RESUMO
MCCs are linear invaginations of the yeast plasma membrane that form stable membrane microdomains. Although over 20 proteins are localized in the MCCs, it is not well understood how these proteins coordinately maintain normal MCC function. Pil1 is a core eisosome protein and is responsible for MCC-invaginated structures. In addition, six-tetraspan membrane proteins (6-Tsp) are localized in the MCCs and classified into two families, the Sur7 family and Nce102 family. To understand the coordinated function of these MCC proteins, single and multiple deletion mutants of Pil1 and 6-Tsp were generated and their MCC structure and growth under various stresses were investigated. Genetic interaction analysis revealed that the Sur7 family and Nce102 function in stress tolerance and normal eisosome assembly, respectively, by cooperating with Pil1. To further understand the role of MCCs/eisosomes in stress tolerance, we screened for suppressor mutants using the SDS-sensitive phenotype of pil1Δ 6-tspΔ cells. This revealed that SDS sensitivity is caused by hyperactivation of Tor kinase complex 2 (TORC2)-Ypk1 signaling. Interestingly, inhibition of sphingolipid metabolism, a well-known downstream pathway of TORC2-Ypk1 signaling, did not rescue the SDS-sensitivity of pil1Δ 6-tspΔ cells. These results suggest that Pil1 and 6-Tsp cooperatively regulate TORC2 signaling during the stress response.
Assuntos
Proteínas de Saccharomyces cerevisiae , Membrana Celular/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
RipAY, an effector protein from the plant bacterial pathogen Ralstonia solanacearum, exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the host cellular glutathione (GSH) when stimulated by host eukaryotic-type thioredoxins (Trxs). Aave_4606 from Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbit plants, shows significant homology to RipAY. Based on its homology, it was predicted that the GGCT activity of Aave_4606 is also stimulated by host Trxs. The GGCT activity of a recombinant Aave_4606 protein was investigated in the presence of various Trxs, such as yeast (ScTrx1), Arabidopsis thaliana (AtTrx-h1, AtTrx-h2, AtTrx-h3, and AtTrx-h5), or watermelon (Cla022460/ClTrx). Unlike RipAY, the GGCT activity of Aave_4606 is stimulated only by AtTrx-h1, AtTrx-h3, AtTrx-h5 and ClTrx from a watermelon, the primary host of A. citrulli, but not by ScTrx1, AtTrx-h2. Interestingly, GGCT activity of Aave_4606 is more efficiently stimulated by AtTrx-h1 and ClTrx than AtTrx-h5. These results suggested that Aave_4606 recognizes host-specific Trxs, which specifically activates the GGCT activity of Aave_4606 to decrease the host cellular GSH. These findings provide new insights into that effector is one of the host-range determinants for pathogenic bacteria via its host-dependent activation.
Assuntos
Arabidopsis , Comamonadaceae , Ralstonia solanacearum , Arabidopsis/metabolismo , Comamonadaceae/metabolismo , Frutas/metabolismo , Glutationa/metabolismo , Plantas/metabolismo , Ralstonia solanacearum/fisiologia , Tiorredoxinas/metabolismoRESUMO
In the fission yeast Schizosaccharomyces pombe, α1,2- and α1,3-linked D-galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases. Although the galactomannans are important for cell-cell communication in S. pombe (e.g., in nonsexual aggregation), the mechanisms underlying galactosylation in cells remain unclear. Schizosaccharomyces pombe has 10 galactosyltransferase-related genes: seven belonging to glycosyltransferase (GT) family 34 and three belonging GT family 8. Disruption of all 10 α-galactosyltransferases (strain Δ10GalT) has been shown to result in a complete lack of α-Gal residues. Here, we have investigated the function and substrate specificities of galactosyltransferases in S pombe by using strains expressing single α-galactosyltransferases in the Δ10GalT background. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides showed that two GT family 34 α1,2-galactosyltransferases (Gma12p and Gmh6p) and two GT family 8 α1,3-galactosyltransferases (Otg2p and Otg3p) are involved in galactosylation of O-linked oligosaccharide. Moreover, 1H-NMR of N-glycans revealed that three GT family 34 α1,2-galactosyltransferases (Gmh1p, Gmh2p and Gmh3p) are required for the galactosylation of N-linked oligosaccharides. Furthermore, HPLC and lectin-blot analysis revealed that Otg1p showed α1,3-galactosyltransferase activity under conditions of co-expression with Gmh6p, indicating that α-1,2-linked galactose is required for the galactosylation activity of Otg1p in S. pombe. In conclusion, eight galactosyltransferases have been shown to have activity in S. pombe with different substrate specificities. These findings will be useful for genetically tailoring the galactosylation of both N- and O-glycans in fission yeast.
