The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital.

The transfer of mannose to seryl and threonyl residues of secretory proteins is catalyzed by a family of protein mannosyltransferases coded for by seven genes (PMT1-7). Mannose dolichylphosphate is the sugar donor of the reaction, which is localized at the endoplasmic reticulum. By gene disruption and crosses all single, double and triple mutants of genes PMT1-4 were constructed. Two of the double and three of the triple mutants were not able to grow under normal conditions; three of these mutants could grow, however, when osmotically stabilized. The various mutants were extensively characterized concerning growth, morphology and their sensitivity to killer toxin K1, caffeine and calcofluor white. O-Mannosylation of gp115/Gas1p was affected only in pmt4 mutants, whereas glycosylation of chitinase was mainly affected in pmt1 and pmt2 mutants. The results show that protein O-glycosylation is essential for cell wall rigidity and cell integrity and that this protein modification, therefore, is vital for Saccharomyces cerevisiae.     

Defective threonine-linked glycosylation of human insulin-like growth factor in mutants of the yeast Saccharomyces cerevisiae

Mutants of the yeast Saccharomyces cerevisiae were identified,in which O-glycosylation at threonine 29 of a heterologous protein,human insulin-like growth factor (hIGF-1), is defective. Inmutant M195, O-glycosylation of hIGF-1, but not of yeast proteinschitinase and a-agglutinin, was reduced; in mutant M577 yeastproteins were affected besides hIGF-1. The mutations of M195and M577 did not affect viability and could not be complementedby the PMT1 or PMT2 genes. The mutant phenorype of strain M195was reconstituted in an in vitro system, in which a hIGF-1-derivedpeptide encompassing residues 24–34 was not used as acceptorfor mannosylation, while unrelated peptides were glycosylatedat wild-type levels. hIGF-1 glycosylation was drastically reducedin pmt1 disruptants and to a lesser extent in pmt2 disruptants,suggesting interaction between the PMT gene products and componentsmutated in M195 and M577 cells. The results suggest that mutationsmay only affect O-glycosylation of a specific subset of secretedproteins in yeast. insulin-like growth factor O-glycosylation protein mannosyltransferase Saccharomyces cerevisiae     

Characterization of the PMT Gene Family in Cryptococcus neoformans

Background

Protein-O-mannosyltransferases (Pmt''s) catalyze the initial step of protein-O-glycosylation, the addition of mannose residues to serine or threonine residues of target proteins.

Methodology/Principal Findings

Based on protein similarities, this highly conserved protein family can be divided into three subfamilies: the Pmt1 sub-family, the Pmt2 sub-family and the Pmt4 sub-family. In contrast to Saccharomyces cerevisiae and Candida albicans, but similar to filamentous fungi, three putative PMT genes (PMT1, PMT2, and PMT4) were identified in the genome of the human fungal pathogen Cryptococcus neoformans. Similar to Schizosaccharomyces pombe and C. albicans, C. neoformans PMT2 is an essential gene. In contrast, the pmt1 and pmt4 single mutants are viable; however, the pmt1/pmt4 deletions are synthetically lethal. Mutation of PMT1 and PMT4 resulted in distinct defects in cell morphology and cell integrity. The pmt1 mutant was more susceptible to SDS medium than wild-type strains and the mutant cells were enlarged. The pmt4 mutant grew poorly on high salt medium and demonstrated abnormal septum formation and defects in cell separation. Interestingly, the pmt1 and pmt4 mutants demonstrated variety-specific differences in the levels of susceptibility to osmotic and cell wall stress. Delayed melanin production in the pmt4 mutant was the only alteration of classical virulence-associated phenotypes. However, the pmt1 and pmt4 mutants showed attenuated virulence in a murine inhalation model of cryptococcosis.

Conclusion/Significance

These findings suggest that C. neoformans protein-O-mannosyltransferases play a crucial role in maintaining cell morphology, and that reduced protein-O-glycosylation leads to alterations in stress resistance, cell wall composition, cell integrity, and survival within the host.     

Characterization of O-mannosyltransferase family in Schizosaccharomyces pombe

Protein O-glycosylation is an essential protein modification in eukaryotic cells. In Saccharomyces cerevisiae, O-mannosylation is initiated in the lumen of the endoplasmic reticulum by O-mannosyltransferase gene products (Pmt1p-7p). A search of the Schizosaccharomyces pombe genome database revealed a total of three O-glycoside mannosyltransferase homologs (ogm1+, ogm2+, and ogm4+), closely related to Saccharomyces cerevisiae PMT1, PMT2, and PMT4. Although individual ogm genes were not found to be essential, ogm1Delta and ogm4Delta mutants exhibited aberrant morphology and failed to agglutinate during mating. The phenotypes of the ogm4Delta mutant were not complemented by overexpression of ogm1+ or ogm2+, suggesting that each of the Ogm proteins does not have overlapping functions. Heterologous expression of a chitinase from S. cerevisiae in the ogm mutants revealed that O-glycosylation of chitinase had decreased in ogm1Delta cells. A GFP-tagged Fus1p from S. cerevisiae was specifically not glycosylated and accumulated in the Golgi in ogm4Delta cells. These results indicate that O-glycosylation initiated by Ogm proteins plays crucial physiological roles and can serve as a sorting determinant for protein transport of membrane glycoproteins in S. pombe.     

Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP-mannose and cis-prenyltransferase activity

Protein O-mannosylation has been postulated to be critical for production and secretion of glycoproteins in fungi. Therefore, understanding the regulation of this process and the influence of heterologous expression of glycoproteins on the activity of enzymes engaged in O-glycosylation are of considerable interest. In this study we expressed cellobiohydrolase II (CBHII) of T. reesei, which is normally highly O-mannosylated, in Saccharomyces cerevisiae pmt mutants partially blocked in O-mannosylation. We found that the lack of Pmt1 or Pmt2 protein O-mannosyltransferase activity limited the glycosylation of CBHII, but it did not affect its secretion. The S. cerevisiae pmt1Δ and pmt2Δ mutants expressing T. reesei cbh2 gene showed a decrease of GDP-mannose level and a very high activity of cis-prenyltransferase compared to untransformed strains. On the other hand, elevation of cis-prenyltransferase activity by overexpression of the S. cerevisiae RER2 gene in these mutants led to an increase of dolichyl phosphate mannose synthase activity, but it did not influence the activity of O-mannosyltransferases. Overexpression of the MPG1 gene increased the level of GDP-mannose and stimulated the activity of mannosyltransferases elongating O-linked sugar chains, leading to partial restoration of CBHII glycosylation.     

Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP-mannose and cis-prenyltransferase activity

Protein O-mannosylation has been postulated to be critical for production and secretion of glycoproteins in fungi. Therefore, understanding the regulation of this process and the influence of heterologous expression of glycoproteins on the activity of enzymes engaged in O-glycosylation are of considerable interest. In this study we expressed cellobiohydrolase II (CBHII) of T. reesei, which is normally highly O-mannosylated, in Saccharomyces cerevisiae pmt mutants partially blocked in O-mannosylation. We found that the lack of Pmt1 or Pmt2 protein O-mannosyltransferase activity limited the glycosylation of CBHII, but it did not affect its secretion. The S. cerevisiae pmt1Delta and pmt2Delta mutants expressing T. reesei cbh2 gene showed a decrease of GDP-mannose level and a very high activity of cis-prenyltransferase compared to untransformed strains. On the other hand, elevation of cis-prenyltransferase activity by overexpression of the S. cerevisiae RER2 gene in these mutants led to an increase of dolichyl phosphate mannose synthase activity, but it did not influence the activity of O-mannosyltransferases. Overexpression of the MPG1 gene increased the level of GDP-mannose and stimulated the activity of mannosyltransferases elongating O-linked sugar chains, leading to partial restoration of CBHII glycosylation.     

Identification of O-mannosylated Virulence Factors in Ustilago maydis

The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens Candida albicans or Cryptococcus neoformans as well as in the phytopathogenic fungus Ustilago maydis. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in pmt4 mutants. Taking advantage of the characteristics described for Pmt4 substrates in Saccharomyces cerevisiae, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus U. maydis and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of pmt4 and msb2 showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in pmt4 mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of pmt4 mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis.     

O-Glycosylation of Axl2/Bud10p by Pmt4p is required for its stability, localization, and function in daughter cells.

Cells of the yeast Saccharomyces cerevisiae choose bud sites in a manner that is dependent upon cell type: a and alpha cells select axial sites; a/alpha cells utilize bipolar sites. Mutants specifically defective in axial budding were isolated from an alpha strain using pseudohyphal growth as an assay. We found that a and alpha mutants defective in the previously identified PMT4 gene exhibit unipolar, rather than axial budding: mother cells choose axial bud sites, but daughter cells do not. PMT4 encodes a protein mannosyl transferase (pmt) required for O-linked glycosylation of some secretory and cell surface proteins (Immervoll, T., M. Gentzsch, and W. Tanner. 1995. Yeast. 11:1345-1351). We demonstrate that Axl2/Bud10p, which is required for the axial budding pattern, is an O-linked glycoprotein and is incompletely glycosylated, unstable, and mislocalized in cells lacking PMT4. Overexpression of AXL2 can partially restore proper bud-site selection to pmt4 mutants. These data indicate that Axl2/Bud10p is glycosylated by Pmt4p and that O-linked glycosylation increases Axl2/ Bud10p activity in daughter cells, apparently by enhancing its stability and promoting its localization to the plasma membrane.     

