Characterization and behaviour of alpha-glucan synthase in Schizosaccharomyces pombe as revealed by electron microscopy

Alpha-1,3-Glucan is a cell wall component in Schizosaccharomyces pombe and is exclusive to budding yeast. We analysed the ultrastructure of the cell wall in the alpha-glucan synthase mutant mok1 and determined the role of alpha-1,3-glucan in cell wall formation of Sz. pombe. The mok1 mutant cell has an abnormal shape, with swelling at the tip or at the site of the septum. The cell wall is thicker and looser than that of wild-type cells, and the layered structure of the cell wall is broken. The glucan fibrils forming the protoplast retain a fine fibril structure, although their development into bundles is abnormal. We also report the localization of Mok1p by immunoelectron microscopy using high-pressure freeze substitution and SDS-digested freeze-fracture replica labelling methods. The Mok1p is localized on the cell membrane and moves from the cell tip to the medial region during the cell cycle. These results confirm that Mok1p plays an important role in the normal construction of the cell wall and in the primary step of glucan bundle formation, and that it is required for new cell wall synthesis during vegetative growth. These findings suggest that alpha-1,3-glucan is an essential component for cell wall formation in fission yeast.     

Mutations in Fks1p affect the cell wall content of beta-1,3- and beta-1,6-glucan in Saccharomyces cerevisiae

Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. Analysis of fks1 delta mutants showed a partial K1 killer toxin-resistant phenotype and a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1 delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. Overexpression of FKS2 suppressed the killer toxin phenotype of fks1 delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1 delta cells. In addition, eight out of 12 fks1ts fks2 delta mutants had altered beta-glucan levels at the permissive temperature: the partial killer resistant FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive allele FKS1T605I M761T increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. We analysed Fks1p and Fks2p in beta-1,6-glucan deficient mutants and found that they were mislocalized and that the mutants had reduced in vitro glucan synthase activity, possibly contributing to the observed beta-1,6-glucan defects.     

Actin patch assembly proteins Las17p and Sla1p restrict cell wall growth to daughter cells and interact with cis-Golgi protein Kre6p

The cytoplasmic tail of Kre6p, a Golgi membrane protein involved in cell wall synthesis, interacts with the actin patch assembly components Las17p and Sla1p in a two-hybrid assay, and Kre6p co-immunoprecipitates with Las17p. Kre6p showed extensive co-localization with Och1p-containing cis-Golgi vesicles. The correct localization of Kre6p requires its cytoplasmic tail, Las17p, Sla1p and Vrp1p, suggesting that the cytoplasmic tail of Kre6p acts as a receptor, linking this cis-Golgi protein to Las17p and Sla1p. The actin patch assembly mutants las17 delta, sla1delta and vrp1 delta showed elevated levels of cell wall beta-1,6-glucan, and mutant cells were capable of only a limited number of cell divisions compared to wild-type. EM image analysis and beta-1,6-glucan localization indicated abnormal wall proliferation in the mother cells of these mutants. The pattern of cell wall hypertrophy indicates a failure to restrict cell wall growth to the bud.     

Glycogen--a covalently linked component of the cell wall in Saccharomyces cerevisiae.

Glycogen in Saccharomyces cerevisiae is present in two pools, one soluble and intracellular, the other present in the cell wall and rendered water-insoluble owing to its covalent linkage to cell wall beta-glucan. The insoluble glycogen fraction was solubilized using beta-1,3-glucanase. The alpha beta-glucan complex obtained showed intense red staining with iodine and was isolated from free beta-glucans by affinity chromatography using concanavalin A sepharose 4B. Further use of molecular sieving has confirmed that glycogen is linked to beta-glucan as the non-retained fraction on Biogel P2 split into two peaks on treatment with amyloglucosidase. Partial acid hydrolysis and subsequent paper chromatography of the alpha beta-glucan complex isolated revealed the presence of gentiobiose and other higher oligosaccharides, indicating that glycogen is linked to beta-1,3-glucan through a beta-1,6 branch. The insoluble glycogen can be extracted in a soluble form by acetic acid treatment and is known as acid-soluble glycogen. The presence of glycogen in the cell wall is confirmed by controlled enzymatic release of alpha beta-glucan complex using lyticase from Arthobacter luteus without disruption of the plasma membrane, as can be visualized using electron microscopy.     