Assuntos
Schizosaccharomyces , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Oligossacarídeos/química , Polissacarídeos/química , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe , Especificidade por SubstratoRESUMO
A class II ChaC protein, RipAY, from phytopathogenic bacterium, Ralstonia solanacearum exhibits γ-glutamylcyclotransferase (GGCT) activity to degrade intracellular glutathione in host cells upon its interaction with host thioredoxins (Trxs). To understand the Trx-dependent activation of RipAY, we constructed various deletion mutants of RipAY and found the determinant region for GGCT activation in the N- and C-terminal sequences of RipAY by analyzing their yeast growth inhibition activity and the interaction with Trxs. Mutational analysis of the active site cysteine residues of Arabidopsis thaliana Trx-h5 (AtTrx-h5), one of the most efficiently stimulating Trxs, revealed that each active site cysteine residue of AtTrx-h5 contributes to efficient RipAY-binding and -activation activity. We also estimated that RipAY and AtTrx-h5 form a complex at a 1:2 M ratio. Furthermore, we found that the constitutive GGCT activity of Gcg1, a yeast class I ChaC protein, is also stimulated by yeast Trx1. These results indicate that class I ChaC proteins can sense the intracellular redox state and interact with Trxs to promote more efficient degradation of glutathione and regulate intracellular redox homeostasis. We hypothesize that RipAY acquired a more efficient and specific Trx-dependent activation mechanism to activate its GGCT activity only in the host eukaryotic cells during the evolution.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Ralstonia solanacearum/fisiologia , Tiorredoxinas/metabolismo , gama-Glutamilciclotransferase/metabolismo , Arabidopsis/metabolismo , Ativação Enzimática , Glutationa/metabolismo , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Ralstonia solanacearum/enzimologiaRESUMO
NAD kinase is a crucial enzyme for production of NADP+. Myxococcus xanthus is a gram-negative soil bacterium that forms fruiting bodies and spores under starvation, and it accumulates polyphosphate (poly(P)) during early development. We found that M. xanthus NAD kinase (PanK) utilized both ATP and poly(P) as phosphoryl donors; therefore, PanK was designated as a poly(P)/ATP-NAD kinase. Unlike other poly(P)/ATP-NAD kinases, PanK hardly exhibited NADH kinase activity. The NAD kinase activity of PanK was inhibited by NADPH, but not NADH. Replacement of Thr-90 in the GGDGT motif of PanK with Asn decreased both ATP- and poly(P)-dependent NAD kinase activities; however, poly(P)-dependent NAD kinase activity was further decreased by approximately 6- to 10-fold compared with ATP-dependent NAD kinase activity, suggesting that Thr-90 in the GGDGT motif of PanK may be important for poly(P) utilization. PanK preferred ATP and short-chain poly(P) as phosphoryl donors. The Km of PanK for ATP, poly(P)4, and poly(P)10-15 was 0.66 mM, 0.08 mM, and 0.71 mM, respectively, and the catalytic efficiency (kcat/Km) for poly(P)4 was 2.4-fold higher than that for ATP, suggesting that M. xanthus under starvation conditions may be able to efficiently generate NADP+ using PanK, ATP, and poly(P).