A new Dol-P-Man:protein O-D-mannosyltransferase activity from Saccharomyces cerevisiae

The deletion of the protein mannosyltransferase 1 gene (PMT1)of Saccharomyces cerevisiae results in viable cells. O-Mannosylationof proteins is reduced to about half of the value in comparisonto wild-type cells. In order to distinguish between the thePMT1 gene product (= Pmt1p) and residual transferase activity,an in vitro assay to measure Dol-P-Man:protein mannosyltransferaseactivity in cells deleted for PMT1 has been developed. The transferaseactivity of these cells exhibits a pH optimum of 6.5 as comparedto pH 7.5 for Pmt1p. The K_$$$ value of the residual enzyme activityfor the hexapeptide YNPTSV is 7 times higher than that of Pmt1pand shows a clear preference for the seryl/residue. Differencesin substrate affinities as well as in seryl/threonyl preferencebetween the two enzymes, however, depend on the specific sequenceof the peptides used in the enzyme assay. The new enzyme activityshows a significantly lower thermal stability as compared toPmt1p. glycoprotein O-glycosylation mannosyltranferase Saccharomyces cerevisiae     

Members of protein O‐mannosyltransferase family in Aspergillus fumigatus differentially affect growth,morphogenesis and viability

O‐mannosylation is an essential protein modification in eukaryotes. It is initiated at the endoplasmic reticulum by O‐mannosyltransferases (PMT) that are evolutionary conserved from yeast to humans. The PMT family is phylogenetically classified into PMT1, PMT2 and PMT4 subfamilies, which differ in protein substrate specificity and number of genes per subfamily. In this study, we characterized for the first time the whole PMT family of a pathogenic filamentous fungus, Aspergillus fumigatus. Genome analysis showed that only one member of each subfamily is present in A. fumigatus, PMT1, PMT2 and PMT4. Despite the fact that all PMTs are transmembrane proteins with conserved peptide motifs, the phenotype of each PMT deletion mutant was very different in A. fumigatus. If disruption of PMT1 did not reveal any phenotype, deletion of PMT2 was lethal. Disruption of PMT4 resulted in abnormal mycelial growth and highly reduced conidiation associated to significant proteomic changes. The double pmt1pmt4 mutant was lethal. The single pmt4 mutant exhibited an exquisite sensitivity to echinocandins that is associated to major changes in the expression of signal transduction cascade genes. These results indicate that the PMT family members play a major role in growth, morphogenesis and viability of A. fumigatus.     

Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants

Protein O mannosylation is a crucial protein modification in uni- and multicellular eukaryotes. In humans, a lack of O-mannosyl glycans causes congenital muscular dystrophies that are associated with brain abnormalities. In yeast, protein O mannosylation is vital; however, it is not known why impaired O mannosylation results in cell death. To address this question, we analyzed the conditionally lethal Saccharomyces cerevisiae protein O-mannosyltransferase pmt2 pmt4Delta mutant. We found that pmt2 pmt4Delta cells lyse as small-budded cells in the absence of osmotic stabilization and that treatment with mating pheromone causes pheromone-induced cell death. These phenotypes are partially suppressed by overexpression of upstream elements of the protein kinase C (PKC1) cell integrity pathway, suggesting that the PKC1 pathway is defective in pmt2 pmt4Delta mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4Delta mutants during exposure to mating pheromone or elevated temperature. Detailed analyses of the plasma membrane sensors of the PKC1 pathway revealed that Wsc1p, Wsc2p, and Mid2p are aberrantly processed in pmt mutants. Our data suggest that in yeast, O mannosylation increases the activity of Wsc1p, Wsc2p, and Mid2p by enhancing their stability. Reduced O mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the PKC1 pathway and finally causes cell death in the absence of osmotic stabilization.     

Morphogenesis, adhesive properties, and antifungal resistance depend on the Pmt6 protein mannosyltransferase in the fungal pathogen candida albicans

Protein mannosyltransferases (Pmt proteins) initiate O glycosylation of secreted proteins in fungi. We have characterized PMT6, which encodes the second Pmt protein of the fungal pathogen Candida albicans. The residues of Pmt6p are 21 and 42% identical to those of C. albicans Pmt1p and S. cerevisiae Pmt6p, respectively. Mutants lacking one or two PMT6 alleles grow normally and contain normal Pmt enzymatic activities in cell extracts but show phenotypes including a partial block of hyphal formation (dimorphism) and a supersensitivity to hygromycin B. The morphogenetic defect can be suppressed by overproduction of known components of signaling pathways, including Cek1p, Cph1p, Tpk2p, and Efg1p, suggesting a specific Pmt6p target protein upstream of these components. Mutants lacking both PMT1 and PMT6 are viable and show pmt1 mutant phenotypes and an additional sensitivity to the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The lack of Pmt6p significantly reduces adherence to endothelial cells and overall virulence in a mouse model of systemic infection. The results suggest that Pmt6p regulates a more narrow subclass of proteins in C. albicans than Pmt1p, including secreted proteins responsible for morphogenesis and antifungal sensitivities.     