Functional,comparative and cell biological analysis of Saccharomyces cerevisiae Kre5p

Saccharomyces cerevisiae kre5delta mutants lack beta-1,6-glucan, a polymer required for proper cell wall assembly and architecture. A functional and cell biological analysis of Kre5p was conducted to further elucidate the role of this diverged protein glucosyltransferase-like protein in beta-1,6-glucan synthesis. Kre5p was found to be a primarily soluble N-glycoprotein of approximately 200 kDa, that localizes to the endoplasmic reticulum. The terminal phenotype of Kre5p-deficient cells was observed, and revealed a severe cell wall morphological defect. KRE6, encoding a glucanase-like protein, was identified as a multicopy suppressor of a temperature-sensitive kre5 allele, suggesting that these proteins may participate in a common beta-1,6-biosynthetic pathway. An analysis of truncated versions of Kre5p indicated that all major regions of the protein are required for viability. Finally, Candida albicans KRE5 was shown to partially restore growth to S. cerevisiae kre5delta cells, suggesting that these proteins are functionally related.     

A single FKS homologue in Yarrowia lipolytica is essential for viability

The synthesis of beta-1,3-glucan, the structural component of the yeast cell wall which gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of beta-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role. FKS1 has been characterized in Saccharomyces cerevisiae, where its function is at least partially redundant with that of FKS2/GSC2. FKS homologues have also been identified in several other fungal species, including Candida albicans, Schizosaccharomyces pombe, Aspergillus nidulans, Cryptococcus neoformans and Paracoccidiodes brasiliensis. In this work, we have used degenerate oligonucleotides derived from the conserved regions of Fks1ps to isolate the possible FKS homologue genes of the strictly aerobic non-conventional yeast Yarrowia lipolytica. Using this approach, we have isolated a single FKS homologue which we have named YlFKS1; this codes a 1961 amino acid protein that shows a high degree of homology with other Fksps. Expression analysis of YlFKS1 under different conditions affecting the cell wall did not reveal significant differences. Finally, attempts to obtain a Y. lipolytica strain containing a disrupted YlFKS1 allele failed, despite having used two different techniques. Taken together, these results suggest that, unlike S. cerevisiae, YlFKS1 is the only FKS1 homologue in Y. lipolytica and is essential for growth.     

Binding assay for incorporation of alkali-extractable proteins in the Saccharomyces cerevisiae cell wall

Yeasts have developed three different ways of attaching proteins to cell wall glucan. Some proteins are bound to beta-1,3-glucan non-covalently, while others are attached covalently, through GPI-anchor and beta-1,6-glucan, or directly to beta-1,3-glucan by alkali-labile ester linkage between the gamma-carboxyl groups of glutamic acid and the hydroxyl groups of glucoses (Pir proteins). In order to obtain further insight into the binding mechanism, a novel, simple binding assay for Pir-family proteins was developed. It has been shown that PIR, as well as SCW4 mutants, can bind externally added Ccw5p to their cell walls. A study of appropriate binding conditions revealed the requirement of the native conformation of Ccw5p. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild-type and mutant cells showed enhanced binding of the Ccw5p in 0.6 M KCl. After disruption of all Pir genes (CCW5, CCW6, CCW7 and CCW8), 67 kDa protein still remained in NaOH extract. SCW4 disruption in the ccw5ccw6ccw7ccw8 mutant resulted in disappearance of the 67 kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall by a so-far unidentified alkali-labile linkage.     

Disruption of LRG1 inhibits mother-daughter separation in Saccharomyces cerevisiae

LRG1, previously characterized as a Rho-GAP that regulates beta-1,3-glucan synthesis, was identified using a filtration screen designed to isolate genes that promote cell aggregation in Sigma1278b Saccharomyces cerevisiae. Disruption of LRG1 in haploid cells resulted in enhanced invasive growth and a strain-specific 'clustered' phenotype that is a consequence of failed separation of mother and daughter cells. Genetic analysis revealed that clustering required functional ROM2, FKS1 and STE12 but not FLO8 or FLO11. Additionally, this phenotype required sufficient nitrogen. beta-1,3-Glucan content was elevated in lrg1 haploids and these cells possessed a thicker cell wall and septum than wild-type strains. Additional glucan at the bud site inhibited separation of lrg1 mutants as the clustered phenotype was repressed by expression of the glucanases ENG1 or EGT2.     

