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.     

Isolation of the MNN9 gene of Yarrowia lipolytica (YlMNN9) and phenotype analysis of a mutant ylmnn9 Delta strain

In this work we describe the isolation of the Yarrowia lipolytica homologue of Saccharomyces cerevisiae MNN9 gene, which we have named YlMNN9, and the phenotype analysis of a Y. lipolytica strain containing the disrupted YlMNN9 allele. YlMNN9 was cloned using degenerate consensus oligonucleotides to generate specific probes that were in turn used to screen mini-gene libraries. The gene is defined by a 1014 bp ORF predicted to encode a protein 337 amino acids long that shares significant homology with the Mnn9ps of S. cerevisiae, Candida albicans and Hansenula polymorpha, including a putative N-terminal transmembrane domain. Disruption of YlMNN9 leads to phenotypes such as resistance to sodium orthovanadate and sensitivity to hygromycin B, compatible with a glycosylation defect, and hypersensitivity to Calcofluor white, Congo red or zymolyase, characteristic of cell wall defects. Analysis of cell wall proteins present in beta-mercaptoethanol and zymolyase extracts showed significant differences between the parental and the ylmnn9 Delta strain. These results suggest that, as has been the case with the mnn9 strain of S. cerevisiae, the ylmnn9 Delta strain we present in this work, could be used to study the cell wall proteins of Y. lipolytica and how they are organized into the cell wall.     

Schizosaccharomyces pombe gmd3(+)/alg11(+) is a functional homologue of Saccharomyces cerevisiae ALG11 which is involved in N-linked oligosaccharide synthesis

The oligosaccharide of glycoproteins in the fission yeast Schizosaccharomyces pombe is unique in containing galactose. We isolated four mutants that had reduced amounts of galactose residues on their cell surface glycoproteins by fluorescence-activated cell sorter. The isolated four recessive mutants, gmd1 to gmd4, showed a defect in glycosylation of acid phosphatase, a cell surface glycoprotein. In gmd3 mutant cells, the amounts of both mannose and galactose residues were decreased on the cell surface galactomannoproteins, suggesting an underglycosylation of galactomannoproteins. The gmd3(+) gene encodes a protein that has significant similarity with Saccharomyces cerevisiae Alg11p and is likely to be involved in N-linked core oligosaccharide synthesis. ALG11 suppressed the gmd3 mutation, indicating that gmd3(+) gene is a functional homologue of the ALG11 gene. We therefore designated gmd3(+) as alg11(+).     

Yeast flocculation: receptor definition by mnn mutants and concanavalin A.

Yeast flocculation involves the binding of surface lectins on flocculent yeasts, to carbohydrate receptors present as constituents of yeast cell walls. Receptors were investigated by coflocculation of flocculent strains of Saccharomyces cerevisiae, of both Flo 1 and NewFlo phenotypes, to known mnn mutants which vary in the wall mannan structure. Strong coflocculation was found with mnn1, mnn4, mnn9 and control strains, while very little coflocculation was found with mnn2 and mnn5 strains. In contrast, aggregation of these mutants by concanavalin A, a lectin with similar sugar inhibition to NewFlo phenotype flocculation, showed strong aggregation of mnn1, mnn4 and mnn5 strains and poor aggregation of mnn2 and mnn9 strains. The mmn mutant data suggested that flocculation receptors were the outer-chain mannan side-branches, two or three mannose residues in length, confirming an earlier theory based on sugar inhibition data. The similarities and differences between flocculation and concanavalin A aggregation are discussed.     

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β‐Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9‐fold higher than that of the wild‐type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild‐type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild‐type strain. The relative value (mnn2 deletion mutant/wild‐type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β‐glucosidase activity using p‐nitrophenyl‐β‐glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high‐molecular‐weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.     

Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9.