Role of protein O-mannosyltransferase Pmt4 in the morphogenesis and virulence of Cryptococcus neoformans

Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen Cryptococcus neoformans. Disruption of the C. neoformans PMT4 gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the pmt4 mutant compared to wild-type cells. The pmt4 mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the pmt4 mutant, as well as a global decrease in protein mannosylation. Heterologous expression of C. neoformans PMT4 in a Saccharomyces cerevisiae pmt1pmt4 mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in C. neoformans was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of C. neoformans.     

酵母PMT1基因参与内质网应激的调控机制

【目的】内质网应激(Endoplasmic reticulum stress,ERS)可激活细胞保护性信号级联反应——未折叠蛋白质反应(Unfolded protein response,UPR)。研究表明,酵母细胞中的UPR信号通路由转录因子Hac1p和ERS感应因子Ire1p共同介导。前期研究发现:蛋白质-O-甘露糖转移酶1(Protein-O-mannosyltransferase 1,PMT1)基因缺失能延长酵母细胞的复制性寿命,其机制与上调UPR通路活性相关。本文进一步探讨PMT1基因缺失在酵母ERS反应中的作用。【方法】观察PMT1基因与IRE1或HAC1基因双缺失酵母菌株(pmt1?hac1?和pmt1?ire1?)在ERS反应条件下的克隆形成能力;通过比色法检测各菌株的细胞增殖活性;RT-PCR检测各菌株UPR通路下游部分靶基因的转录水平。【结果】与对照菌株比较,PMT1基因缺失菌株(pmt1?)在ERS反应条件下生长较慢,而HAC1和IRE1单基因缺失菌株(hac1?和ire1?)在ERS反应条件下无法存活;在hac1?或ire1?菌株的基础上进一步缺失PMT1基因,可以改善hac1?菌株在ERS反应条件下的生长状态;但缺失PMT1基因没有上调hac1?菌株UPR通路靶基因的转录水平。【结论】缺失PMT1基因可增强hac1?菌株对ERS诱导剂衣霉素的抗性,机制与已知的UPR通路不相关,提示可能存在其它途径参与ERS反应的调控。     

Molecular Cloning and Characterization of a cDNA Clone That Encodes a Cdc2 Homolog from Nicotiana tabacum

We have isolated a cDNA clone (cdc2Nt1) that encodes a homologof p34^cdc2/CDC28 kinase from tobacco (Nicotiana tabacum). Thecdc2Ntl protein showed extensive similarity to other homologsof Cdc2 from plants. Complementation studies showed that thecdc2Ntl gene was able to overcome cell cycle arrest at boththe G1/S and the G2/M transitions of cdc28^ts mutants of buddingyeast, demonstrating that the cdc2Ntl protein was able to replacethe Cdc28 kinase at both the G1/S and the G2/M transitions.Analysis of gene expression demonstrated that the cdc2Ntl genewas transcribed constitutively throughout the cell cycle butthat it was preferentially expressed in actively dividing tobaccoBY-2 cells. (Received July 13, 1995; Accepted February 15, 1996)     

O-Mannosyltransferase 1 in Aspergillus fumigatus (AfPmt1p) is crucial for cell wall integrity and conidium morphology, especially at an elevated temperature

Protein O-mannosyltransferases initiate O mannosylation of secretory proteins, which are of fundamental importance in eukaryotes. In this study, the PMT gene family of the human fungal pathogen Aspergillus fumigatus was identified and characterized. Unlike the case in Saccharomyces cerevisiae, where the PMT family is highly redundant, only one member of each PMT subfamily, namely, Afpmt1, Afpmt2, and Afpmt4, is present in A. fumigatus. Mutants with a deletion of Afpmt1 are viable. In vitro and in vivo activity assays confirmed that the protein encoded by Afpmt1 acts as an O-mannosyltransferase (AfPmt1p). Characterization of the ΔAfpmt1 mutant showed that a lack of AfPmt1p results in sensitivity to elevated temperature and defects in growth and cell wall integrity, thereby affecting cell morphology, conidium formation, and germination. In a mouse model, Afpmt1 was not required for the virulence of A. fumigatus under the experimental conditions used.