Saccharomyces cerevisiae Big1p,a putative endoplasmic reticulum membrane protein required for normal levels of cell wall beta-1,6-glucan

Deletion of Saccharomyces cerevisiae BIG1 causes an approximately 95% reduction in cell wall beta-1,6-glucan, an essential polymer involved in the cell wall attachment of many surface mannoproteins. The big1 deletion mutant grows very slowly, but growth can be enhanced if cells are given osmotic support. We have begun a cell biological and genetic analysis of its product. We demonstrate, using a Big1p-GFP fusion construct, that Big1p is an N-glycosylated integral membrane protein with a Type I topology that is located in the endoplasmic reticulum (ER). Some phenotypes of a big1Delta mutant resemble those of strains disrupted for KRE5, which encodes another ER protein affecting beta-l,6-glucan levels to a similar extent. In a big1Deltakre5Delta double mutant, both the growth and alkali-soluble beta-l,6-glucan levels were reduced as compared to either single mutant. Thus, while Big1p and Kre5p may have similar effects on beta-l,6-glucan synthesis, these effects are at least partially distinct. Residual beta-l,6-glucan levels in the big1Deltakre5Delta double mutant indicate that these gene products are unlikely to be beta-l,6-glucan synthase subunits, but rather may play some ancillary roles in beta-l,6-glucan synthase assembly or function, or in modifying proteins for attachment of beta-l,6-glucan.     

SKG1, a suppressor gene of synthetic lethality of kex2Deltagas1Delta mutations, encodes a novel membrane protein that affects cell wall composition

The fungal GAS1-related genes encode GPI-anchored beta-1,3-glucanosyltransferase, and their loss causes a defect in the assembly of the cell wall. The KEX2 gene encodes a processing protease in the late Golgi compartment and its loss also results in defects in the cell wall. Simultaneous mutations of these genes are lethal in Saccharomyces cerevisiae. To understand the basis of this synthetic lethality, we screened for multicopy suppressors and identified 13 SKG (suppressor of kex2 gas1 synthetic lethality) genes. SKG1 encodes a transmembrane protein that localizes on the inner surface of the plasma membrane at the bud and in the daughter cell. The multicopy SKG1 increases the sensitivity of cells to zymolyase, and the skg1Delta null mutation increases resistance to it. This zymolyase susceptibility corresponds to an increase of alkali-soluble beta-1,3-glucan and a decrease of chitin in the cell wall. Thus SKG1 encodes a novel protein that affects the cell wall polymer composition in the growing region of the cell.     

Characterization of a Schizosaccharomyces pombe mutant deficient in UDP-galactose transport activity.

In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist of galactomannan, unlike in Saccharomyces cerevisiae. We previously found that the disruption of gms1+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, led to the complete defect of cell surface galactosylation in Sz. pombe. The Deltagms1 strain is therefore useful for the analysis of physiological properties of galactose residues in Sz. pombe. The deletion strain of gms1+ was viable; however, itshowed an aberrant cell morphology and increased sensitivities to digestion with beta-glucanase and to various drugs, such as hygromycin B, sodium orthovanadate and Calcofluor white. A reduction of galactomannan layers of the cell wall in the Deltagms1 strain was observed by scanning and transmission electron microscopic analyses. The addition of osmotic stabilizer suppressed the morphologic defect of the Deltagms1 cells, while other phenotypes were weakly suppressed. The Deltagms1 (h90) strain was incapable of sexual conjugation during nutritional starvation. These results suggest that the cell surface galactosylation is required not only for non-sexual flocculation but also for sexual conjugation in Sz. pombe.     