Mannoproteins were isolated from Saccharomyces cerevisiae mnn9 mutant cell walls by laminarinase digestion and purified by affinity and anion-exchange chromatography. The purified mannoprotein fraction contained three predominant proteins with molecular masses of 300 kDa, 220 kDa and 160 kDa. These compounds were absent in an SDS extract of cell walls or in a hot-citrate extract of mnn9 cells. The carbohydrate part of the purified mannoproteins consisted of (N-acetyl)glucosamine, mannose and glucose in a molar ratio of 1:53:4. O-Glycosidically linked chains, containing 70% of the mannose, were released by mild beta-elimination. N-Glycosidically linked chains, representing 80% of the (N-acetyl)glucosamine and 20% of the mannose, were released by peptide N-glycosidase F (PNGase F) digestion. Complete degradation of protein by alkaline hydrolysis released besides the N- and O-glycosidically linked chains, another type of carbohydrate chain containing the residual (N-acetyl)glucosamine, mannose and most of the glucose in a molar ratio of 1:17:18. Glucose was beta-glycosidically linked. The results indicate that beta-glucose is linked to PNGase F-resistant N-linked chains present on cell wall mannoproteins. We propose that these chains are responsible for the linkage between mannoproteins and glucan in the cell wall.     

KlSEC53 is an essential Kluyveromyces lactis gene and is homologous with the SEC53 gene of Saccharomyces cerevisiae

Phosphomannomutase (PMM) is a key enzyme, which catalyses one of the first steps in the glycosylation pathway, the conversion of D-mannose-6-phosphate to D-mannose-1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for the glycosylation reactions in eukaryotic cells. In the yeast Saccharomyces cerevisiae PMM is encoded by the gene SEC53 (ScSEC53) and the deficiency of PMM activity leads to severe defects in both protein glycosylation and secretion. We report here on the isolation of the Kluyveromyces lactis SEC53 (KlSEC53) gene from a genomic library by virtue of its ability to complement a Saccharomyces cerevisiae sec53 mutation. The sequenced DNA fragment contained an open reading frame of 765 bp, coding for a predicted polypeptide, KlSec53p, of 254 amino acids. The KlSec53p displays a high degree of homology with phosphomannomutases from other yeast species, protozoans, plants and humans. Our results have demonstrated that KlSEC53 is the functional homologue of the ScSEC53 gene. Like ScSEC53, the KlSEC53 gene is essential for K. lactis cell viability. Phenotypic analysis of a K. lactis strain overexpressing the KlSEC53 gene revealed defects expected for impaired cell wall integrity.     

vga Mutants of Kluyveromyces lactis show cell integrity defects

We studied the cell wall alterations that occur in mutants of Kluyveromyces lactis impaired in glycosylation. The mutants belong to four complementation groups named vga1 to vga4 (vanadate glycosylation affected), characterized by sodium orthovanadate resistance and alteration of the glycosylation profile of native invertase. A drastic reduction of the alkali-soluble fraction of the beta-D-glucan was observed in vga1, vga2 and vga3 cells, accompanied by an increase in the chitin content of the cell wall. In vga4 cells, both beta-D-glucan fractions (alkali-soluble and alkali-insoluble) were reduced to about half of the corresponding wild-type value but the chitin content was normal. A protein related to Fks1p, the catalytic subunit of the major 1,3-beta-D-glucan synthase of S. cerevisiae, was detected in K. lactis. The amount of this Fks1p-like protein increased 7-10 times in vga1, vga2 and vga3 mutants as compared to wild-type cells; the same strains released significant amounts of beta-D-glucan in the culture supernatant. These mutations also resulted in abnormally thick cell walls with conspicuous irregularities in the structure, as revealed by electron microscopy and by an altered resistance to Zymolyase. The observed high responsiveness of cell wall phenotypes to alterations of glycosylation make K. lactis an attractive system for studying the interconnections between these processes.     

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.     

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae MNN9 gene

A gene homologous to Saccharomyces cerevisiae MNN9 has been cloned and characterized in the methylotrophic yeast Hansenula polymorpha. This gene was cloned from a H. polymorpha genomic DNA library using the S. cerevisiae MNN9 gene as a probe. The H. polymorpha MNN9 homologue (HpMNN9) contained a 1062 bp open reading frame encoding a predicted protein of 354 amino acids. The deduced amino acid sequence showed 58% and 51% identity, respectively, with the S. cerevisiae and Candida albicans Mnn9 proteins. Disruption of HpMNN9 leads to phenotypic effects suggestive of cell wall defects, including detergent sensitivity and hygromycin B sensitivity. The hygromycin B sensitivity of S. cerevisiae mnn9 null mutant was complemented in the presence of the HpMNN9 gene. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession No. AF264786.     