Assuntos
Proteínas de Bactérias/metabolismo , Myxococcus xanthus/enzimologia , NADP/metabolismo , Fosfotransferases/metabolismo , Proteínas de Bactérias/genética , Cinética , Myxococcus xanthus/genética , Fosfotransferases/genéticaRESUMO
The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to â¼30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other γ-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ralstonia solanacearum/genética , Sistemas de Secreção Tipo III/genética , Fatores de Virulência/genética , gama-Glutamilciclotransferase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Redes Reguladoras de Genes , Glutationa/metabolismo , Interações Hospedeiro-Patógeno , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oxirredução , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Filogenia , Plantas/microbiologia , Estrutura Terciária de Proteína , Ralstonia solanacearum/classificação , Ralstonia solanacearum/enzimologia , Ralstonia solanacearum/patogenicidade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo , gama-Glutamilciclotransferase/química , gama-Glutamilciclotransferase/metabolismoRESUMO
Schizosaccharomyces pombe has eight hexose transporter genes, ght1 (+) to ght8 (+). Here we report that ght2 (+), which is highly expressed in the presence of glucose, is essential for UDP-galactose synthesis from extracellular galactose when cells grow on glucose. The galactosylation defect of a uge1Δ mutant defective in synthesis of UDP-galactose from glucose was suppressed in galactose-containing medium, but disruption of ght2 (+) in the uge1Δ mutant reversed suppression of the galactosylation defect. Expression of Saccharomyces cerevisiae GAL2 in uge1Δght2Δ cells suppressed the defective galactosylation phenotype in galactose-containing medium. These results indicate that galactose is transported from the medium to the cytosol in a Ght2-dependent manner, and is then converted into UDP-galactose.
Assuntos
Galactose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Schizosaccharomyces/metabolismo , Uridina Difosfato Galactose/biossíntese , Meios de Cultura/química , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimentoRESUMO
Although various mutant strains of the fission yeast Schizosaccharomyces pombe exhibit non-sexual flocculation, little is known about the mechanistic basis for this phenomenon, nor have genes encoding the implicated flocculin been identified. In the budding yeast Saccharomyces cerevisiae, the transcription factor Flo8 controls expression of some of the genes involved in non-sexual flocculation. We have found that overexpression of S. cerevisiae FLO8 induced non-sexual flocculation in S. pombe. This non-sexual flocculation was Ca(2+) -dependent, and was inhibited by addition of galactose, but not by mannose, glucose or sucrose. In the FLO8-overexpressing strain, a gene designated gsf2(+) (galactose-specific flocculation) was specifically induced. The gsf2(+) gene was also highly expressed in lkh1Δ, tup12Δ and gsf1 mutants, all of which exhibited non-sexual flocculation dependent on gsf2(+) . We show that the N-terminal region of Gsf2 recognizes galactose in mediating cell-cell interaction. Disruption of gsf2(+) also abolished the adhesion phenotype and invasive growth of the wild-type strain cultured in low ammonium medium. The newly identified flocculin Gsf2 in fission yeast was not only required for non-sexual flocculation but was also required for adhesion and filamentous growth through recognition of galactose residues on cell surface glycoconjugates.
Assuntos
Galactose/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/química , Schizosaccharomyces/crescimento & desenvolvimento , Motivos de Aminoácidos , Sequência de Bases , Floculação , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
In Saccharomyces cerevisiae, three classes of sphingolipids contain myo-inositol--inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)(2)C]. No fission yeast equivalent of Ipt1p, the inositolphosphotransferase that synthesizes M(IP)(2)C from MIPC, has been found in the Schizosaccharomyces pombe genome. Analysis of the sphingolipid composition of wild-type cells confirmed that MIPC is the terminal and most abundant complex sphingolipid in S. pombe. Three proteins (Sur1p, Csg2p and Csh1p) have been shown to be involved in the synthesis of MIPC from IPC in S. cerevisiae. The S. pombe genome has three genes (SPAC2F3.01, SPCC4F11.04c and SPAC17G8.11c) that are homologues of SUR1, termed imt1(+), imt2(+) and imt3(+), respectively. To determine whether these genes function in MIPC synthesis in S. pombe, single and multiple gene disruptants were constructed. Single imt disruptants were found to be viable. MIPC was not detected and IPC levels were increased in the triple disruptant, indicating that the three SUR1 homologues are involved in the synthesis of MIPC. GFP-tagged Imt1p, Imt2p and Imt3p localized to Golgi apparatus membranes. The MIPC-deficient mutant exhibited pleiotropic phenotypes, including defects in cellular and vacuolar morphology, and in localization of ergosterols. MIPC seemed to be required for endocytosis of a plasma-membrane-localized amino acid transporter, because sorting of the transporter from the plasma membrane to the vacuole was severely impaired in the MIPC-deficient mutant grown under nitrogen-limiting conditions. These results suggest that MIPC has multiple functions not only in the maintenance of cell and vacuole morphology but also in vesicular trafficking in fission yeast.
