Search

Competing Interests: The authors have declared that no competing interests exist.

Innate immunity is the first line of defense against pathogens and predators. To initiate a response, it relies on the detection of invaders, where lectin-carbohydrate interactions play a major role. O-Methylated glycans were previously identified as non-self epitopes and conserved targets for defense effector proteins belonging to the tectonin superfamily. Here, we present two crystal structures of Tectonin 2 from the mushroom Laccaria bicolor in complex with methylated ligands, unraveling the molecular basis for this original specificity. Furthermore, they revealed the formation of a ball-shaped tetramer with 24 binding sites distributed at its surface, resembling a small virus capsid. Based on the crystal structures, a methylation recognition motif was identified and found in the sequence of many tectonins from bacteria to human. Our results support a key role of tectonins in innate defense based on a distinctive and conserved type of lectin-glycan interaction., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Among the invaluable benefits of basidiomycete genomics is the dramatically enhanced insight into the potential capacity to biosynthesize natural products. This study focuses on adenylate-forming reductases, which is a group of natural product biosynthesis enzymes that resembles non-ribosomal peptide synthetases, yet serves to modify one substrate, rather than to condense two or more building blocks. Phylogenetically, these reductases fall in four classes. The phylogeny of Heterobasidion annosum (Russulales) and Serpula lacrymans (Boletales) adenylate-forming reductases was investigated. We identified a previously unrecognized phylogenetic branch within class III adenylate-forming reductases. Three representatives were heterologously produced and their substrate preferences determined in vitro: NPS9 and NPS11 of S. lacrymans preferred l-threonine and benzoic acid, respectively, while NPS10 of H. annosum accepted phenylpyruvic acid best. We also investigated two class IV adenylate-forming reductases of Coprinopsis cinerea, which each were active with l-alanine, l-valine, and l-serine as substrates. Our results show that adenylate-forming reductases are functionally more diverse than previously recognized. As none of the natural products known from the species investigated in this study includes the identified substrates of their respective reductases, our findings may help further explore the diversity of these basidiomycete secondary metabolomes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Peptide backbone N-methylation, as seen in cyclosporin A, has been considered to be exclusive to nonribosomal peptides. We have identified the first post-translationally modified peptide or protein harboring internal α-N-methylations through discovery of the genetic locus for the omphalotins, cyclic N-methylated peptides produced by the fungus Omphalotus olearius. We show that iterative autocatalytic activity of an N-methyltransferase fused to its peptide substrate is the signature of a new family of ribosomally encoded metabolites.

Lectins are used as defense effector proteins against predators, parasites and pathogens by animal, plant and fungal innate defense systems. These proteins bind to specific glycoepitopes on the cell surfaces and thereby interfere with the proper cellular functions of the various antagonists. The exact cellular toxicity mechanism is in many cases unclear. Lectin CCL2 of the mushroom Coprinopsis cinerea was previously shown to be toxic for Caenorhabditis elegans and Drosophila melanogaster. This toxicity is dependent on a single, high-affinity binding site for the trisaccharide GlcNAc(Fucα1,3)β1,4GlcNAc, which is a hallmark of nematode and insect N-glycan cores. The carbohydrate-binding site is located at an unusual position on the protein surface when compared to other β-trefoil lectins. Here, we show that CCL2 forms a compact dimer in solution and in crystals. Substitution of two amino acid residues at the dimer interface, R18A and F133A, interfered with dimerization of CCL2 and reduced toxicity but left carbohydrate-binding unaffected. These results, together with the positioning of the two carbohydrate-binding sites on the surface of the protein dimer, suggest that crosslinking of N-glycoproteins on the surface of intestinal cells of invertebrates is a crucial step in the mechanism of CCL2-mediated toxicity. Comparisons of the number and positioning of carbohydrate-binding sites among different dimerizing fungal β-trefoil lectins revealed a considerable variability in the carbohydrate-binding patterns of these proteins, which are likely to correlate with their respective functions., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Biofilm formation on fungal hyphae and production of antifungal molecules are strategies of bacteria in their competition with fungi for nutrients. Since these strategies are often coordinated and under control of quorum sensing by the bacteria, interference with this bacterial communication system can be used as a counter-strategy by the fungi in this competition. Hydrolysis of N-acyl-homoserine lactones (HSL), a quorum sensing molecule used by Gram-negative bacteria, by fungal cultures has been demonstrated. However, the enzymes that are responsible for this activity, have not been identified. In this study, we identified and characterized two paralogous HSL hydrolyzing enzymes from the coprophilous fungus Coprinopsis cinerea. The C. cinerea HSL lactonases belong to the metallo-β-lactamase family and show sequence homology to and a similar biochemical activity as the well characterized lactonase AiiA from Bacillus thuringiensis. We show that the fungal lactonases, similar to the bacterial enzymes, are kept intracellularly and act as a sink for the bacterial quorum sensing signals both in C. cinerea and in Saccharomyces cerevisiae expressing C. cinerea lactonases, due to the ability of these signal molecules to diffuse over the fungal cell wall and plasma membrane. The two isogenes coding for the C. cinerea HSL lactonases are arranged in the genome as a tandem repeat and expressed preferentially in vegetative mycelium. The occurrence of orthologous genes in genomes of other basidiomycetes appears to correlate with a saprotrophic lifestyle., (Copyright © 2016 Elsevier Inc. All rights reserved.)

