta cells have reduced glucan synthase activity and diminished levels of Fks1p, the glucan synthase catalytic subunit. In addition, activation of Slt2p, the downstream effector of the protein kinase C (PKC)-activated cell integrity pathway, was defective in pgs1Delta. The kre5W1166X suppressor restored Slt2p activation and dramatically increased (>10-fold) mRNA levels of FKS2, the alternate catalytic subunit of glucan synthase, partially restoring glucan synthase activity. Consistent with these results, up-regulation of PKC-Slt2 signaling and overexpression of FKS1 or FKS2 alleviated sensitivity of pgs1Delta to cell wall-perturbing agents and restored growth at elevated temperature. These findings demonstrate that functional Pgs1p is essential for cell wall biogenesis and activation of the PKC-Slt2 signaling pathway.     

Complementing yeast rho1 mutation groups with distinct functional defects

Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1A and rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-beta-glucan synthase, respectively. Heteroallelic diploid strains with rho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementing rho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-beta-glucan synthase activity.     

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

Hyperpolarized growth of Saccharomyces cerevisiae cak1P212S and cla4 mutants weakens cell walls and renders cells dependent on chitin synthase 3.

In a screen for cell wall defects in Saccharomyces cerevisiae, we isolated a strain carrying a mutation in the Cdc28-activating kinase CAK1. The cak1P212S mutant cells exhibit multiple, elongated and branched buds, beta(1,3)glucan-poor regions of the cell periphery and lysed upon osmotic shock after treatment with the chitin synthase III inhibitor Nikkomycin Z. Ultrastructural examination of cak1P212S mutants revealed a thin, uneven cell wall and marked abnormalities in septum formation. In all of the above aspects, the cak1P212S mutants are similar to previously described cla4 mutants, suggesting that the cell wall defects are common to mutants with hyperpolarized growth. In cak1P212S mutants, chitin accumulates all over the surface of the cells and glucan synthase activity is located preferentially to the tips of elongated buds. We conclude that the cell wall weakness in cak1P212S mutants is caused by hyperpolarized secretion of glucan synthase and lack of reinforcement of the lateral cell walls. Showing that the defect depends at least in part on Cdc28, the cak1P212S hyperpolarized growth phenotype can be suppressed by a Cak1-independent Cdc28-allele. The results underline the importance of a minor cell wall component, the chitin of lateral walls, for the integrity of the cell in a stress situation.