Assuntos
Glicoesfingolipídeos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/metabolismo , Cromatografia em Camada Fina , Endocitose/efeitos dos fármacos , Ergosterol/farmacologia , Genes Fúngicos/genética , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Manosiltransferases/deficiência , Manosiltransferases/genética , Microdomínios da Membrana/enzimologia , Proteínas de Membrana/química , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Transporte Proteico/efeitos dos fármacos , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.
Assuntos
Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Polissacarídeos/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Substituição de Aminoácidos , Retículo Endoplasmático/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Saccharomyces cerevisiae LIP1 encodes a regulatory subunit that forms a complex with the ceramide synthase catalytic subunits, Lag1/Lac1, which is localized on the membrane of endoplasmic reticulum. To understand the underlying regulatory mechanism of sphingolipid biosynthesis, we generated strains upon replacing the chromosomal LIP1 promoter with a Tet-off promoter, which enables the expression in Dox-dependent manner. The lip1-1 strain, obtained through the promoter substitution, exhibits severe growth inhibition and remarkable decrease in sphingolipid synthesis in the presence of Dox. Using this strain, we investigated the effect of a decrease in ceramide synthesis on TOR complex 2 (TORC2)-Ypk1 signaling, which senses the complex sphingolipid level at the plasma membrane and promotes sphingolipid biosynthesis. In lip1-1 cells, Ypk1 was activated via both upstream kinases, TORC2 and yeast PDK1 homologues, Pkh1/2, thereby inducing hyperphosphorylation of Lag1, but not of another Ypk1-substrate, Orm1, which is a known negative regulator of the first step of sphingolipid metabolism, in the presence of Dox. Therefore, our data suggest that the metabolic enzyme activities at each step of the sphingolipid biosynthetic pathway are controlled through a fine regulatory mechanism.
Assuntos
Quinase 3 da Glicogênio Sintase/genética , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Esfingolipídeos/biossíntese , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Domínio Catalítico/genética , Membrana Celular/genética , Retículo Endoplasmático/genética , Regulação Fúngica da Expressão Gênica/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Oxirredutases/genética , Oxirredutases/ultraestrutura , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Esfingolipídeos/genéticaRESUMO
In the fission yeast Schizosaccharomyces pombe, galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases in the lumen of the Golgi apparatus. In S. pombe, the major in vitro α1,2-galactosyltransferase activity has been purified, the gma12(+) gene has been cloned, and three α-galactosyltransferase genes (gmh1(+)-gmh3(+)) have also been partially characterized. In this study, we found three additional uncharacterized genes with homology to gmh1(+) (gmh4(+)-gmh6(+)) in the fission yeast genome sequence. All possible single disruption mutants and the septuple disruption strain were constructed and characterized. The electrophoretic mobility of acid phosphatase prepared from gma12Δ, gmh2Δ, gmh3Δ and gmh6Δ mutants was higher than that from wild type, indicating that Gma12p, Gmh2p, Gmh3p and Gmh6p are required for the galactosylation of N-linked oligosaccharides. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides from each single mutant showed that Gma12p, Gmh2p and Gmh6p are involved in galactosylation of O-linked oligosaccharides. The septuple mutant exhibited similar drug and temperature sensitivity as a gms1Δ mutant that is incapable of galactosylation. Oligosaccharide structural analysis based on HPLC and methylation analysis revealed that the septuple mutant still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating that an unknown α1,3-galactosyltransferase activity was still present in the septuple mutant.