PSL1a is a lectin from the mushroom Polyporus squamosus that binds to sialylated glycans and glycoconjugates with high specificity and selectivity. In addition to its N-terminal carbohydrate-binding domain, PSL1a possesses a Ca2+-dependent proteolytic activity in the C-terminal domain. In the present study, we demonstrate that PSL1a has cytotoxic effects on mammalian cancer cells, and we show that the cytotoxicity is dependent on the cysteine protease activity. PSL1a treatment leads to cell rounding and detachment from the substratum, concomitant with disruption of vinculin complexes in focal adhesions. We also demonstrate that PSL1a inhibits protein synthesis and induces apoptosis in HeLa cells, in a time- and concentration-dependent manner., Competing Interests: The authors have declared that no competing interests exist.

Selenoglycosides are used as reactive glycosyl donors in the syntheses of oligosaccharides. In addition, such heavy atom analogs of natural glycosides are useful tools for structure determination of their lectin receptors using X-ray crystallography. Some lectins, e.g., members of the tectonin family, only bind to carbohydrate epitopes with O-alkylated ring hydroxy groups. In this context, we report the first synthesis of an O -methylated selenoglycoside, specifically methyl 2- O -methyl-L-selenofucopyranoside, a ligand of the lectin tectonin-2 from the mushroom Laccaria bicolor . The synthetic route required a strategic revision and further optimization due to the intrinsic lability of alkyl selenoglycosides, in particular for the labile fucose. Here, we describe a successful synthetic access to methyl 2- O -methyl-L-selenofucopyranoside in 9 linear steps and 26% overall yield starting from allyl L-fucopyranoside.

Background: Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a lectin from the fairy ring mushroom with specificity for Galα1-3Gal containing carbohydrates. This lectin is composed of an N-terminal carbohydrate-binding domain and a C-terminal dimerization domain. The dimerization domain of MOA shows in addition calcium-dependent cysteine protease activity, similar to the calpain family., Methods: Cell detachment assay, cell viability assay, immunofluorescence, live cell imaging and Western blot using MDCKII cell line., Results: In this study, we demonstrate in MDCKII cells that after internalization, MOA protease activity induces profound physiological cellular responses, like cytoskeleton rearrangement, cell detachment and cell death. These changes are preceded by a decrease in FAK phosphorylation and an internalization and degradation of β1-integrin, consistent with a disruption of integrin-dependent cell adhesion signaling. Once internalized, MOA accumulates in late endosomal compartments., Conclusion: Our results suggest a possible toxic mechanism of MOA, which consists of disturbing the cell adhesion and the cell viability., General Significance: After being ingested by a predator, MOA might exert a protective role by diminishing host cell integrity., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The ideal prehospital management of patients with severe traumatic brain injury (TBI) including the impact of endotracheal intubation (ETI) and physicians on scene is unclear. Prehospital management differs substantially in Switzerland and the United States: in Switzerland, there is usually a physician on scene who may provide ETI and other advanced life support procedures, whereas in the United States, prehospital management (including ETI) is performed by paramedics., Methods: This is a retrospective cohort-matched study of patients with isolated blunt severe TBI (head Abbreviated Injury Scale [AIS] score, 4-5) and no major extracranial injuries, using Bern University Hospital data from the Swiss PEBITA [Patient-relevant Endpoints after Brain Injury from Traumatic Accidents] (TBI-specific) database and the US National Trauma Data Bank from 2009 to 2010. A 1:4 cohort matching of Bern and US patients was performed. Matching criteria were sex, age (±10 years), exact field Glasgow Coma Scale (GCS) score, exact head AIS score, and injury type (subdural hematoma, epidural hematoma, intraparenchymal hemorrhage, intraventricular hemorrhage, brain edema/swelling, brain stem injury). The matched cohorts were compared with univariable analysis (Fisher's exact test and Mann-Whitney U-test)., Results: Matching of the Bern (n = 128) and US (n = 86,375) cohort resulted in 355 matched cases (71 Bern and 284 US patients). Bern patients had significantly longer scene times (median, 23.0 minutes vs. 9.0 minutes, p < 0.001) and more frequent prehospital ETI (31.0% vs. 18.7%, p = 0.034) and air transportation (39.4% vs. 19.4%, p < 0.001). No significant difference in procedures (craniotomy/craniectomy, intracranial pressure monitoring, tracheotomy), intensive care unit and total hospital lengths of stay, ventilator days, and in-hospital mortality (14.1% vs. 15.8%, p = 0.855) was found between the two cohorts., Conclusion: When taking into account the limitation that patient- and injury-related factors, but not in-hospital treatment variables, were matched, the more frequent prehospital ETI and presence of a physician on scene in the Swiss cohort compared with the US cohort had no significant effect on outcomes, including intensive care unit and total hospital lengths of stay, ventilator days, and in-hospital mortality., Level of Evidence: Therapeutic study, level IV.

