Complementation of Essential Yeast GPI Mannosyltransferase Mutations Suggests a Novel Specificity for Certain Trypanosoma and Plasmodium PigB Proteins

The glycosylphosphatidylinositol (GPI) anchor is an essential glycolipid that tethers certain eukaryotic proteins to the cell surface. The core structure of the GPI anchor is remarkably well conserved across evolution and consists of NH₂-CH₂-CH₂-PO₄-6Manα1,2Manα1,6Manα1,4-GlcNα1,6-myo-inositol-PO₄-lipid. The glycan portion of this structure may be modified with various side-branching sugars or other compounds that are heterogeneous and differ from organism to organism. One such modification is an α(1,2)-linked fourth mannose (Man-IV) that is side-branched to the third mannose (Man-III) of the trimannosyl core. In fungi and mammals, addition of Man-III and Man-IV occurs by two distinct Family 22 α(1,2)-mannosyltransferases, Gpi10/PigB and Smp3/PigZ, respectively. However, in the five protozoan parasite genomes we examined, no genes encoding Smp3/PigZ proteins were observed, despite reports of tetramannosyl-GPI structures (Man₄-GPIs) being produced by some parasites. In this study, we tested the hypothesis that the Gpi10/PigB proteins produced by protozoan parasites have the ability to add both Man-III and Man-IV to GPI precursors. We used yeast genetics to test the in vivo specificity of Gpi10/PigB proteins from several Plasmodium and Trypanosoma species by examining their ability to restore viability to Saccharomyces cerevisiae strains harboring lethal defects in Man-III (gpi10Δ) or Man-IV (smp3Δ) addition to GPI precursor lipids. We demonstrate that genes encoding PigB enzymes from T. cruzi, T. congolense and P. falciparum are each capable of separately complementing essential gpi10Δ and smp3Δ mutations, while PIGB genes from T. vivax and T. brucei only complement gpi10Δ. Additionally, we show the ability of T. cruzi PIGB to robustly complement a gpi10Δ/smp3Δ double mutant. Our data suggest that certain Plasmodium and Trypanosoma PigB mannosyltransferases can transfer more than one mannose to GPI precursors in vivo, and suggest a novel biosynthetic mechanism by which Man₄-GPIs may be synthesized in these organisms.     

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins.     

Chemical Organization of the Cell Wall Polysaccharide Core of Malassezia restricta

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% β-(1,3)-glucan, and 70% β-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and β-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is β-(1,6)-glucans, which are large molecules composed of a linear β-(1,6)-glucan chains with β-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both β-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date.     

Posttranslational modifications required for cell surface localization and function of the fungal adhesin Aga1p

Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts. Adhesins are cell surface proteins that mediate these different cell adhesion interactions. In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation. The processing of some fungal GPI anchors, creating links to cell wall β-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions. By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. A portion of the cellular pool of Aga1p was determined to be cell wall resident. Aga1p and the α-agglutinin Agα1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATα cells for single mutants but in both cell types when both genes are absent. We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity. Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.     

Mutational Effect of Fission Yeast Polα on Cell Cycle Events

Polα is the principal DNA polymerase for initiation of DNA replication and also functions in postinitiation DNA synthesis. In this study, we investigated the cell cycle responses induced by mutations in polα⁺. Germinating spores carrying either a deletion of polα⁺ (polαΔ) or a structurally intact but catalytically dead polα mutation proceed to inappropriate mitosis with no DNA synthesis. This suggests that the catalytic function, and not the physical presence of Polα, is required to generate the signal that prevents the cells from entering mitosis prematurely. Cells with a polαts allele arrest the cell cycle near the hydroxyurea arrest point, but, surprisingly, polαts in cdc20 (polε mutant) background arrested with a cdc phenoytpe, not a polαts-like phenotype. At 25°C, replication perturbation caused by polαts alleles induces Cds1 kinase activity and requires the checkpoint Rads, Cds1, and Rqh1, but not Chk1, to maintain cell viability. At 36°C, replication disruption caused by polαts alleles induces the phosphorylation of Chk1; however, mutant cells arrest with heterogeneous cell sizes with a population of the cells entering aberrant mitosis. Together, our results indicate that the initiation DNA structure synthesized by Polα is required to bring about the S phase to mitosis checkpoint, whereas replication defects of different severity caused by polαts mutations induce differential downstream kinase responses.     

