Secondary literature sources for SMI1_KNR4
The following references were automatically generated.
- Kimura T et al.
- A novel yeast gene, RHK1, is involved in the synthesis of the cell wallreceptor for the HM-1 killer toxin that inhibits beta-1,3-glucansynthesis.
- Mol Gen Genet. 1997; 254: 139-47
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The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wallbeta-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitivestrains of yeast. A number of mutants of Saccharomyces cerevisiae thatshow resistance to this toxin were isolated in order to clarify thekilling mechanism of the toxin. These mutants, designated rhk (resistantto Hansenula killer), were classified into three complementation groups. Anovel gene RHK1, which complements the killer-resistant phenotype of thelargest complementation group rhk1, was isolated. DNA sequence analysisrevealed an open reading frame that encodes a hydrophobic protein composedof 458 amino acids. Gene disruption followed by tetrad analysis showedthat RHK1 is not essential and loss of RHK1 function endowed S. cerevisiaecells with complete killer resistance. A biochemical analysis suggestedthat RHK1 does not participate directly in the synthesis ofbeta-1,3-glucan but is involved in the synthesis of the receptor for theHM-1 killer toxin.
- Mio T et al.
- Cloning of the Candida albicans homolog of Saccharomyces cerevisiaeGSC1/FKS1 and its involvement in beta-1,3-glucan synthesis.
- J Bacteriol. 1997; 179: 4096-105
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Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also calledFKS2) have been identified as the genes for putative catalytic subunits ofbeta-1,3-glucan synthase. We have cloned three Candida albicans genes,GSC1, GSL1, and GSL2, that have significant sequence homologies with S.cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA ofAspergillus nidulans at both nucleotide and amino acid levels. Like S.cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicansGSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminalends, but each product possessed 10 to 16 potential transmembrane heliceswith a relatively long cytoplasmic domain in the middle of the protein.Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2mRNAs were expressed in the growing yeast-phase cells. Three copies ofGSC1 were found in the diploid genome of C. albicans CAI4. Although wecould not establish the null mutation of C. albicans GSC1, disruption oftwo of the three GSC1 alleles decreased both GSC1 mRNA and cell wallbeta-glucan levels by about 50%. The purified C. albicans beta-1,3-glucansynthase was a 210-kDa protein as judged by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and all sequences determinedwith peptides obtained by lysyl endopeptidase digestion of the 210-kDaprotein were found in the deduced amino acid sequence of C. albicansGsc1p. Furthermore, the monoclonal antibody raised against the purifiedbeta-1,3-glucan synthase specifically reacted with the 210-kDa protein andcould immunoprecipitate beta-1,3-glucan synthase activity. These resultsdemonstrate that C. albicans GSC1 is the gene for a subunit ofbeta-1,3-glucan synthase.
- Kondoh O, Tachibana Y, Ohya Y, Arisawa M, Watanabe T
- Cloning of the RHO1 gene from Candida albicans and its regulation ofbeta-1,3-glucan synthesis.
- J Bacteriol. 1997; 179: 7734-41
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The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weightGTPase. One of its recently identified functions is the regulation ofbeta-1,3-glucan synthase, which synthesizes the main component of thefungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazurand W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota etal., Science 272:279-281, 1996). From the opportunistic pathogenic fungusCandida albicans, we cloned the RHO1 gene by the PCR andcross-hybridization methods. Sequence analysis revealed that the CandidaRHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDapeptide. The deduced amino acid sequence predicts that Candida albicansRho1p is 82.9% identical to Saccharomyces Rho1p and contains all thedomains conserved among Rho-type GTPases from other organisms. The Candidaalbicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1deletion. Furthermore, recombinant Candida albicans Rho1p could reactivatethe beta-1,3-glucan synthesis activities of both C. albicans and S.cerevisiae membranes in which endogenous Rho1p had been depleted byTergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified withthe beta-1,3-glucan synthase putative catalytic subunit, Candida albicansGsc1p, by product entrapment. Candida albicans Rho1p was shown to interactdirectly with Candida albicans Gsc1p in a ligand overlay assay and across-linking study. These results indicate that Candida albicans Rho1pacts in the same manner as Saccharomyces cerevisiae Rho1p to regulatebeta-1,3-glucan synthesis.
- Tentler S et al.
- Inhibition of Neurospora crassa growth by a glucan synthase-1 antisenseconstruct.