Fruiting bodies or sporocarps of dikaryotic (ascomycetous and basidiomycetous) fungi, commonly referred to as mushrooms, are often rich in entomotoxic and nematotoxic proteins that include lectins and protease inhibitors. These protein toxins are thought to act as effectors of an innate defense system of mushrooms against animal predators including fungivorous insects and nematodes. In this review, we summarize current knowledge about the structures, target molecules, and regulation of the biosynthesis of the best characterized representatives of these fungal defense proteins, including galectins, beta-trefoil-type lectins, actinoporin-type lectins, beta-propeller-type lectins and beta-trefoil-type chimerolectins, as well as mycospin and mycocypin families of protease inhibitors. We also present an overview of the phylogenetic distribution of these proteins among a selection of fungal genomes and draw some conclusions about their evolution and physiological function. Finally, we present an outlook for future research directions in this field and their potential applications in medicine and crop protection.

The dung of herbivores, the natural habitat of the model mushroom Coprinopsis cinerea, is a nutrient-rich but also very competitive environment for a saprophytic fungus. We showed previously that C. cinerea expresses constitutive, tissue-specific armories against antagonists such as animal predators and bacterial competitors. In order to dissect the inducible armories against such antagonists, we sequenced the poly(A)-positive transcriptome of C. cinerea vegetative mycelium upon challenge with fungivorous and bacterivorous nematodes, Gram-negative and Gram-positive bacteria and mechanical damage. As a response to the fungivorous nematode Aphelenchus avenae, C. cinerea was found to specifically induce the transcription of several genes encoding previously characterized nematotoxic lectins. In addition, a previously not characterized gene encoding a cytoplasmic protein with several predicted Ricin B-fold domains, was found to be strongly upregulated under this condition. Functional analysis of the recombinant protein revealed a high toxicity toward the bacterivorous nematode Caenorhabditis elegans. Challenge of the mycelium with A. avenae also lead to the induction of several genes encoding putative antibacterial proteins. Some of these genes were also induced upon challenge of the mycelium with the bacteria Escherichia coli and Bacillus subtilis. These results suggest that fungi have the ability to induce specific innate defense responses similar to plants and animals., (Copyright © 2016 Plaza et al.)

Background: Lectins are carbohydrate-binding proteins that are involved in fundamental intra- and extracellular biological processes. They occur ubiquitously in nature and are especially abundant in plants and fungi. It has been well established that certain higher fungi produce lectins in their fruiting bodies and/or sclerotia as a part of their natural resistance against free-living fungivorous nematodes and other pests. Despite relatively high diversity of the glycan structures in nature, many of the glycans targeted by fungal lectins are conserved among organisms of the same taxon and sometimes even among different taxa. Such conservation of glycans between free-living and parasitic nematodes is providing us with a useful tool for discovery of novel chemotherapeutic and vaccine targets. In our study, a subset of fungal lectins emanating from toxicity screens on Caenorhabditis elegans was tested for their potential to inhibit larval development of Haemonchus contortus., Methods: The effect of Coprinopsis cinerea lectins - CCL2, CGL2, CGL3; Aleuria aurantia lectin - AAL; Marasmius oreades agglutinin - MOA; and Laccaria bicolor lectin - Lb-Tec2, on cultivated Haemonchus contortus larval stages was investigated using a larval development test (LDT). To validate the results of the toxicity assay and determine lectin binding capacity to the nematode digestive tract, biotinylated versions of lectins were fed to pre-infective larval stages of H. contortus and visualized by fluorescent microscopy. Lectin histochemistry on fixed adult worms was performed to investigate the presence and localisation of lectin binding sites in the disease-relevant developmental stage., Results: Using an improved larval development test we found that four of the six tested lectins: AAL, CCL2, MOA and CGL2, exhibited a dose-dependent toxicity in LDT, as measured by the number of larvae developing to the L3 stage. In the case of AAL, CGL2 and MOA lectin, doses as low as 5 μg/ml caused >95 % inhibition of larval development while 40 μg/ml were needed to achieve the same inhibition by CCL2 lectin. MOA was the only lectin tested that caused larval death while other toxic lectins had larvistatic effect manifesting as L1 growth arrest. Using lectin histochemistry we demonstrate that of all lectins tested, only the four toxic ones displayed binding to the larvae's gut and likewise were found to interact with glycans localized to the gastrodermal tissue of adults., Conclusion: The results of our study suggest a correlation between the presence of target glycans of lectins in the digestive tract and the lectin-mediated toxicity in Haemonchus contortus. We demonstrate that binding to the structurally conserved glycan structures found in H. contortus gastrodermal tissue by the set of fungal lectins has detrimental effect on larval development. Some of these glycan structures might represent antigens which are not exposed to the host immune system (hidden antigens) and thus have a potential for vaccine or drug development. Nematotoxic fungal lectins prove to be a useful tool to identify such targets in parasitic nematodes.