The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length,Immune Recognition and Virulence of Candida albicans

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.     

Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast

DNA polymerase epsilon (Pol ε) is a multi-subunit enzyme required for the initiation of chromosomal DNA replication. Here, we report the cloning of two fission yeast genes, called dpb3⁺ and dpb4⁺ that encode proteins homologous to the two smallest subunits of Pol ε. Although Dpb4 is not required for cell viability, Δdpb4 mutants are synthetically lethal with mutations in four genes required for DNA replication initiation, cdc20⁺ (encoding DNA Pol ε), cut5⁺ (homologous to DPB11/TopBP1), sna41⁺ (homologous to CDC45) and cdc21⁺ (encoding Mcm4, a component of the pre-replicative complex). In contrast to Dpb4, Dpb3 is essential for cell cycle progression. A glutathione S-transferase pull-down assay indicates that Dpb3 physically interacts with both Dpb2 and Dpb4, suggesting that Dpb3 associates with other members of the Pol ε complex. Depletion of Dpb3 leads to an accumulation of cells in S phase consistent with Dpb3 having a role in DNA replication. In addition, many of the cells have a bi-nucleate or multinucleate phenotype, indicating that cell separation is also inhibited. Finally, we have examined in vivo localization of green fluorescent protein (GFP)-tagged Dpb3 and Dpb4 and found that both proteins are localized to the nucleus consistent with their proposed role in DNA replication. However, in the absence of Dpb3, GFP-Dpb4 appears to be more dispersed throughout the cell, suggesting that Dpb3 may be important in establishing or maintaining normal localization of Dpb4.     

α-1,6-Mannosylation of N-Linked Oligosaccharide Present on Cell Wall Proteins Is Required for Their Incorporation into the Cell Wall in the Filamentous Fungus Neurospora crassa