- Curr Microbiol. 1997; 34: 303-8
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We have used the filamentous fungus, Neurospora crassa, as a model systemto test the concept that antisense targeting of the cell-wall assemblyenzyme, (1,3)beta-glucan synthase [E.C. 18.104.22.168; UDPglucose:1,3-beta-D-glucan 3-beta-D-glucosyltransferase], leads to a correspondingdecrease in growth of the organism. Previously, our laboratory isolated agene (glucan synthase-1, gs-1) that is required for (1,3)beta-glucansynthase activity. Wild-type cells were transformed with DNA vectorsencoding various RNAs complementary to the gs-1 messenger RNA (antisenseRNA) cloned downstream from an inducible promoter (quinic acid-2[qa-2p]).Stable transformants, expressing a partially inverted antisense message ofgs-1 (pMYX107), exhibited dramatic reduction ingrowth compared with emptyvector controls. Hyphal measurements of these transformants grown on racetubes indicated that all of the transformants showed various degrees ofinhibition. Microscopic observations of transformants revealed shorterhyphal lengths when grown under conditions expressing antisense. Furthercharacterization revealed that the specific activities of (1,3)beta-glucansynthase were decreased by as much as 63% relative to empty vectorcontrols. Together, these observations suggest that antisense against(1,3)beta-glucan synthase led to a reduction in enzyme levels thatresulted in altered cell-wall morphology and inhibition of growth. It ispossible that antisense oligonucleotides against gs-1 may be usefulantifungal agents.
- Ishiguro J, Saitou A, Duran A, Ribas JC
- cps1+, a Schizosaccharomyces pombe gene homolog of Saccharomycescerevisiae FKS genes whose mutation confers hypersensitivity tocyclosporin A and papulacandin B.
- J Bacteriol. 1997; 179: 7653-62
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The Schizosaccharomyces pombe cps1-12 (for chlorpropham supersensitive)mutant strain was originally isolated as hypersensitive to the spindlepoison isopropyl N-3-chlorophenyl carbamate (chlorpropham) (J. Ishiguroand Y. Uhara, Jpn. J. Genet. 67:97-109, 1992). We have found that thecps1-12 mutation also confers (i) hypersensitivity to theimmunosuppressant cyclosporin A (CsA), (ii) hypersensitivity to the drugpapulacandin B, which specifically inhibits 1,3-beta-D-glucan synthesisboth in vivo and in vitro, and (iii) thermosensitive growth at 37 degreesC. Under any of these restrictive treatments, cells swell up and finallylyse. With an osmotic stabilizer, cells do not lyse, but at 37 degrees Cthey become multiseptated and multibranched. The cps1-12 mutant, grown ata restrictive temperature, showed an increase in sensitivity to lysis byenzymatic cell wall degradation, in in vitro 1,3-beta-D-glucan synthaseactivity (173% in the absence of GTP in the reaction), and in cell wallbiosynthesis (130% of the wild-type amount). Addition of Ca2+ suppresseshypersensitivity to papulacandin B and septation and branching phenotypes.All of these data suggest a relationship between the cps1+ gene and cellwall synthesis. A DNA fragment containing the cps1+ gene was cloned, andsequence analysis indicated that it encodes a predicted membrane proteinof 1,729 amino acids with 15 to 16 transmembrane domains. S. pombe cps1phas overall 55% sequence identity with Fks1p or Fks2p, proposed to becatalytic or associated subunits of Saccharomyces cerevisiae1,3-beta-D-glucan synthase. Thus, the cps1+ product might be a catalyticor an associated copurifying subunit of the fission yeast1,3-beta-D-glucan synthase that plays an essential role in cell wallsynthesis.
- Lai MH, Silverman SJ, Gaughran JP, Kirsch DR
- Multiple copies of PBS2, MHP1 or LRE1 produce glucanase resistance andother cell wall effects in Saccharomyces cerevisiae.
