Protein-O-glycosylation in yeast: protein-specific mannosyltransferases

S.cerevisiae contains at least six genes (PMT1–6) fordolicholphosphate-D-mannose: protein-O-D-mannosyltransferases.The in vivo mannosylation of seven O-mannosylated yeast proteinshas been analyzed in a number of pmt mutants. The results clearlyindicate that the various protein O-mannosyltransferases havedifferent specificities for protein substrates. Five of theproteins tested (chitinase, a-agglutinin, Kre9p, Bar1p, Pir2p/hsp150)are mainly underglycosylated in pmt1 and pmt2 mutants, wherebyqualitative differences exist among the various proteins. Twoof the O-mannosylated proteins (Ggp1p and Kex2p) are not atall affected in pmt1 and pmt2 mutants but are clearly underglycosylatedwhen PMT4 is mutated. Although the PMT4 gene product is shownto be responsible for O-mannosylating a Ser-rich region of Ggp1pin vivo, a penta-seryl-peptide is not an in vitro substratefor this transferase. A PMT3 mutation does affect O-manno-sylationof chitinase only in the genetic background of a pmt1pmt2 doublemutation, indicating that PMT1 and PMT2 can compensate for adeleted PMT3 gene. dolichol-phosphate PMT gene family protein glycosylation S. 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.     

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。     

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.     

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.     

Arabidopsis extra-large G proteins (XLGs) regulate root morphogenesis

As in mammalian systems, heterotrimeric G proteins, composed of alpha, beta and gamma subunits, are present in plants and are involved in the regulation of development and cell signaling. Besides the sole prototypical G protein alpha subunit gene, GPA1, the Arabidopsis thaliana genome has three extra-large GTP-binding protein (XLG)-encoding genes: XLG1 (At2g23460), XLG2 (At4g34390) and XLG3 (At1g31930). The C-termini of the XLGs are Galpha domains that are homologous to GPA1, whereas their N-termini each contain a cysteine-rich region and a putative nuclear localization signal (NLS). GFP fusions with each XLG confirmed nuclear localization. All three XLG genes are expressed in essentially all plant organs, with strong expression in vascular tissues, primary root meristems and lateral root primordia. Analysis of single, double and triple T-DNA insertional mutants of the XLG genes revealed redundancy in XLG function. Dark-grown xlg1-1 xlg2-1 xlg3-1 triple mutant plants showed markedly increased primary root length compared with wild-type plants. This phenotype was not observed in dark-grown xlg single mutants, and was suppressed upon complementation of the xlg triple mutant with each XLG. Root cell sizes of the xlg triple mutant and root morphology were highly similar to those of wild-type roots, suggesting that XLGs may regulate cell proliferation. Dark-grown roots of the xlg triple mutants also showed altered sensitivity to sugars, ABA hyposensitivity and ethylene hypersensitivity, whereas seed germination in xlg triple mutants was hypersensitive to osmotic stress and ABA. As plant-specific proteins, regulatory mechanisms of XLGs may differ from those of conventional Galphas.     

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.     

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     

Multiple loss-of-function of Arabidopsis gibberellin receptor AtGID1s completely shuts down a gibberellin signal

Arabidopsis carries three receptor genes for the phytohormone gibberellin (GA), AtGID1a, AtGID1b and AtGID1c. Expression of each gene in the rice gid1-1 mutant for GA receptors causes reversion of its severely dwarfed phenotype and GA insensitivity to a normal level, even though each loss-of-function mutant shows no clear phenotype in Arabidopsis (Nakajima et al., 2006). In this paper, we report the functional redundancy and specificity of each AtGID1 by analyzing the multiple mutants for loss of function. Seeds of the double knockout mutants atgid1a atgid1b, atgid1a atgid1c and atgid1b atgid1c germinated normally. The double knockout mutant atgid1a atgid1c showed a dwarf phenotype, while other double mutants were of normal height compared to the wild-type. The stamens of the double knockout mutant atgid1a atgid1b were significantly shorter than those of the wild-type, and this leads to low fertility. A severe disarrangement of the pattern on its seed surface was also observed. The triple knockout mutant atgid1a atgid1b atgid1c did not germinate voluntarily, and only started to grow when the seed coat was peeled off after soaking. Seedlings of the triple knockout mutants were severe dwarfs, only a few millimeters high after growing for 1 month. Moreover, the triple knockout seedlings completely lost their ability to respond to exogenously applied GA. These results show that all AtGID1s function as GA receptors in Arabidopsis, but have specific role(s) for growth and development.     