The enzyme α-1,6-mannosyltransferase (OCH-1) is required for the synthesis of galactomannans attached to the N-linked oligosaccharides of Neurospora crassa cell wall proteins. The Neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. A carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. Analysis of the integral cell wall protein from wild-type and och-1 mutant cells showed that the mutant cell wall had reduced protein content. The och-1 mutant was found to secrete 18-fold more protein than wild-type cells. Proteomic analysis of the proteins released by the mutant into the growth medium identified seven of the major cell wall proteins. Western blot analysis of ACW-1 and GEL-1 (two glycosylphosphatidylinositol [GPI]-anchored proteins that are covalently integrated into the wild-type cell wall) showed that high levels of these proteins were being released into the medium by the och-1 mutant. High levels of ACW-1 and GEL-1 were also released from the och-1 mutant cell wall by subjecting the wall to boiling in a 1% SDS solution, indicating that these proteins are not being covalently integrated into the mutant cell wall. From these results, we conclude that N-linked mannosylation of cell wall proteins by OCH-1 is required for their efficient covalent incorporation into the cell wall.The fungal cell wall is an important organelle that protects the cell from various environmental stresses. It is a dynamic structure that interacts with the environment and is modified to accommodate growth, cell division, and development. Fungal cell walls have been shown to contain β-1,3-glucan, α-1,3-glucan, β-1,6-glucan, mixed β-1,3/β-1,4-glucans, chitin, and mannan/galactomannan (6, 19). These polysaccharide polymers constitute 80 to 85% of the cell wall mass, while glycoproteins constitute the remaining 15 to 20% (6). The cell wall glycoproteins are required for vital functions, like structural support, signal transduction, biofilm formation, and cell wall biosynthesis. In the case of pathogenic fungi, the cell wall is critical for the invasion of host tissues (8). Because of their accessibility and the crucial functions they perform, cell wall proteins could be important targets for the development of antifungal therapeutics.The glucan and chitin cell wall polymers are synthesized by enzyme complexes (glucan synthases and chitin synthases) that are associated with the plasma membrane. Glucan and chitin are vectorially passed into the cell wall space during synthesis and cross-linked together in the cell wall space. The mannan and galactomannan present in the cell wall are found as glycoconjugates on cell wall proteins. Mannosylation of cell wall proteins occurs in the endoplasmic reticulum (ER) and Golgi apparatus at O-linked and N-linked glycosylation sites. In Saccharomyces cerevisiae, mannosylation of N-linked glycosylation is initiated by the addition of an α-1,6-linked mannose residue by Och1p (33). In the filamentous fungus Neurospora crassa, the structure of the galactomannan associated with N-linked sites has not been definitively determined, but N. crassa has most of the enzymes defined in yeast for the mannosylation of N-linked oligosaccharides (14). An analysis of N-linked oligosaccharides from N. crassa glycoproteins showed that the glycoproteins are modified by the addition of short α-1,6-mannans with short α-1,2-mannose branches that are terminated by galactofuranose residues (31, 32). The N. crassa posttranslational modifications appear to differ from those found in S. cerevisiae by having shorter mannan chains and by the presence of terminal galactofuranose residues.Mannosylation of glycoproteins has been extensively studied in yeast. In S. cerevisiae, OCH1 encodes the α-1,6-mannosyltransferase enzyme that mediates the addition of the initial α-1,6-mannose in the synthesis of long mannans which are attached to the N-linked oligosaccharides (22, 33). Knockout mutants of OCH1 are viable but exhibit a temperature-sensitive growth pattern and are sensitive to cell wall perturbation reagents (34). Mutants for Candida albicans homologs of OCH1 had near-normal growth rates but were much less virulent (3). Mass spectrometry analysis of glycoproteins from the S. cerevisiae och1 and C. albicans och1 mutants showed that the α-1,6-mannose core was absent (3, 33). In Kluyveromyces lactis, the KlOCH1 gene has been shown to be important for cell wall organization and to give a hypersecretion phenotype (37). OCH1 mutants have also been identified in Pichia angusta, Yarrowia lipolytica, Pichia pastoris, and Schizosaccharomyces pombe, and these mutants have cell wall-related phenotypes (2, 9, 17, 38). However, a recent report of OCH1 knockout mutants of Aspergillus fumigatus indicates that these mutants do not have a cell wall-defective phenotype (18).Mannosylation of cell wall proteins has not been extensively studied in filamentous fungi. We report on the characterization of the N. crassa knockout mutant of the α-1,6-mannosyltransferase, och-1. The mutant was generated by the Neurospora genome knockout project (10). The N. crassa och-1 mutant has a severe growth defect and exhibits a tight colonial phenotype. We demonstrate that the och-1 mutant exhibits a defect in cell wall biosynthesis. A carbohydrate analysis of the mutant cell wall showed a drastic reduction in mannose and galactose content with a compensatory increase in the glucose content. The och-1 cell wall also showed a reduced cell wall protein content as assessed by a Coomassie brilliant blue dye binding assay and by proteomic analysis. Protein secretion assays showed that the mutant releases large amounts of cell wall protein into the growth medium. We demonstrate that the och-1 mutant is defective in covalently cross-linking known cell wall proteins into the cell wall matrix. Our data demonstrate that the N-linked galactomannan, which is built upon the mannose residue added by OCH-1, is required for the incorporation of cell wall proteins into the cell wall matrix.     

Decrease in Cell Surface Galactose Residues of Schizosaccharomyces pombe Enhances Its Coflocculation with Pediococcus damnosus

Pediococcus damnosus can coflocculate with Saccharomyces cerevisiae and cause beer acidification that may or may not be desired. Similar coflocculations occur with other yeasts except for Schizosaccharomyces pombe which has galactose-rich cell walls. We compared coflocculation rates of S. pombe wild-type species TP4-1D, having a mannose-to-galactose ratio (Man:Gal) of 5 to 6 in the cell wall, with its glycosylation mutants gms1-1 (Man:Gal = 5:1) and gms1Δ (Man:Gal = 1:0). These mutants coflocculated at a much higher level (30 to 45%) than that of the wild type (5%). Coflocculation of the mutants was inhibited by exogenous mannose but not by galactose. The S. cerevisiae mnn2 mutant, with a mannan content similar to that of gms1Δ, also showed high coflocculation (35%) and was sensitive to mannose inhibition. Coflocculation of P. damnosus and gms1Δ (or mnn2) also could be inhibited by gms1Δ mannan (with unbranched α-1,6-linked mannose residues), concanavalin A (mannose and glucose specific), or NPA lectin (specific for α-1,6-linked mannosyl units). Protease treatment of the bacterial cells completely abolished coflocculation. From these results we conclude that mannose residues on the cell surface of S. pombe serve as receptors for a P. damnosus lectin but that these receptors are shielded by galactose residues in wild-type strains. Such interactions are important in the production of Belgian acid types of beers in which mixed cultures are used to improve flavor.     