- Yeast. 1997; 13: 199-213
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Five sequences were isolated by selection for multiple copy plasmids thatconferred resistance to laminarinase, an enzyme that specifically degradescell wall beta(1-3) glucan linkages. Strains carrying three of theseplasmids showed alterations in cell wall glucan labelling. One of theseplasmids carried PBS2, a previously identified, non-essential gene whichproduces a variety of phenotypes and encodes a mitogen-activated proteinkinase kinase analogue (Boguslawski and Polazzi, 1987). Cells carryingPBS2 at multiple copy show a small decrease in cell wall beta(1-6)glucans. Measurements of beta(1-3) glucan synthase activity in multi-copyPBS2 cells showed an approximate 30-45% increase in enzyme specificactivity while a pbs2 delta disruption strain showed a decrease in glucansynthase activity of approximately 45% relative to control. A pbs2 deltadisruption strain was laminarinase super-sensitive and supersensitive toK1 killer toxin while a strain carrying PBS2 at multiple copy wasresistant to killer toxin. A second plasmid carried a portion of the MHP1gene which has been reported to encode a microtubule-interacting protein(Irminger-Finger et al., 1996). The MHP1 gene product is a predicted 1398amino acid protein and only approximately 80% of the amino portion of thisprotein is required for laminarinase resistance. Cells carrying the aminoportion of MHP1 at multiple copy show a decrease in high molecular weightcell wall beta(1-6) glucans and were killer toxin resistant while adisruption strain was viable and killer toxin super-sensitive. Cellscarrying this plasmid showed decreased levels of high molecular weightbeta(1-6) glucans and increased glucan synthase activity. The laminarinaseresistance conferred by the third plasmid mapped to the previouslyuncharacterized YCL051W open reading frame and this gene was thereforenamed LRE1 (laminarinase resistance). The LRE1 gene encodes anon-essential 604 amino acid hydrophilic protein. Unexpectedly, cellscarrying LRE1 at multiple copy show no alteration in cell wall glucans orglucan synthase activity. Subcloning experiments demonstrated that theproduction of these cell wall effects requires the presence of both LRE1and YCL052C (PBN1), a second open reading frame present on the originalplasmid. Cells carrying multiple copies of PBN1 alone show no significantalterations in cell wall glucans or glucan synthase activity, indicatingthat these effects require the presence of multiple copies of both genes.
- Kelly R, Register E, Hsu MJ, Kurtz M, Nielsen J
- Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillusnidulans and purification of the corresponding protein.
- J Bacteriol. 1996; 178: 4381-91
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Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2,which encode interchangeable putative catalytic subunits of1,3-beta-glucan synthase (GS), an enzyme that synthesizes an essentialpolymer of the fungal cell wall. To determine if GS in Aspergillus speciesis similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans bycross-hybridization, and the corresponding protein was purified. Sequenceanalysis revealed a 5,716-nucleotide coding region interrupted by two56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa,FksAp, that shows a remarkable degree of conservation in size, charge,amino acid identity, and predicted membrane topology with the S.cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity toFks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis ofFksAp suggests an integral membrane protein with 16 transmembrane helicesthat coincide with the transmembrane helices of the Saccharomyces Fksps.The sizes of the nontransmembrane domains are strikingly similar to thoseof Fks1p. The region of FksAp most homologous to the Saccharomyces FKSpolypeptides is a large hydrophilic domain of 578 amino acids that ispredicted to be cytoplasmic. This domain is 86% identical to thecorresponding region of Fks1p and is a good candidate for the location ofthe catalytic site. Antibodies raised against a peptide derived from theFksAp sequence recognize a protein of approximately 200 kDa in crudemembranes and detergent-solubilized active extracts. This protein isenriched approximately 300-fold in GS purified by product entrapment.Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GSactivity in crude or purified extracts when Staphylococcus aureus cellsare used to precipitate the antibodies, although it does not inhibitenzymatic activity when added to extracts. The purified GS is inhibited byechinocandins with a sensitivity equal to that displayed by whole cells.Thus, the product of fksA is important for the activity of highly purifiedpreparations of GS, either as the catalytic subunit itself or as anassociated copurifying subunit that mediates susceptibility of enzymaticactivity to echinocandin inhibition.
- Castro C, Ribas JC, Valdivieso MH, Varona R, del Rey F, Duran A
- Papulacandin B resistance in budding and fission yeasts: isolation andcharacterization of a gene involved in (1,3)beta-D-glucan synthesis inSaccharomyces cerevisiae.
- J Bacteriol. 1995; 177: 5732-9
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Papulacandin B, an antifungal agent that interferes with the synthesis ofyeast cell wall (1,3)beta-D-glucan, was used to isolate resistant mutantsin Schizosaccharomyces pombe and Saccharomyces cerevisiae. The resistanceto papulacandin B always segregated as a recessive character that definesa single complementation group in both yeasts (pbr1+ and PBR1,respectively). Determination of several kinetic parameters of(1,3)beta-D-glucan synthase activity revealed no differences between S.pombe wild-type and pbr1 mutant strains except in the 50% inhibitoryconcentration for papulacandin B of the synthases (about a 50-foldincrease in mutant activity). Inactivation of the synthase activity ofboth yeasts after in vivo treatment with the antifungal agent showed thatmutant synthases were more resistant than the corresponding wild-typeones. Detergent dissociation of the S. pombe synthase into soluble andparticulate fractions and subsequent reconstitution indicated that theresistance character of pbr1 mutants resides in the particulate fractionof the enzyme. Cloning and sequencing of PBR1 from S. cerevisiae revealeda gene identical to others recently reported (FKS1, ETG1, CWH53, andCND1). Its disruption leads to reduced levels of both (1,3)beta-D-glucansynthase activity and the alkali-insoluble cell wall fraction.Transformants containing the PBR1 gene reverse the defect in(1,3)beta-D-glucan synthase. It is concluded that Pbr1p is probably partof the (1,3)beta-D-glucan synthase complex.