Lectins are non-immunoglobulin carbohydrate-binding proteins without enzymatic activity towards the bound carbohydrates. Many lectins of e.g. plants or fungi have been suggested to act as toxins to defend the host against predators and parasites. We have previously shown that the Coprinopsis cinerea lectin 2 (CCL2), which binds to α1,3-fucosylated N-glycan cores, is toxic to Caenorhabditis elegans and results in developmental delay and premature death. In this study, we investigated the underlying toxicity phenotype at the cellular level by electron and confocal microscopy. We found that CCL2 directly binds to the intestinal apical surface and leads to a highly damaged brush border with loss of microvilli, actin filament depolymerization, and invaginations of the intestinal apical plasma membrane through gaps in the terminal web. We excluded several possible toxicity mechanisms such as internalization and pore-formation, suggesting that CCL2 acts directly on intestinal apical plasma membrane or glycocalyx proteins. A genetic screen for C. elegans mutants resistant to CCL2 generated over a dozen new alleles in bre 1, ger 1, and fut 1, three genes required for the synthesis of the sugar moiety recognized by CCL2. CCL2-induced intestinal brush border defects in C. elegans are similar to the damage observed previously in rats after feeding the dietary lectins wheat germ agglutinin or concanavalin A. The evolutionary conserved reaction of the brush border between mammals and nematodes might allow C. elegans to be exploited as model organism for the study of dietary lectin-induced intestinal pathology in mammals.

Organisms which rely solely on innate defense systems must combat a large number of antagonists with a comparably low number of defense effector molecules. As one solution of this problem, these organisms have evolved effector molecules targeting epitopes that are conserved between different antagonists of a specific taxon or, if possible, even of different taxa. In order to restrict the activity of the defense effector molecules to physiologically relevant taxa, these target epitopes should, on the other hand, be taxon-specific and easily accessible. Glycans fulfill all these requirements and are therefore a preferred target of defense effector molecules, in particular defense proteins. Here, we review this defense strategy using the example of the defense system of multicellular (filamentous) fungi against microbial competitors and animal predators.

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/β-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Interactions between fungi and prokaryotes are abundant in many ecological systems. A wide variety of biomolecules regulate such interactions and many of them have found medicinal or biotechnological applications. However, studying a fungal-bacterial system at a cellular level is technically challenging. New microfluidic devices provided a platform for microscopic studies and for long-term, time-lapse experiments. Application of these novel tools revealed insights into the dynamic interactions between the basidiomycete Coprinopsis cinerea and the bacterium Bacillus subtilis. Direct contact was mediated by polar attachment of bacteria to only a subset of fungal hyphae suggesting a differential competence of fungal hyphae and thus differentiation of hyphae within a mycelium. The fungicidal activity of B. subtilis was monitored at a cellular level and showed a novel mode of action on fungal hyphae.

Unlabelled: Lectins are carbohydrate-binding proteins present in all organisms. Some cytoplasmic lectins from fruiting bodies of dikaryotic fungi are toxic against a variety of parasites and predators. We have isolated, cloned and expressed a novel, single domain lectin from Macrolepiota procera, designated MpL. Determination of the crystal structure revealed that MpL is a ricin B-like lectin with a β-trefoil fold. Biochemical characterization, site-directed mutagenesis, co-crystallization with carbohydrates, isothermal titration calorimetry and glycan microarray analyses show that MpL forms dimers with the carbohydrate-binding site at the α-repeat, with the highest specificity for terminal N-acetyllactosamine and other β-galactosides. A second putative carbohydrate-binding site with a low affinity for galactose is present at the γ-repeat. In addition, a novel hydrophobic binding site was detected in MpL with specificity for molecules other than carbohydrates. The tissue specific distribution of MpL in the stipe and cap tissue of fruiting bodies and its toxicity towards the nematode Caenorhabditis elegans indicate a function of MpL in protecting fruiting bodies against predators and parasites., Database: Nucleotide sequence data have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers HQ449738 and HQ449739. Structural data have been deposited in the Protein Data Bank under accession codes 4ION, 4IYB, 4IZX and 4J2S., (© 2014 FEBS.)

Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of β-propeller lectins conserved from bacteria to mammals that have been shown to bind bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans, suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1, coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus, showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.