Candida glabrata Ste20 is involved in maintaining cell wall integrity and adaptation to hypertonic stress, and is required for wild-type levels of virulence

The conserved family of fungal Ste20 p21-activated serine-threonine protein kinases regulate several signalling cascades. In Saccharomyces cerevisiae Ste20 is involved in pheromone signalling, invasive growth, the hypertonic stress response, cell wall integrity and binds Cdc42, a Rho-like small GTP-binding protein required for polarized morphogenesis. We have cloned the STE20 homologue from the fungal pathogen Candida glabrata and have shown that it is present in a single copy in the genome. Translation of the nucleotide sequence predicts that C. glabrata Ste20 contains a highly conserved p21-activated serine-threonine protein kinase domain, a binding site for G-protein beta subunits and a regulatory Rho-binding domain that enables the kinase to interact with Cdc42 and/or Rho-like small GTPases. C. glabrata Ste20 has 53% identity and 58% predicted amino acid similarity to S. cerevisiae Ste20 and can complement both the nitrogen starvation-induced filamentation and mating defects of S. cerevisiae ste20 mutants. Analysis of ste20 null and disrupted strains suggest that in C. glabrata Ste20 is required for a fully functional hypertonic stress response and intact cell wall integrity pathway. C. glabrata Ste20 is not required for nitrogen starvation-induced filamentation. Survival analysis revealed that C. glabrata ste20 mutants, while still able to cause disease, are mildly attenuated for virulence compared to reconstituted STE20 cells.     

The 'SUN' family: yeast SUN4/SCW3 is involved in cell septation

SUN4 is the fourth member of the SUN gene family from S. cerevisiae, whose products display high homology in their 258 amino acid C-terminal domain. SIM1, UTH1, NCA3 (the founding members) are involved in different cellular processes (DNA replication, ageing, mitochondrial biogenesis) and it is shown herein that SUN4 plays a role in the cell septation process. sun4 delta cells are larger than wild-type and begin a new cell cycle before they have separated from their mother cell. This phenotype is more pronounced in sun4Delta cells also deleted for UTH1. FACS analysis shows apparent polyploidy which disappears when the cell cycle is arrested by mating factor or nocodazole, indicating that cell septation is delayed without modification of the doubling time. Elutriated sun4 delta uth1 delta daughter cells are born larger, and therefore enter S phase sooner than their wild-type counterpart. S phase duration, as well as timing of Clb2 degradation, is normal, but cell septation is delayed. Sun4p/Scw3p was recently described as a cell wall protein (Cappellaro et al., 1998) and, consistent with this notion, electron micrographs of sun4 delta cells show defects in the final steps of cell wall septation. Our data suggest that Sun4p and Uth1p might contribute to the regulated process of cell wall morphogenesis and septation.     

Mutation of the homologue of GDP-mannose pyrophosphorylase alters cell wall structure, protein glycosylation and secretion in Hansenula polymorpha

A Hansenula polymorpha mutant with enhanced ability to secrete a heterologous protein has been isolated. The mutation defines a gene, designated OPU24, which encodes a protein highly homologous to GDP-mannose pyrophosphorylase Psa1p/Srb1p/Vig9p of Saccharomyces cerevisiae and CaSrb1p of Candida albicans. The opu24 mutant manifests phenotypes similar to those of S. cerevisiae mutants depleted for GDP-mannose, such as cell wall fragility and defects in N- and O-glycosylation of secreted proteins. The influence of the opu24 mutation on endoplasmic reticulum-associated protein degradation is discussed. The GenBank Accession No. for the OPU24 sequence is AF234177.     

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.     