- Roemer T, Paravicini G, Payton MA, Bussey H
- Characterization of the yeast (1-->6)-beta-glucan biosynthetic components,Kre6p and Skn1p, and genetic interactions between the PKC1 pathway andextracellular matrix assembly.
- J Cell Biol. 1994; 127: 567-79
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A characterization of the S. cerevisiae KRE6 and SKN1 gene productsextends previous genetic studies on their role in (1-->6)-beta-glucanbiosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucansynthesis: KRE6 encodes a predicted type II membrane protein required forglucan synthesis in vivo and for glucan synthase activity in vitro. Proc.Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H.Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encodingputative membrane proteins involved in beta-glucan synthesis. Mol. Cell.Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologousproteins that participate in assembly of the cell wall polymer(1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membraneglycoproteins, with Kre6p likely localized within a Golgi subcompartment.Deletion of both these genes is shown to result in a dramaticdisorganization of cell wall ultrastructure. Consistent with their directrole in the assembly of this polymer, both Kre6p and Skn1p possessCOOH-terminal domains with significant sequence similarity to two recentlyidentified glucan-binding proteins. Deletion of the yeast protein kinase Chomolog, PKC1, leads to a lysis defect (Levin, D. E., and E.Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display acell cycle-specific osmotic stability defect. J. Cell Biol.116:1221-1229). Kre6p when even mildly overproduced, can suppress thispkc1 lysis defect. When mutated, several KRE pathway genes and members ofthe PKC1-mediated MAP kinase pathway have synthetic lethal interactions asdouble mutants. These suppression and synthetic lethal interactions, aswell as reduced beta-glucan and mannan levels in the pkc1 null wall,support a role for the PKC1 pathway functioning in cell wall assembly.PKC1 potentially participates in cell wall assembly by regulating thesynthesis of cell wall components, including (1-->6)-beta-glucan.
- Kasahara S et al.
- Cloning of the Saccharomyces cerevisiae gene whose overexpressionovercomes the effects of HM-1 killer toxin, which inhibits beta-glucansynthesis.
- J Bacteriol. 1994; 176: 1488-99
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A gene whose overexpression can endow Saccharomyces cerevisiae cells withresistance to HM-1 killer toxin was cloned from an S. cerevisiae genomiclibrary. This gene, designated HKR1 (Hansenula mrakii killertoxin-resistant gene 1), contains a 5.4-kb open reading frame. Thepredicted amino acid sequence of the protein specified by HKR1 indicatesthat the protein consists of 1,802 amino acids and is very rich in serineand threonine, which could serve as O-glycosylation sites. The proteinalso contains two hydrophobic domains at the N-terminal end and in theC-terminal half, which could function as a signal peptide andtransmembrane domain, respectively. Hkr1p is found to contain an EF handmotif of the calcium-binding consensus sequence in the C-terminalcytoplasmic domain. Thus, Hkr1p is expected to be a calcium-binding,glycosylated type I membrane protein. Southern and Northern (RNA) analysesdemonstrated that there is a single copy of the HKR1 gene in the S.cerevisiae genome, and the transcriptional level of HKR1 is extremely low.Gene disruption followed by tetrad analysis showed that HKR1 is anessential gene. Overexpression of the truncated HKR1 encoding theC-terminal half of Hkr1p made the cells more resistant to HM-1 killertoxin than the full-length HKR1 did, demonstrating that the C-terminalhalf of Hkr1p is essential for overcoming the effect of HM-1 killer toxin.Furthermore, overexpression of HKR1 increased the beta-glucan content inthe cell wall without affecting in vitro beta-glucan synthase activity,suggesting that HKR1 regulates beta-glucan synthesis in vivo.
- Diaz M et al.
- The Schizosaccharomyces pombe cwg2+ gene codes for the beta subunit of ageranylgeranyltransferase type I required for beta-glucan synthesis.