RHO1 (YlRHO1) is a non-essential gene in Yarrowia lipolytica and complements rho1Delta lethality in Saccharomyces cerevisiae

The synthesis of beta-1,3-glucan, the structural component of the yeast cell wall that gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of beta-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role (Yamochi et al., 1994). RHO1 has been characterized in Saccharomyces cerevisiae (Yamochi et al., 1994), and in several other fungal species. In this work, we have used degenerate oligonucleotides derived from the conserved regions of Rho1ps to isolate the RHO1 gene of Yarrowia lipolytica. The gene isolated in this way, which we have named YlRHO1, encodes a 204 amino acid protein that shows a high degree of homology with other Rho1ps. However, unlike S. cerevisiae, the ylrho1Delta disruptant strain in Y. lipolytica is viable, although it exhibits an increased sensitivity to Calcofluor white and Congo red. Also, YlRHO1 complements rho1 lethality in S. cerevisiae at both 28 degrees C and 37 degrees C. The complete sequence of YlRHO1 can be obtained from GenBank under Accession No. AF279915.     

Functional analysis of the cysteine residues and the repetitive sequence of Saccharomyces cerevisiae Pir4/Cis3: the repetitive sequence is needed for binding to the cell wall beta-1,3-glucan

Identification of PIR/CIS3 gene was carried out by amino-terminal sequencing of a protein band released by beta-mercaptoethanol (beta-ME) from S. cerevisiae mnn9 cell walls. The protein was released also by digestion with beta-1,3-glucanases (laminarinase or zymolyase) or by mild alkaline solutions. Deletion of the two carboxyterminal Cys residues (Cys(214)-12aa-Cys(227)-COOH), reduced but did not eliminate incorporation of Pir4 (protein with internal repeats) by disulphide bridges. Similarly, site-directed mutation of two other cysteine amino acids (Cys(130)Ser or Cys(197)Ser) failed to block incorporation of Pir4; the second mutation produced the appearance of Kex2-unprocessed Pir4. Therefore, it seems that deletion or mutation of individual cysteine molecules does not seem enough to inhibit incorporation of Pir4 by disulphide bridges. In fks1Delta and gsc2/fks2Delta cells, defective in beta-1,3-glucan synthesis, modification of the protein pattern found in the supernatant of the growth medium, as well as the material released by beta-ME or laminarinase, was evident. However, incorporation of Pir4 by both disulphide bridges and to the beta-1,3-glucan of the cell wall continued. Deletion of the repetitive sequence (QIGDGQVQA) resulted in the secretion and incorporation by disulphide bridges of Pir4 in reduced amounts together with substantial quantities of the Kex2-unprocessed Pir4 form. Pir4 failed to be incorporated in alkali-sensitive linkages involving beta-1,3-glucan when the first repetitive sequence was deleted. Therefore, this suggests that this sequence is needed in binding Pir4 to the beta-1,3-glucan.     

Deletion of MCD 4 involved in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an increase in beta-1,6-glucan level and a decrease in GPI-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae

Most proteins involved in the synthesis of the GPI core structure of Saccharomyces cerevisiae are essential for growth. To explore the relationship between the GPI anchor structure and beta-1,6-glucan synthesis, we screened deletion mutants in genes involved in GPI synthesis for osmotic remedial growth. Heterozygous diploid strains were dissected on medium with osmotic support and slow growth of the mcd 4 deletion mutant was observed. The mcd 4 mutant showed abnormal morphology and cell aggregation, and was hypersensitive to SDS, hygromycin B and K1 killer toxin. Incorporation of GPI cell wall proteins was examined using a GPI-Flo 1 fusion protein. The result suggested that the mcd 4 deletion causes a decrease in GPI cell wall proteins levels. The mutation also caused a decrease in mannan levels and an increase in alkali-insoluble beta-1,6-glucan and chitin levels in the cell wall.     

