Importance of cell wall mannoproteins for septum formation in Saccharomyces cerevisiae

The mannosyltransferase mutants mnn9 and mnn10 were isolated in a genetic screen for septation defects in Saccharomyces cerevisiae. Ultrastructural examination of mutant cell walls revealed markedly thin septal structures and occasional failure to construct trilaminar septa, which then led to the formation of bulky default septa at the bud neck. In the absence of a functional septation apparatus, mnn10 mutants are unable to complete cytokinesis and die as cell chains with incompletely separated cytoplasms, indicating that mannosylation defects impair the ability to form remedial septa. We could not detect N-linked glycosylation of the beta(1,3)glucan synthase Fks1p and mnn10 defects do not change the molecular weight or abundance of the protein. We discuss a model explaining the pleiotropic effects of impaired N-linked protein glycosylation on septation in S. cerevisiae.     

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.     

Candida albicans homologue of GGP1/GAS1 gene is functional in Saccharomyces cerevisiae and contains the determinants for glycosylphosphatidylinositol attachment

The GGP1/GAS1/CWH52 gene of Saccharomyces cerevisiae encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gp115 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. PHR1 gene isolated from Candida alibicans is homologous to the GGP1 gene. In this report we have analysed the ability of PHR1 to complement a ggp1Δ mutation in S. cerevisiae. The expression of PHR1 controlled by its natural promoter or by the GGP1 promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that PHR1 in budding yeast gives rise to a 75–80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. albicans gene product is functional in S. cerevisiae.     

Cooperative functions of the mannoprotein-encoding genes in the biogenesis and maintenance of the cell wall in Saccharomyces cerevisiae.

To elucidate the roles of genes involved in the cell wall biogenesis and function in Saccharomyces cerevisiae, we isolated and characterized mutants that were lethal in a strain in which the SED1 gene encoding a cell wall mannoprotein was disrupted. Thus, double mutants of SED1 and either MNN9 or MNN10 were unable to grow and YOL155c on a multicopy plasmid could suppress their synthetic lethality. A Yol155cp-GFP fusion protein was found to localize to the cell wall, suggesting that it might also be a cell wall mannoprotein. Subsequently, we analysed the effects of the shut-off of SED1 in a sed1 and mnn9 double mutant: cells after the shut-off showed anomalous cellular morphology and died in the mitotic M phase. From these and other results, we postulate that these genes function cooperatively with each other and in a cell cycle-dependent manner in the biogenesis and maintenance of cell wall in S. cerevisiae.     

A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae

A reliable acid hydrolysis method for quantitative determination of the proportion of β-glucan, mannan and chitin in Saccharomyces cerevisiae cell wall is reported together with a simple extraction procedure to quantify within a standard error of less than 2% the proportion of the wall per gram of cell dry mass. This method is an optimized version of Saeman's procedure based on sulfuric acid hydrolysis of complex polysaccharides. It resulted in an almost complete release of glucose, mannose and glucosamine residues from cell wall polysaccharides. After complete removal of sulfate ions by precipitation with barium hydroxide, the liberated monosaccharides were separated and quantified by high performance anion-exchange chromatography with pulsed amperometric detection. The superiority of this method over the hydrolysis in either trifluoroacetic or hydrochloric acid resides in its higher efficiency regarding the release of glucose from β1,6-glucan and of glucosamine from chitin. The sulfuric acid method was successfully applied to determine the β-glucan, mannan and chitin contents in cell walls of genetically well-characterized yeast mutants defective in cell wall biosynthesis, and in Schizosaccharomyces pombe cell walls. The simplicity and reliability of this procedure make it the method of choice for the characterization of cell walls from S. cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological and biotechnological applications. © 1998 John Wiley & Sons, Ltd.     

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.     