- EMBO J. 1993; 12: 5245-54
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The product of the Schizosaccharomyces pombe cwg2+ gene is involved in thebiosynthesis of beta-D-glucan. When grown at the non-permissivetemperature, cwg2-1 mutant cells lyse in the absence of an osmoticstabilizer and display a reduced (1-3) beta-D-glucan content and (1-3)beta-D-glucan synthase activity. The cwg2+ gene was cloned by the rescueof the cwg2-1 mutant phenotype using an S. pombe genomic library andsubsequently verified by integration of the appropriate insert into the S.pombe genome. Determination of the nucleotide sequence of this generevealed a putative open reading frame of 1065 bp encoding a polypeptideof 355 amino acids with a calculated M(r) of 40,019. The cwg2+ DNAhybridizes to a main transcript, the 5' end of which maps to a position469 bp upstream of the predicted start of translation. The sequencebetween the transcription and the translation start sites is unusuallylong and has several short open reading frames which suggest atranslational control of the gene expression. Comparative analysis of thepredicted amino acid sequence shows that it possesses significantsimilarity to three Saccharomyces cerevisiae proteins, encoded by theDPR1/RAM1, CDC43/CAL1 and ORF2/BET2 genes respectively, which are betasubunits of different prenyltransferases. When grown at 37 degrees C,cwg2-1 mutant extracts were specifically deficient ingeranylgeranyltransferase type I activity, as measured in vitro. Multiplecopies of the CDC43 gene can partially suppress the growth and (1-3)beta-D-glucan synthase defect of the cwg2-1 mutant at the restrictivetemperature. In a similar manner, the cwg2+ gene can partially suppressthe cdc43-2 growth defect. These results indicate that cwg2+ is thestructural gene for the beta subunit of geranylgeranyltransferase type Iin S. pombe and that this enzyme is required for (1-3) beta-D-glucansynthase activity. The functional homology of Cwg2 with Cdc43, which hasbeen implicated in the control of cell polarity, suggests a link betweentwo morphogenetic events such as establishment of cell polarity and cellwall biosynthesis.
- Brown JL, Kossaczka Z, Jiang B, Bussey H
- A mutational analysis of killer toxin resistance in Saccharomycescerevisiae identifies new genes involved in cell wall (1-->6)-beta-glucansynthesis.
- Genetics. 1993; 133: 837-49
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Recessive mutations leading to killer resistance identify the KRE9, KRE10and KRE11 genes. Mutations in both the KRE9 and KRE11 genes lead toreduced levels of (1-->6)-beta-glucan in the yeast cell wall. The KRE11gene encodes a putative 63-kD cytoplasmic protein, and disruption of theKRE11 locus leads to a 50% reduced level of cell wall (1-->6)-glucan.Structural analysis of the (1-->6)-beta-glucan remaining in a kre11 mutantindicates a polymer smaller in size than wild type, but containing asimilar proportion of (1-->6)- and (1-->3)-linkages. Genetic interactionsamong cells harboring mutations at the KRE11, KRE6 and KRE1 loci indicatelethality of kre11 kre6 double mutants and that kre11 is epistatic tokre1, with both gene products required to produce the mature glucanpolymer at wild-type levels. Analysis of these KRE genes should extendknowledge of the beta-glucan biosynthetic pathway, and of cell wallsynthesis in yeast.
- Roemer T, Bussey H
- Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membraneprotein required for glucan synthesis in vivo and for glucan synthaseactivity in vitro.
- Proc Natl Acad Sci U S A. 1991; 88: 11295-9
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The KRE6 gene product is required for synthesis of the major beta-glucansof the yeast cell wall, as mutations in this gene confer reduced levels ofboth the (1----6)- and (1----3)-beta-D-glucan polymers. Cloning andsequencing of KRE6 reveals a gene encoding a predicted 80-kDa protein witha central transmembrane domain and the topology of a type II membraneprotein. Null mutants of KRE6 grow slowly, have larger cells, and show areduction in alkali-insoluble wall glucans. The mutants show goodviability and are not osmotically sensitive, but they are more susceptibleto beta-glucanase digestion and mechanical stress than wild-type cells.The specific activity of the GTP-dependent, membrane-associated, in vitro(1----3)-beta-glucan synthase is reduced 50% in kre6 null mutants, andthis reduction correlates with the mutation in meiotic tetrads.Transformants of kre6 null mutants with a KRE6 gene expressed from acentomere-based vector show a 4- to 5-fold increase in in vitro(1----3)-beta-glucan synthase activity over transformants with the vectoralone. The phenotype and structure of the KRE6 product, Kre6p, suggestthat Kre6p may be a beta-glucan synthase, and if so, it implies thatbeta-glucan synthases are functionally redundant in yeast. Alternatively,Kre6p may be part of a single multiprotein glucan synthase or modulate itsactivity. Use of KRE6 should permit a genetic analysis of eukaryotic(1----3)-beta-glucan synthesis.