Induction of TNF-alpha production from human peripheral blood monocytes with beta-1,3-glucan oligomer prepared from laminarin with beta-1,3-glucanase from Bacillus clausii NM-1

We prepared a beta-1,3-glucan oligomer (DP> or = 4) from laminarin (DP: 25-30) derived from Laminaria digitata with beta-1,3-glucanase, and examined its effect on human peripheral blood monocytes. Conditioned medium prepared by incubating monocytes (MC-CM) with the beta-1,3-glucan oligomer showed strong inhibitory activity against the proliferation of human leukemic U937 cells. Since the beta-1,3-glucan oligomer had no direct cytotoxic effect on U937 cells up to 1000 microg/ml, the cytotoxicity of the MC-CM may be due to cytotoxic cytokines produced from monocytes stimulated by the beta-1,3-glucan oligomer. On the other hand, the MC-CM prepared with original laminarin had little effect on the growth of U937 cells. The cytotoxicity of the MC-CM prepared with the beta-1,3-glucan oligomer was significantly reduced by an anti-TNF-alpha antibody, but the anti-TNF-beta antibody had no effect. Our results suggest that the enzymatically depolymerized beta-1,3-glucan oligomer induces TNF-alpha production from human monocytes.     

Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions

Evidence is presented that temperature-sensitive Saccharomyces cerevisiae mutants, impaired in dolichol kinase (Sec59p) or dolichyl phosphate mannose synthase (Dpm1p) activity have an aberrant cell wall composition and ultrastructure. The mutants were oversensitive to Calcofluor white, an agent interacting with the cell wall chitin. In accordance with this, chemical analysis of the cell wall alkali-insoluble fraction indicated an increased amount of chitin and changes in the quantity of beta1,6- and beta1,3-glucan in sec59-1 and dpm1-6 mutants. In order to unravel the link between the formation of dolichyl phosphate and dolichyl phosphate mannose and the cell wall assembly, we screened a yeast genomic library for a multicopy suppressors of the thermosensitive phenotype. The RER2 and SRT1 genes, encoding cis-prenyltransferases, were isolated. In addition, the ROT1 gene, encoding protein involved in beta1,6-glucan synthesis (Machi et al., 2004) and protein folding (Takeuchi et al., 2006) acted as a multicopy suppressor of the temperature-sensitive phenotype of the sec59-1 mutant. The cell wall of the mutants and of mutants bearing the multicopy suppressors was analysed for carbohydrate and mannoprotein content. We also examined the glycosylation status of the plasma membrane protein Gas1p, a beta1,3-glucan elongase, and the degree of phosphorylation of the Mpk1/Slt2 protein, involved in the cell wall integrity pathway.     

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1-dependent cell wall integrity pathway.

The Saccharomyces cerevisiae cwh43-2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1-MPK1 pathway, including a growth defect and increased release of beta-1,6-glucan and beta-glucosylated proteins into the growth medium at increased temperatures. The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14-16 transmembrane segments containing several putative phosphorylation and glycosylation sites. The N-terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the fibroblast growth factor receptor of rat osteosarcoma (FGFR2-ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanogaster. The C-terminus of Cwh43p shows low similarities with a xylose permease of Bacillus megaterium and with putative sugar transporter from D. melanogaster, and has 52% similarity with a protein sequence from a Schizosaccharomyces pombe cDNA. A Cwh43-GFP fusion protein suggested a plasma membrane localization, although localization to the internal structure of the cells could not be excluded, and it concentrates to the bud tip of small budded cells and to the neck of dividing cells. Deletion of CWH43 resulted in cell wall defects less pronounced than those of the cwh43-2 mutant. This allele-specific phenotype appears to be due to a G-R substitution at position 57 in a highly conserved region of the protein. Genetic analysis places CWH43 upstream of the BCK2 branch of the PKC1 signalling pathway, since cwh43 mutations were synthetic lethal with pkc1 deletion, whereas the cwh43 defects could be rescued by overexpression of BCK2 and not by high-copy-number expression of genes encoding downstream proteins of the PKC1 pathway However, unlike BCK2, whose disruption in a cln3 mutant resulted in growth arrest in G(1), no growth defect was observed in a double cwh43 cln3 mutants. Taken together, it is proposed that CWH43 encodes a protein with putative sensor and transporter domains acting in parallel to the main PKC1-dependent cell wall integrity pathway, and that this gene has evolved into two distinct genes in higher eukaryotes.     