Expression,purification, and characterization of N-acetylglucosaminylphosphatidylinositol de-N-acetylase (ScGpi12), the enzyme that catalyses the second step of GPI biosynthesis in S. cerevisiae

ScGpi12 is a 304 amino residue long endoplasmic reticulum membrane protein, which participates in the de-N-acetylation of N-acetylglucosaminyl phosphatidylinositol to produce glucosaminyl phosphatidylinositol in the second step of GPI anchor biosynthesis pathway in Saccharomyces cerevisiae. ScGpi12 was cloned in a pMAL-c2x vector and expressed heterologously in Rosetta-gami (DE3) strain of E. coli. Affinity purification of the protein yielded low amounts of the MBP-tagged enzyme, which was active. To the best of our knowledge, this is the first successful purification of full-length Gpi12 enzyme, without the accompanying GroEL that was seen in other studies. The presence of the tag did not greatly alter the activity of the enzyme. ScGpi12 was optimally active in the pH range of 6.5–8.5 and at 30 °C. It was not sensitive to treatment with EDTA but was stimulated by multiple divalent cations. The divalent cation did not alter the pH profile of the enzyme, suggesting no role of the divalent metal in creating a nucleophile for catalysis. Divalent cations did, however, enhance the turnover number of the enzyme for its substrate, suggesting that they are probably required for the production of a catalytically competent active site by bringing the active site residues within optimum distance of the substrate for catalysis.     

Kluyveromyces lactis SSO1 and SEB1 genes are functional in Saccharomyces cerevisiae and enhance production of secreted proteins when overexpressed

The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae. Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion. Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S. cerevisiae, which suggests that they may be rate-limiting factors in this process. Here we report isolation of Kluyveromyces lactis homologues of these genes. KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature. The encoded Kluyveromyces proteins are up to 70% identical with the S. cerevisiae homologues at the amino acid level and can functionally replace them. Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S. cerevisiae when overexpressed. In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S. cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K. lactis and Pichia pastoris, the latter also in Candida utilis. The GenBank Accession Nos are AF307983 (K. lactis SSO1) and AF318314 (K. lactis SEB1).     

Identification of multicopy suppressors of cell cycle arrest at the G1-S transition in Saccharomyces cerevisiae

Inactivation of HAL3 in the absence of SIT4 function leads to cell cycle arrest at the G(1)-S transition. To identify genes potentially involved in the control of this phase of the cell cycle, a screening for multicopy suppressors of a conditional sit4 hal3 mutant (strain JC002) has been developed. The screening yielded several genes known to perform key roles in cell cycle events, such as CLN3, BCK2 or SWI4, thus proving its usefulness as a tool for this type of studies. In addition, this approach allowed the identification of additional genes, most of them not previously related to the regulation of G(1)-S transition or even without known function (named here as VHS1-3, for viable in a hal3 sit4 background). Several of these gene products are involved in phospho-dephosphorylation processes, including members of the protein phosphatase 2A and protein phosphatases 2C families, as well as components of the Hal5 protein kinase family. The ability of different genes to suppress sit4 phenotypes (such as temperature sensitivity and growth on non-fermentable carbon sources) or to mimic the functions of Hal3 was evaluated. The possible relationship between the known functions of these suppressor genes and the progress through the G(1)-S transition is discussed.     

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.     

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.     

The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene

We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology. The complete nucleotide sequence was submitted to the EMBL Data Library under Accession Number X83690.     

Structure-function analysis of Knr4/Smi1, a newly member of intrinsically disordered proteins family, indispensable in the absence of a functional PKC1-SLT2 pathway in Saccharomyces cerevisiae

The coordination between cell wall synthesis and cell growth in the yeast Saccharomyces cerevisiae implicates the PKC1-dependent MAP kinase pathway. KNR4, encoding a 505 amino acid long protein, participates in this coordination, since it displays synthetic lethality with all the members of the PKC1 pathway and shows physical interaction with Slt2/Mpk1. The recent finding that KNR4 interacts genetically or physically with more than 100 partners implicated in different cellular processes raised the question of how these interactions may occur and their physiological significance. This called for an in-depth structure-function analysis of the Knr4 protein, which is reported in the present paper. Computational analysis supported by biochemical and biophysical data characterize Knr4 as a newly identified member of the growing family of intrinsically disordered proteins. Despite disordered regions that are located at the N- and C-termini and are probably responsible for fine regulatory function; this protein contains a structured central core (amino acid residues 80-340) that is able to restore wild-type phenotypes of knr4Delta mutant in stress conditions. However, this fragment was unable to complement synthetic lethality between knr4 mutations and deletions of genes encoding protein kinases of the PKC1-dependent pathway. For these crucial events to occur, the presence of the N-terminal part of Knr4 protein is indispensable. Moreover, we demonstrate that this protein is essential for cell viability in the absence of a functional Pkc1-Slt2 pathway, since the lethality caused by KNR4 deletion in such a genetic background could not be compensated by overexpression of any gene from yeast genomic libraries.     

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.