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.     

Mutants of Serratia marcescens lacking cyclic nucleotide phosphodiesterase activity and requiring cyclic 3',5'-AMP for the utilization of various carbohydrates.

Adenine requiring mutants of Serratia marcescens SM-6-F'lac+ have been found to grow well in minimal-glucose medium solely supplemented with cAMP. From one of these ade strains double mutants (called ade cpd) were isolated which could no longer utilize cAMP but which still grew on 5'AMP. Dialyzed cell extracts (soluble fraction) of the double mutants, assayed for cAMP phosphodiesterase, were unable to hydrolyze cAMP whereas cell extracts of the parental strains yielded 5'AMP at a rate of 1.6-2.0 mumoles min-1 mg-1 protein. The loss of the phosphodiesterase activity in S. marcescens cpd W 1181 did not cause an accumulation of large amounts of cAMP as was found for the diesterase-negative mutant AB257pc-1 of Escherichia coli. The induced synthesis of beta-galactosidase in mutant cpd W 1181 showed about the same sensitivity to transient and permanent catabolite (glucose) repression as the corresponding cpd+ strain. Starting from S. marcescens cpd W 1182 three independent double mutants (called cpd cya) were isolated which required exogenous cAMP for utilizing various carbohydrates as carbon source, for motility and for the formation of extracellular lipase and the red pigment prodigiosine. The intracellular concentration of cAMP in these mutants, grown in nutrient broth, was 40-60% of that of the parental strain which is about 4 x 10(-4) M. However, the adenylate cyclase in cell extracts of the mutants W 1237 and W 1270 was like that of the corresponding cya+ strain (about 2 x 10(-2) mumoles min-1 mg-1 protein).     

New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1

We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose. The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose-ammonium medium and selecting for mutants able to grow in the non-permissive medium. HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired. REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1. Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4. ESC3-1 did not bypass the requirement of Snf1 for derepression. We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation.     

Revelation of ancestral roles of KNOX genes by a functional analysis of <Emphasis Type="Italic">Physcomitrella</Emphasis> homologues

KNOX genes are indispensable elements of indeterminate apical growth programmes of vascular plant sporophytes. Since little is known about the roles of such genes in non-vascular plants, functional analysis of moss KNOX homologues (MKN genes) was undertaken using the genetically amenable model plant, Physcomitrella patens. Three MKN genes were inactivated by targeted gene knockout to produce single, double and triple mutants. MKN2 (a class 1 KNOX gene) mutants were characterised by premature sporogenesis, abnormal sporophyte ontogeny and irregular spore development. MKN4 (a second class 1 gene) mutants were phenotypically normal. MKN1-3 (a class 2 KNOX gene) mutants exhibited defects in spore coat morphology. Analysis of double and triple mutants revealed that the abnormal sporophytic phenotype of MKN2 mutants was accentuated by mutating MKN4 and to a lesser degree by mutating MKN1-3. The aberrant spore phenotype of MKN1-3 and MKN2 mutants was exacerbated by mutating MKN4. This study provides the first instance in which an abnormal phenotype has been associated with the disruption of a class 2 KNOX gene as well as the first demonstrated case of functional redundancy between a class 1 and a class 2 KNOX gene. We conclude that KNOX genes play significant roles in programming sporophytic development in moss and we provide evidence that ancestral function(s) of this gene family were instrumental in the successful transition of plants to a terrestrial environment.