Background: It is well known that mushrooms produce defense proteins and secondary metabolites against predators and competitors; however, less is known about the correlation between the tissue-specific expression and the target organism (antagonist) specificity of these molecules. In addition, conserved transcriptional circuitries involved in developing sexual organs in fungi are not characterized, despite the growing number of gene expression datasets available from reproductive and vegetative tissue. The aims of this study were: first, to evaluate the tissue specificity of defense gene expression in the model mushroom Coprinopsis cinerea and, second, to assess the degree of conservation in transcriptional regulation during sexual development in basidiomycetes., Results: In order to characterize the regulation in the expression of defense loci and the transcriptional circuitries controlling sexual reproduction in basidiomycetes, we sequenced the poly (A)-positive transcriptome of stage 1 primordia and vegetative mycelium of C. cinerea A43mutB43mut. Our data show that many genes encoding predicted and already characterized defense proteins are differentially expressed in these tissues. The predicted specificity of these proteins with regard to target organisms suggests that their expression pattern correlates with the type of antagonists these tissues are confronted with. Accordingly, we show that the stage 1 primordium-specific protein CC1G&#95;11805 is toxic to insects and nematodes. Comparison of our data to analogous data from Laccaria bicolor and Schizophyllum commune revealed that the transcriptional regulation of nearly 70 loci is conserved and probably subjected to stabilizing selection. A Velvet domain-containing protein was found to be up-regulated in all three fungi, providing preliminary evidence of a possible role of the Velvet protein family in sexual development of basidiomycetes. The PBS-soluble proteome of C. cinerea primordia and mycelium was analyzed by shotgun LC-MS. This proteome data confirmed the presence of intracellular defense proteins in primordia., Conclusions: This study shows that the exposure of different tissues in fungi to different types of antagonists shapes the expression pattern of defense loci in a tissue-specific manner. Furthermore, we identify a transcriptional circuitry conserved among basidiomycetes during fruiting body formation that involves, amongst other transcription factors, the up-regulation of a Velvet domain-containing protein.

The pathogenic fungus Cryptococcus neoformans synthesizes a complex family of glycosylinositolphosphoceramide (GIPC) structures. These glycosphingolipids (GSLs) consist of mannosylinositolphosphoceramide (MIPC) extended by β1-6-linked galactose, a unique structure that has to date only been identified in basidiomycetes. Further extension by up to five mannose residues and a branching xylose has been described. In this study, we identified and determined the gene structure of the enzyme Ggt1, which catalyzes the transfer of a galactose residue to MIPC. Deletion of the gene in C. neoformans resulted in complete loss of GIPCs containing galactose, a phenotype that could be restored by the episomal expression of Ggt1 in the deletion mutant. The entire annotated open reading frame, encoding a C-terminal GT31 galactosyltransferase domain and a large N-terminal domain of unknown function, was required for complementation. Notably, this gene does not encode a predicted signal sequence or transmembrane domain. The demonstration that Ggt1 is responsible for the transfer of a galactose residue to a GSL thus raises questions regarding the topology of this biosynthetic pathway and the function of the N-terminal domain. Phylogenetic analysis of the GGT1 gene shows conservation in hetero- and homobasidiomycetes but no homologs in ascomycetes or outside of the fungal kingdom.

The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Tamavidins are fungal biotin-binding proteins (BBPs) displaying antifungal activity against phytopathogens. Here we show high toxicity of tamavidins toward nematodes, insects, and amoebae. As these organisms represent important phyla of fungal predators and parasites, we propose that BBPs are part of the chemical defense system of fungi.

The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose ("GalFuc" epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N'-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency.

Cospin (PIC1) from Coprinopsis cinerea is a serine protease inhibitor with biochemical properties similar to those of the previously characterized fungal serine protease inhibitors, cnispin from Clitocybe nebularis and LeSPI from Lentinus edodes, classified in the family I66 of the MEROPS protease inhibitor classification. In particular, it exhibits a highly specific inhibitory profile as a very strong inhibitor of trypsin with K(i) in the picomolar range. Determination of the crystal structure revealed that the protein has a β-trefoil fold. Site-directed mutagenesis and mass spectrometry results have confirmed Arg-27 as the reactive binding site for trypsin inhibition. The loop containing Arg-27 is positioned between the β2 and β3 strands, distinguishing cospin from other β-trefoil-fold serine protease inhibitors in which β4-β5 or β5-β6 loops are involved in protease inhibition. Biotoxicity assays of cospin on various model organisms revealed a strong and specific entomotoxic activity against Drosophila melanogaster. The inhibitory inactive R27N mutant was not entomotoxic, associating toxicity with inhibitory activity. Along with the abundance of cospin in fruiting bodies of C. cinerea and the lack of trypsin-like proteases in the C. cinerea genome, these results suggest that cospin and its homologs are effectors of a fungal defense mechanism against fungivorous insects that function by specific inhibition of serine proteases in the insect gut.

Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAcβ1,4[Fucα1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a β-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to α1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity.

Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a Galα1,3Gal/GalNAc-specific lectin from the fairy ring mushroom that consists of an N-terminal ricin B-type lectin domain and a C-terminal dimerization domain. The latter domain shows structural similarity to catalytically active proteins, suggesting that, in addition to its carbohydrate-binding activity, MOA has an enzymatic function. Here, we demonstrate toxicity of MOA toward the model nematode Caenorhabditis elegans. This toxicity depends on binding of MOA to glycosphingolipids of the worm via its lectin domain. We show further that MOA has cysteine protease activity and demonstrate a critical role of this catalytic function in MOA-mediated nematotoxicity. The proteolytic activity of MOA was dependent on high Ca(2+) concentrations and favored by slightly alkaline pH, suggesting that these conditions trigger activation of the toxin at the target location. Our results suggest that MOA is a fungal toxin with intriguing similarities to bacterial binary toxins and has a protective function against fungivorous soil nematodes.

Coprinopsis cinerea is a model organism for fruiting body development in homobasidiomycetes. Here, we focused on N-linked oligosaccharides (NLO) of cell wall proteins in the hyphae of two developmental stages, vegetative mycelium and fruiting body. High mannose-type glycans were the most commonly found structures. In addition, we observed a novel glycan, predominantly present in fruiting body. This oligosaccharide structure was of the high mannose type with at least five mannoses and a bisecting alpha1-4 N-acetylglucosamine (GlcNAc) at the beta-mannose of the N-glycan core. The transferase responsible for this modification, CcGnt1 (C. cinerea GlcNAc transferase 1), was identified and expressed in insect cells. In vitro activity of CcGnt1 was demonstrated. This novel glycosyltransferase belongs to the glycosyltransferase family 8 (GT8) and is predicted to be a type II membrane protein. Expression of the CcGnt1 locus was up-regulated in fruiting body, but down-regulation of expression by means of RNAi decreased the level of bisected NLO; however had no apparent effect on fruiting body formation.

Recent studies suggest that a specific class of fungal lectins, commonly referred to as fruiting body lectins, play a role as effector molecules in the defense of fungi against predators and parasites. Hallmarks of these fungal lectins are their specific expression in reproductive structures, fruiting bodies, and/or sclerotia and their synthesis on free ribosomes in the cytoplasm. Fruiting body lectins are released upon damage of the fungal cell and bind to specific carbohydrate structures of predators and parasites, which leads to deterrence, inhibition of growth, and development or even killing of these organisms. Here, we describe assays to assess the toxicity of such lectins and other cytoplasmic proteins toward three different model organisms: the insect Aedes aegypti, the nematode Caenorhabditis elegans, and the amoeba Acanthamoeba castellanii. All three assays are based on heterologous expression of the examined proteins in the cytoplasm of Escherichia coli and feeding of these recombinant bacteria to omnivorous and bacterivorous organisms., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galbeta1,4Fucalpha1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galbeta1,4Fucalpha1,6GlcNAc trisaccharide at 1.5 A resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.

Galectin CGL2 from the ink cap mushroom Coprinopsis cinerea displays toxicity toward the model nematode Caenorhabditis elegans. A mutation in a putative glycosyltransferase-encoding gene resulted in a CGL2-resistant C. elegans strain characterized by N-glycans lacking the beta1,4-galactoside linked to the alpha1,6-linked core fucose. Expression of the corresponding GALT-1 protein in insect cells was used to demonstrate a manganese-dependent galactosyltransferase activity. In vitro, the GALT-1 enzyme showed strong selectivity for acceptors with alpha1,6-linked N-glycan core fucosides and required Golgi- dependent modifications on the oligosaccharide antennae for optimal synthesis of the Gal-beta1,4-fucose structure. Phylogenetic analysis of the GALT-1 protein sequence identified a novel glycosyltransferase family (GT92) with members widespread among eukarya but absent in mammals.

Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G(1) phase of the cell cycle. Ste5 scaffold protein is essential for pheromone response because it couples pheromone receptor stimulation to activation of the appropriate mitogen-activated protein kinase (MAPK) cascade. In naïve cells, Ste5 resides primarily in the nucleus. Upon pheromone treatment, Ste5 is rapidly exported from the nucleus and accumulates at the tip of the mating projection via its association with multiple plasma membrane-localized molecules. We found that concomitant with its nuclear export, the rate of Ste5 turnover is markedly reduced. Preventing nuclear export destabilized Ste5, whereas preventing nuclear entry stabilized Ste5, indicating that Ste5 degradation occurs mainly in the nucleus. This degradation is dependent on ubiquitin and the proteasome. We show that Ste5 ubiquitinylation is mediated by the SCF(Cdc4) ubiquitin ligase and requires phosphorylation by the G(1) cyclin-dependent protein kinase (cdk1). The inability to efficiently degrade Ste5 resulted in pathway activation and cell cycle arrest in the absence of pheromone. These findings reveal that maintenance of this MAPK scaffold at an appropriately low level depends on its compartment-specific and cell cycle-dependent degradation. Overall, this mechanism provides a novel means for helping to prevent inadvertent stimulus-independent activation of a response and for restricting and maximizing the signaling competence of the cell to a specific cell cycle stage, which likely works hand in hand with the demonstrated role that G(1) Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.

Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in beta-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of beta1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAc beta 1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities.

The ink cap Coprinopsis cinerea is a model organism for studying fruiting body (mushroom) formation in homobasidiomycetes. Mutant screens and expression studies have implicated a number of genes in this developmental process. Functional analysis of these genes, however, is hampered by the lack of reliable reverse genetics tools for C. cinerea. Here, we report the applicability of gene targeting by RNA silencing for this organism. Efficient silencing of both an introduced GFP expression cassette and the endogenous cgl1 and cgl2 isogenes was achieved by expression of homologous hairpin RNAs. In latter case, silencing was the result of a hairpin construct containing solely cgl2 sequences, demonstrating the possibility of simultaneous silencing of whole gene families by a single construct. Expression of the hairpin RNAs reduced the mRNA levels of the target genes by at least 90%, as determined by quantitative real-time PCR. The reduced mRNA levels were accompanied by cytosine methylation of transcribed and nontranscribed DNA at both silencing and target loci in the case of constitutive high-level expression of the hairpin RNA but not in the case of transient expression. These results suggest the presence of both posttranscriptional and transcriptional gene silencing mechanisms in C. cinerea and demonstrate the applicability of targeted gene silencing as a powerful reverse genetics approach in this organism.

Galectin-1 is a component of the extracellular matrix as well as a ligand of cell surface counter receptors such as beta-galactoside-containing glycolipids, however, the molecular mechanism of galectin-1 secretion has remained elusive. Based on a nonbiased screen for galectin-1 export mutants we have identified 26 single amino acid changes that cause a defect of both export and binding to counter receptors. When wild-type galectin-1 was analyzed in CHO clone 13 cells, a mutant cell line incapable of expressing functional galectin-1 counter receptors, secretion was blocked. Intriguingly, we also find that a distant relative of galectin-1, the fungal lectin CGL-2, is a substrate for nonclassical export from Chinese hamster ovary (CHO) cells. Alike mammalian galectin-1, a CGL-2 mutant defective in beta-galactoside binding, does not get exported from CHO cells. We conclude that the beta-galactoside binding site represents the primary targeting motif of galectins defining a galectin export machinery that makes use of beta-galactoside-containing surface molecules as export receptors for intracellular galectin-1.

The basidiomycete Coprinopsis cinerea (Coprinus cinereus) expresses two fruiting body-specific isolectins (CGL1 and CGL2) that belong to the family of galectins. Understanding the role of these beta-galactoside binding lectins is still in the beginning. Even though the prerequisites for substrate binding are well understood, it is not known how discrimination between potential substrates is achieved and what kind of influence this has on the function in a distinct cellular context. Precise knowledge of the expression of galectins and their ligands will aid in elucidating their function. In Coprinopsis, the developmentally regulated ligands for galectins co-localise with galectin expression in the veil surrounding the developing primordium and the outer cells of the young stipe. In addition, galectin ligands are observed in the hymenium. The subcellular localisation of the galectin ligands suggests these to be present in cellular compartments distinct from galectin transport. The sensitivity of the in situ interactions with exogenous galectin towards detergents and organic solvents infers that these ligands are lipid-borne. Accordingly, lipid fractions from primordia are shown to contain galectin-binding compounds. Based on these results and the determined binding specificity towards substituted beta-galactosides we hypothesise that beta-galactoside-containing lipids (basidiolipids) found in mushrooms are physiological ligands for the galectins in C. cinerea.

In the homobasidiomycete Coprinopsis cinerea, expression of the two fruiting body-specific galectins, CGL1 and CGL2, is controlled by nutrients, light and darkness and the A mating type genes. In this study, we analyzed the promoter of the cgl2 gene by measuring transcript levels by quantitative real-time PCR and show that regulation of CGL2 expression occurs at the transcriptional level. A minimal promoter sufficient to confer regulated expression of a heterologous reporter gene and comprising 627 base pairs from the start codon was defined. On the minimal promoter we identified a 120 bp sequence mediating induction of the cgl2 gene in constant darkness. Along with direct repeats (TGGAAG/TGGAAG/GGAA), the sequence contains a CRE consensus site (cAMP-responsive element, TGCGTCA) suggesting the involvement of cAMP signaling in cgl2 activation. No specific elements responsible for light repression and mating type regulation were found in the promoter.