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

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

Induction of a Mitosis Delay and Cell Lysis by High-Level Secretion of Mouse α-Amylase from Saccharomyces cerevisiae

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse α-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in α-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of α-amylase. Our data also suggest that high levels of α-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous α-amylase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis.     

Deletion of GPI7, a yeast gene required for addition of a side chain to the glycosylphosphatidylinositol (GPI) core structure, affects GPI protein transport, remodeling, and cell wall integrity.

Gpi7 was isolated by screening for mutants defective in the surface expression of glycosylphosphatidylinositol (GPI) proteins. Gpi7 mutants are deficient in YJL062w, herein named GPI7. GPI7 is not essential, but its deletion renders cells hypersensitive to Calcofluor White, indicating cell wall fragility. Several aspects of GPI biosynthesis are disturbed in Deltagpi7. The extent of anchor remodeling, i.e. replacement of the primary lipid moiety of GPI anchors by ceramide, is significantly reduced, and the transport of GPI proteins to the Golgi is delayed. Gpi7p is a highly glycosylated integral membrane protein with 9-11 predicted transmembrane domains in the C-terminal part and a large, hydrophilic N-terminal ectodomain. The bulk of Gpi7p is located at the plasma membrane, but a small amount is found in the endoplasmic reticulum. GPI7 has homologues in Saccharomyces cerevisiae, Caenorhabditis elegans, and man, but the precise biochemical function of this protein family is unknown. Based on the analysis of M4, an abnormal GPI lipid accumulating in gpi7, we propose that Gpi7p adds a side chain onto the GPI core structure. Indeed, when compared with complete GPI lipids, M4 lacks a previously unrecognized phosphodiester-linked side chain, possibly an ethanolamine phosphate. Gpi7p contains significant homology with phosphodiesterases suggesting that Gpi7p itself is the transferase adding a side chain to the alpha1,6-linked mannose of the GPI core structure.     

Translation Initiation Requires Cell Division Cycle 123 (Cdc123) to Facilitate Biogenesis of the Eukaryotic Initiation Factor 2 (eIF2)

The eukaryotic translation initiation factor 2 (eIF2) is central to the onset of protein synthesis and its modulation in response to physiological demands. eIF2, a heterotrimeric G-protein, is activated by guanine nucleotide exchange to deliver the initiator methionyl-tRNA to the ribosome. Here we report that assembly of the eIF2 complex in vivo depends on Cdc123, a cell proliferation protein conserved among eukaryotes. Mutations of CDC123 in budding yeast reduced the association of eIF2 subunits, diminished polysome levels, and increased GCN4 expression indicating that Cdc123 is critical for eIF2 activity. Cdc123 bound the unassembled eIF2γ subunit, but not the eIF2 complex, and the C-terminal domain III region of eIF2γ was both necessary and sufficient for Cdc123 binding. Alterations of the binding site revealed a strict correlation between Cdc123 binding, the biological function of eIF2γ, and its ability to assemble with eIF2α and eIF2β. Interestingly, high levels of Cdc123 neutralized the assembly defect and restored the biological function of an eIF2γ mutant. Moreover, the combined overexpression of eIF2 subunits rescued an otherwise inviable cdc123 deletion mutant. Thus, Cdc123 is a specific eIF2 assembly factor indispensable for the onset of protein synthesis. Human Cdc123 is encoded by a disease risk locus, and, therefore, eIF2 biogenesis control by Cdc123 may prove relevant for normal cell physiology and human health. This work identifies a novel step in the eukaryotic translation initiation pathway and assigns a biochemical function to a protein that is essential for growth and viability of eukaryotic cells.     

A WD Repeat Protein Controls the Cell Cycle and Differentiation by Negatively Regulating Cdc2/B-Type Cyclin Complexes

In the fission yeast Schizosaccharomyces pombe, p34^cdc2 plays a central role controlling the cell cycle. We recently isolated a new gene named srw1⁺, capable of encoding a WD repeat protein, as a multicopy suppressor of hyperactivated p34^cdc2. Cells lacking srw1⁺ are sterile and defective in cell cycle controls. When starved for nitrogen source, they fail to effectively arrest in G₁ and die of accelerated mitotic catastrophe if regulation of p34^cdc2/Cdc13 by inhibitory tyrosine phosphorylation is compromised by partial inactivation of Wee1 kinase. Fertility is restored to the disruptant by deletion of Cig2 B-type cyclin or slight inactivation of p34^cdc2. srw1⁺ shares functional similarity with rum1⁺, having abilities to induce endoreplication and restore fertility to rum1 disruptants. In the srw1 disruptant, Cdc13 fails to be degraded when cells are starved for nitrogen. We conclude that Srw1 controls differentiation and cell cycling at least by negatively regulating Cig2- and Cdc13-associated p34^cdc2 and that one of its roles is to down-regulate the level of the mitotic cyclin particularly in nitrogen-poor environments.     