Characterization of a gene similar to BIK1 in the yeast Kluyveromyces lactis

In Saccharomyces cerevisiae, Bik1p is a microtubule plus-end-tracking protein that plays several roles in mitosis and ploidy. KlBik1p (from Kluyveromyces lactis) maintains the same structural-domain organization as does S. cerevisiae Bik1p. As part of its characterization, we constructed a stable klbik1 mutant which is sensitive to benomyl only at 14 degrees C and has a higher frequency of crescent-shaped nuclei than S. cerevisiae bik1 mutants. This phenotype is partially rescued by S. cerevisiae BIK1. Other phenotypes associated with bik1 are not present in the K. lactis mutant. By fusion to GFP we were able to show the functionality of the KlBik1p CAP-Gly domain and found that the fusion protein changes its cellular location during the cell cycle.     

A genetic screen for Saccharomyces cerevisiae mutants affecting proteasome function, using a ubiquitin-independent substrate

The great majority of proteasome substrates are marked for degradation by the attachment of polyubiquitin chains. Ornithine decarboxylase is degraded by the proteasome in the absence of this modification. We previously showed that this mechanism of degradation was conserved in eukaryotic cells. Here we use a reporter destabilized by mouse ornithine decarboxylase to screen non-essential Saccharomyces cerevisiae deletion mutants. We identified novel mutants that affect both ubiquitin-dependent and -independent proteasome degradation pathways. YLR021W (IRC25/POC3) and YPL144W (POC4) encode interacting proteins that function in proteasome assembly, with putative homologues widespread among eukaryotes. Several additional mutants suffered from defects in proteasome-mediated proteolysis. These included mutants in the urmylation pathway of protein modification (but not the Urm1 modifier itself) and the Reg1 regulatory subunit of protein phosphatase 1. Finally, we noted increased rates of ornithine decarboxylase turnover in an rpn10Delta mutant in which the degradation of certain ubiquitinated substrates is impaired. Together, these results highlight the utility of a ubiquitin-independent degron in uncovering novel factors affecting general and substrate-specific proteasome function.     

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.     

The immunodetected yeast purine—cytosine permease is not N-linked glycosylated,nor are glycosylation sequences required to have a functional permease

The purine-cytosine permease (PCP) of the yeast Saccharomyces cerevisiae was detected by immunological methods. Using antibodies directed against synthetic peptides, whose sequences were derived from the primary structure of the PCP, immunoprecipitation of [³⁵S]methionine-labelled PCP was achieved either from cellular extracts or from in vitro translation mixtures. Non-labelled PCP was also detected on Western blots of membrane proteins. Similar migration rates were observed for PCP originating both from immunoprecipitated cellular extracts and from in vitro translation mixtures. Hence, post-translational processing, if any, only slightly affects the size of the protein. Also no evidence was found for N-linked core-glycosylation: identical migration rates were observed when immunoprecipitated PCP molecules were extracted from cells labelled for 10 min with [³⁵S]methionine, pretreated or not with tunicamycin. On the other hand, the suppresion of the two potential N-linked glycosylation sequences in the DNA did not lead to inactivation of the transport activity, confirming that N-linked glycosylation is not required for the permease activity.     

Hut1 proteins identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe are functional homologues involved in the protein-folding process at the endoplasmic reticulum

The Saccharomyces cerevisiae HUT1 gene (scHUT1) and the Schizosaccharomyces pombe hut1(+) gene (sphut1(+)) encode hydrophobic proteins with approximately 30% identity to a human UDP-galactose transporter-related gene (UGTrel1) product. These proteins show a significant similarity to the nucleotide sugar transporter and are conserved in many eukaryotic species, but their physiological functions are not known. Both scHUT1 and sphut1(+) genes are non-essential for cell growth under normal conditions, and their disruptants show no defects in the modification of O- and N-linked oligosaccharides, but are sensitive to a membrane-permeable reducing agent, dithiothreitol (DTT). Consistent with this phenotype, scHUT1 has genetic interaction with ERO1, which plays an essential role in the oxidation of secretory proteins at the endoplasmic reticulum (ER). Overexpression of the MPD1 or MPD2 genes, which were isolated as multicopy suppressors of protein disulphide isomerase (PDI) depletion, could not replace the essential function of PDI in Delta hut1 S. cerevisiae cells. Our results indicate that scHut1p and spHut1p are functional homologues, and their physiological function is to maintain the optimal environment for the folding of secretory pathway proteins in the ER.