Molecular and structural characterization of the spindle pole bodies in the fission yeast Schizosaccharomyces japonicus var japonicus

The structure and localization of the microtubule organization centres (MTOCs) of the fission yeast Schizosaccharomyces japonicus var. japonicus were examined by fluorescence microscopy and electron microscopy. Spindle pole bodies (SPBs), which are the fungal equivalent of centrosomes, of Sz. japonicus were visualized by immunofluorescent staining using a monoclonal anti-gamma-tubulin antibody. The behaviour of the SPBs during the cell cycle mostly coincided with previous reports on the most widely used fission yeast Schizosaccharomyces pombe. We cloned the gamma-tubulin gene from Sz. japonicus by PCR using redundant sets of primers corresponding to conserved regions of known gamma-tubulins. The predicted amino acid sequence of Sz. japonicus gamma-tubulin was most similar to the Sz. pombe gamma-tubulin. Under the electron microscope, the SPBs of Sz. japonicus were detected as electron-dense multilayered structures located just outside the nuclear envelope. The SPBs of Sz. japonicus were composed of three electron-dense layers and were surrounded by fuzzy material. Each layer showed structural changes according to the progression of the cell cycle. In mitotic cells, the SPBs were located on the fenestrae of the nuclear envelopes through which the mitotic spindle microtubules ran into the nucleoplasm. Our results show that Sz. japonicus is a very potent and attractive organism for the investigation of the microtubule nucleation system and morphogenesis in yeasts. The Accession No. for the nucleotide sequence of the Sz. japonicus gtb1(+) gene is AF159163.     

Mass spectrometric identification of covalently bound cell wall proteins from the fission yeast Schizosaccharomyces pombe

The cell wall of Schizosaccharomyces pombe is bilayered, consisting of an inner layer of mainly polysaccharides and an outer layer of galactomannoproteins. We present a detailed analysis of the cell wall proteome. Six covalently-bound cell wall proteins (CWPs) were identified using tandem mass spectrometry, including four predicted GPI-dependent CWPs (Gas1p, Gas5p, Ecm33p and Pwp1p) and two alkali-sensitive CWPs (Psu1p and Asl1p). Gas1p and Gas5p belong to glycoside hydrolase family 72, and are believed to be involved in 1,3-beta-glucan elongation. Ecm33p belongs to a ubiquitous fungal protein family with an unknown but crucial function in cell wall integrity. Pwp1p is an abundant protein with an unknown but probably non-enzymatic function. All four CWPs were present in HF-pyridine extracts, indicating that they are linked via a phosphodiester bridge to the glucan network. Psu1p is a homologue of the Saccharomyces cerevisiae Sun family, whereas Asl1p has no homologues in S. cerevisiae but is related to Aspergillus fumigatus and Ustilago maydis proteins. Finally, although the protein content of Sz. pombe cell walls is only slightly less than in S. cerevisiae and Candida albicans, the amount of carbohydrate added to the proteins was found to be two- to three-fold decreased, consistent with earlier reported differences in outer chain N-glycosylation.     

A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white

To study cell wall assembly, a simple screening method was devised for isolating cell wall mutants. Mutagenized cells were screened for hypersensitivity to Calcofluor White, which interferes with cell wall assembly. The rationale is that Calcofluor White amplifies the effect of cell wall mutations. As a result, the cells stop growing at lower concentrations of Calcofluor White than cells with normal cell wall. In this way, 63 Calcofluor White-hypersensitive (cwh), monogenic mutants were obtained, ordered into 53 complementation groups. The mannose/glucose ratios of the mutant cell walls varied from 0.15 to 3.95, while wild-type cell walls contained about equal amounts of mannose and glucose. This indicates that both low-mannose and low-glucose cell wall mutants had been obtained. Further characterization showed the presence of three low-mannose cell wall mutants with a mnn9-like phenotype, affected, however, in different genes. In addition, four new killer-resistant (kre) mutants were found, which are presumably affected in the synthesis of β1,6-glucan. Most low-glucose cell wall mutants were not killer resistant, indicating that they might be defective in the synthesis of β1,3-glucan. Eleven cwh mutants were found to be hypersensitive to papulacandin B, which is known to interfere with β1,3-glucan synthesis, and four cwh mutants were temperature-sensitive and lysed at the restrictive temperature. Finally, nine cwh mutants were hypersensitive to caffeine, suggesting that these were affected in signal transduction related to cell wall assembly.