A Cytochemical Study of Extracellular Sheaths Associated with Rigidoporus lignosus during Wood Decay

An ultrastructural and cytochemical investigation of the development of Rigidoporus lignosus, a white-rot fungus inoculated into wood blocks, was carried out to gain better insight into the structure and role of the extracellular sheaths produced by this fungus during wood degradation. Fungal sheaths had a dense or loose fibrillar appearance and were differentiated from the fungal cell wall early after wood inoculation. Close association between extracellular fibrils and wood cell walls was observed at both early and advanced stages of wood alteration. Fungal sheaths were often seen deep in host cell walls, sometimes enclosing residual wood fragments. Specific gold probes were used to investigate the chemical nature of R. lignosus sheaths. While labeling of chitin, pectin, β-1,4- and β-1,3-glucans, β-glucosides, galactosamine, mannose, sialic acid, RNA, fucose, and fimbrial proteins over fungal sheaths did not succeed, galactose residues and laccase (a fungal phenoloxidase) were found to be present. The positive reaction of sheaths with the PATAg test indicates that polysaccharides such as β-1,6-glucans are important components. Our data suggest that extracellular sheaths produced by R. lignosus during host cell colonization play an important role in wood degradation. Transportation of lignin-degrading enzymes by extracellular fibrils indicates that alteration of plant polymers may occur within fungal sheaths. It is also proposed that R. lignosus sheaths may be involved in recognition mechanisms in fungal cell-wood surface interactions.     

beta-d-Glucan Antibodies Inhibit Auxin-Induced Cell Elongation and Changes in the Cell Wall of Zea Coleoptile Segments

Antiserum was raised against the Avena sativa L. caryopsis β-d-glucan fraction with an average molecular weight of 1.5 × 10⁴. Polyclonal antibodies recovered from the serum after Protein A-Sepharose column chromatography precipitated when cross-reacted with high molecular weight (1→3), (1→4)-β-d-glucans. These antibodies were effective in suppression of cell wall autohydrolytic reactions and auxin-induced decreases in noncellulosic glucose content of the cell wall of maize (Zea mays L.) coleoptiles. The results indicate antibody-mediated interference with in situ β-d-glucan degradation. The antibodies at a concentration of 200 micrograms per milliliter also suppress auxin-induced elongation by about 40% and cell wall loosening (measured by the minimum stress-relaxation time of the segments) of Zea coleoptiles. The suppression of elongation by antibodies was imposed without a lag period. Auxin-induced elongation, cell wall loosening, and chemical changes in the cell walls were near the levels of control tissues when segments were subjected to antibody preparation precipitated by a pretreatment with Avena caryopsis β-d-glucans. These results support the idea that the degradation of (1→3), (1→4)-β-d-glucans by cell wall enzymes is associated with the cell wall loosening responsible for auxin-induced elongation.     

GPI anchor biosynthesis in yeast: phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid

Cells synthesize the GPI anchor carbohydrate core by successively adding N-acetylglucosamine, three mannoses, and phosphoethanolamine (EtN-P) onto phosphatidylinositol, thus forming the complete GPI precursor lipid which is then added to proteins. Previously, we isolated a GPI deficient yeast mutant accumulating a GPI intermediate containing only two mannoses, suggesting that it has difficulty in adding the third, alpha1,2-linked Man of GPI anchors. The mutant thus displays a similar phenotype as the mammalian mutant cell line S1A-b having a mutation in the PIG-B gene. The yeast mutant, herein named gpi10-1 , contains a mutation in YGL142C, a yeast homolog of the human PIG-B. YGL142C predicts a highly hydrophobic integral membrane protein which by sequence is related to ALG9, a yeast gene required for adding Man in alpha1,2 linkage to N-glycans. Whereas gpi10-1 cells grow at a normal rate and make normal amounts of GPI proteins, the microsomes of gpi10-1 are completely unable to add the third Man in an in vitro assay. Further analysis of the GPI intermediate accumulating in gpi10 shows it to have the structure Manalpha1-6(EtN-P-)Manalpha1-4GlcNalpha1- 6(acyl) Inositol-P-lipid. The presence of EtN-P on the alpha1,4-linked Man of GPI anchors is typical of mammalian and a few other organisms but had not been observed in yeast GPI proteins. This additional EtN-P is not only found in the abnormal GPI intermediate of gpi10-1 but is equally present on the complete GPI precursor lipid of wild type cells. Thus, GPI biosynthesis in yeast and mammals proceeds similarly and differs from the pathway described for Trypanosoma brucei in several aspects.      