Recognition of and discrimination between potential glyco-substrates is central to the function of galectins. Here we dissect the fundamental parameters responsible for such selectivity by the fungal representative, CGL2. The 2.1 A crystal structure of CGL2 and five substrate complexes reveal that this prototype galectin achieves increased substrate specificity by accommodating substituted oligosaccharides of the mammalian blood group A/B type in an extended binding cleft. Kinetic studies on wild-type and mutant CGL2 proteins demonstrate that the tetrameric organization is essential for functionality. The geometric constraints due to the orthogonal orientation of the four binding sites have important consequences on substrate binding and selectivity.

The Ran GTPase plays a key role in nucleocytoplasmic transport. In its GTP-bound form, it directly interacts with members of the importin beta family of nuclear transport receptors and modulates their association with cargo. Work in cell-free higher-eukaryote systems has demonstrated additional roles for Ran in spindle and nuclear envelope formation during mitosis. However, until recently, no Ran-target proteins in these cellular processes were known. Several groups have now identified importin beta as one important target of Ran during mitotic spindle formation. This finding suggests that Ran uses the same effectors to regulate different cellular processes.

We identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (alpha-importin) and several beta-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Delta and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.

The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex. Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm. Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD). By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP. Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1. Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo.

Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-beta-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast.

CSE1 is essential for yeast cell viability and has been implicated in chromosome segregation. Based on its sequence similarity, Cse1p has been grouped into the family of importin beta-like nucleocytoplasmic transport receptors with highest homology to the recently identified human nuclear export receptor for importin alpha, CAS. We demonstrate here that Cse1p physically interacts with yeast Ran and yeast importin alpha (Srp1p) in the yeast two-hybrid system and that recombinant Cse1p, Srp1p and Ran-GTP form a trimeric complex in vitro. Re-export of Srp1p from the nucleus into the cytoplasm and nuclear uptake of a reporter protein containing a classical NLS are inhibited in a cse1 mutant strain. These findings suggest that Cse1p is the exportin of importin alpha in yeast.

Although serine and glycine are ubiquitous amino acids the genetic and biochemical regulation of their synthesis has not been studied in detail. The SER1 gene encodes 3-phosphoserine aminotransferase which catalyzes the formation of phosphoserine from 3-phosphohydroxy-pyruvate, which is obtained by oxidation of 3-phosphoglycerate, an intermediate of glycolysis. Saccharomyces cerevisiae cells provided with fermentable carbon sources mainly use this pathway (glycolytic pathway) to synthesize serine and glycine. We report the isolation of the SER1 gene by complementation and the disruption of the chromosomal locus. Sequence analysis revealed an open reading frame encoding a protein with a predicted molecular weight of 43,401 Da. A previously described mammalian progesterone-induced protein shares 47% similarity with SER1 over the entire protein, indicating a common function for both proteins. We demonstrate that SER1 transcription is regulated by the general control of amino-acid biosynthesis mediated by GCN4. Additionally, DNaseI protection experiments proved the binding of GCN4 protein to the SER1 promoter in vitro and three GCN4 recognition elements (GCREs) were identified. Furthermore, there is evidence for an additional regulation by serine end product repression.

The ARO3 gene of Saccharomyces cerevisiae codes for the phenylalanine-inhibited 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 4.1.2.15) and is regulated by the general control system of amino acid biosynthesis through a single GCN4-binding site in its promoter. A combined deletion and mutation analysis of the ARO3 promoter region in a delta gcn4-background revealed two additional regulatory systems involved in ARO3 transcription. The ARO3 gene is (i) activated through a sequence element which binds the multifunctional DNA-binding protein ABF1 in vitro and (ii) repressed through an URS1 element, which binds the same protein in vitro as the URS1 element in the CAR1 promoter. Since both the ABF1-binding site and the URS1 element represent cis-acting elements of global transcription regulatory systems in yeast, the ARO3 gene is the first example of a GCN4-regulated gene which is both activated and repressed by global transcription factors. Activation of the ARO3 gene through the ABF1-binding site and repression through the URS1 element seem to be independent of each other and independent of activation by the GCN4 protein.

In Saccharomyces cerevisiae, two differently regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (DAHPS; EC 4.1.2.15) isoenzymes carry out the first step in the shikimate pathway. Mutations in both genes are necessary to cause aromatic amino acid (aa) auxotrophy, since one isoenzyme alone is sufficient to produce enough DAHP for normal growth of the cells. The phenylalanine-inhibited DAHPS is encoded by the previously isolated and characterized ARO3 gene. Here, we report the cloning and characterization of the ARO4 gene, encoding the second DAHPS, which is inhibited by tyrosine. The aa sequence of the ARO4 gene product reveals 76% similarity to the ARO3-encoded isoenzyme and 66 and 73% to the three DAHPS isoenzymes from Escherichia coli. ARO4 gene expression is regulated by the general control system of aa biosynthesis. As in the case of the ARO3 gene, a single GCN4-recognition element in the promoter is responsible for derepression of the ARO4 gene under aa starvation conditions. However, in contrast to the situation in the isogene, ARO3, GCN4 does not contribute to the basal level of ARO4 transcription under nonderepressing conditions.