Analyses of Extracellular Carbohydrates in Oomycetes Unveil the Existence of Three Different Cell Wall Types

Some of the most devastating plant and animal pathogens belong to the oomycete class. The cell walls of these microorganisms represent an excellent target for disease control, but their carbohydrate composition is elusive. We have undertaken a detailed cell wall analysis in 10 species from 2 major oomycete orders, the Peronosporales and the Saprolegniales, thereby unveiling the existence of 3 clearly different cell wall types: type I is devoid of N-acetylglucosamine (GlcNAc) but contains glucuronic acid and mannose; type II contains up to 5% GlcNAc and residues indicative of cross-links between cellulose and 1,3-β-glucans; type III is characterized by the highest GlcNAc content (>5%) and the occurrence of unusual carbohydrates that consist of 1,6-linked GlcNAc residues. These 3 cell wall types are also distinguishable by their cellulose content and the fine structure of their 1,3-β-glucans. We propose a cell wall paradigm for oomycetes that can serve as a basis for the establishment of cell wall architectural models and the further identification of cell wall subtypes. This paradigm is complementary to morphological and molecular criteria for taxonomic grouping and provides useful information for unraveling poorly understood cell wall carbohydrate biosynthetic pathways through the identification and characterization of the corresponding enzymes.     

Fission Yeast Cdc24 Is a Replication Factor C- and Proliferating Cell Nuclear Antigen-Interacting Factor Essential for S-Phase Completion

At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. The cdc24⁺ gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1⁺, rfc1⁺ (a fission yeast homologue of RFC1), and hhp1⁺, which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition, cdc24⁺ genetically interacts with the gene encoding the catalytic subunit of DNA polymerase , which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.     

Two Cell Division Cycle Mutants of SACCHAROMYCES CEREVISIAE Are Defective in Transmission of Mitochondria to Zygotes

Mutations in CDC genes of S. cerevisiae disrupt the cell cycle at specific stages. The experiments reported here demonstrate that two CDC genes, CDC5 and CDC27, are necessary for mitochondrial segregation as well as for nuclear division. The defect in the transmission of mitochondria was revealed by the examination of uninucleate and binucleate progeny of transient heterokaryons generated by using the kar1-1 mutation that disrupts nuclear fusion. One of the parents lacked mitochondrial DNA (ρ⁰) whereas the other parent had functional mitochondria (ρ⁺). When the parents of the heterokaryon were both wild-type (CDC), nearly all progeny received mitochondria at 21° and at 34°. Thirty-four of the 36 cdc mutations tested had no defect in transmission of mitochondria to zygotic progeny in crosses in which one parent was a cdc mutant and the other parent was not (CDC). However, the cdc5 and cdc27 mutations prevented the transmission of mitochondria to cdc progeny at 34° but not at 21°; CDC progeny received mitochondria at either temperature. This defect was observed in crosses of cdc5 or cdc27 by wild-type cells regardless of which parent donated mitochondria to the zygote. The defect in mitochondrial transmission cosegregated in meiotic tetrads with the defect in mitosis demonstrating that both are likely to be caused by the same temperature-sensitive mutation. These results indicate that the CDC5 and CDC27 gene products are essential in two motility-related processes: mitochondrial movement from the zygote to the progeny and in mitosis.—Furthermore, the results suggest that the function performed by the CDC5 and CDC27 gene products for mitochondrial transmission differ in some fundamental way from the function performed for mitosis. The function necessary for mitosis can be supplied to the cdc5 (or cdc27) nucleus by the CDC5 (or CDC27) nucleus in the same heterokaryon but the function necessary for mitochondrial transmission cannot. Perhaps the function needed for mitochondrial transmission must be performed in the cell cycle preceding the actual segregation of mitochondria whereas the function needed for nuclear segregation can be performed at the time that mitosis occurs.