Assuntos
Galactose/química , Galactosiltransferases/genética , Oligossacarídeos/química , Schizosaccharomyces/química , Fosfatase Ácida/química , Sequência de Aminoácidos , Deleção de Genes , Glicosilação , Dados de Sequência Molecular , Fenótipo , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/genética , Alinhamento de SequênciaRESUMO
The gmn2 mutant of Schizosaccharomyces pombe has previously been shown to exhibit defects in protein glycosylation of N-linked oligosaccharides (Ballou, L. and Ballou, CE., Proc. Natl. Acad. Sci. USA, 92, 2790-2794 (1995)). Like most glycosylation-defective mutants, the S. pombe gmn2 mutant was found to be sensitive to hygromycin B, an aminoglycoside antibiotic. As a result of complementation analysis, the gmn2+ gene was found to be a single open reading frame that encodes a polypeptide of 373 amino acids consisting of multiple membrane-spanning regions. The Gmn2 protein shares sequence similarity with Kluyveromyces lactis and Saccharomyces cerevisiae Erd1 proteins, which are required for retention of luminal endoplasmic reticulum (ER) proteins. Although disruption of the gmn2+ gene is not lethal, the secreted glycoprotein showed a significant glycosylation defect with destabilization of the glycosyltransferase responsible for N-glycan elongation. It was also shown that a significant amount of BiP was missorted to the cell surface according to ADEL receptor destabilization. Fluorescent microscopy revealed that the functional Gmn2-EGFP fusion protein is mainly localized in the Golgi membrane. These results indicate that the Gmn2 protein is required for protein glycosylation and for retention of ER-resident proteins in S. pombe cells.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Glicosilação , Glicosiltransferases/metabolismo , Complexo de Golgi/metabolismo , Kluyveromyces/genética , Proteínas de Membrana/genética , Mutação , Fases de Leitura Aberta , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Schizosaccharomyces species are currently the only known organisms with two types of genes encoding UDP-glucose/-galactose 4-epimerase, uge1(+) and gal10(+). A strain deleted for uge1(+) exhibited a severe galactosylation defect and a decrease in activity and in UDP-galactose content when grown in glucose-rich medium (2 % glucose), indicating that Uge1p is a major UDP-glucose/-galactose 4-epimerase under these growth conditions. In contrast, gal10(+) was efficiently expressed and involved in galactosylation of cell-surface proteins in low-glucose medium (0.1 % glucose and 2 % glycerol), but not in galactose-containing medium. In a uge1Deltagal10Delta strain, the galactosylation defect was suppressed and UDP-galactose content restored to wild-type levels in galactose-containing medium. Disruption of gal7(+), encoding galactose-1-phosphate uridylyltransferase, in the uge1Deltagal10Delta strain reversed suppression of the galactosylation defect and reduced levels of UDP-galactose, indicating that galactose is transported from the medium to the cytosol and is converted into UDP-galactose via galactose 1-phosphate by Gal7p in Sch. pombe.
Assuntos
Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , UDPglucose 4-Epimerase/metabolismo , Galactosefosfatos/metabolismo , Regulação Fúngica da Expressão Gênica , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , UDPglucose 4-Epimerase/genética , Uridina Difosfato Galactose/metabolismoRESUMO
The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone [DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1 (+)), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1 Delta cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1 (+) is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1 (+) and dak2 (+) encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1 Delta strain showed a more severe reduction of growth on glycerol and DHA than the dak2 Delta strain because the expression of dak1 (+) mRNA was higher than that of dak2 (+). In wild-type S. pombe, expression of the gld1 (+), dak1 (+), and dak2 (+) genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1 (+) was regulated by glucose repression and that it was derepressed in scr1 Delta and tup12 Delta strains.
Assuntos
Proteínas Fúngicas/metabolismo , Glicerol/metabolismo , Schizosaccharomyces/enzimologia , Desidrogenase do Álcool de Açúcar/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Glucose/metabolismo , Dados de Sequência Molecular , Schizosaccharomyces/química , Schizosaccharomyces/genética , Alinhamento de Sequência , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/genéticaRESUMO
The cell walls of yeast cells possess a large mannan structure mainly comprising of a linear α1,6-linked mannose oligomer on the N-linked glycans. The biosynthesis of the mannan is initiated by ScOch1p α1,6-mannosyltransfease, and elongated by the mannan polymerase complexes M-Pol I and II in the Golgi of Saccharomyces cerevisiae. Here, we functionally characterized SpMnn9 and SpAnp1 proteins in the fission yeast Schizosaccharomyces pombe; these proteins are homologs of S. cerevisiae M-Pol II complex proteins ScMnn9p and ScAnp1p. Cells harboring disruptions in Spmnn9+ and Spanp1+ genes showed slower growth at 37°C and an increased sensitivity to hygromycin B, characteristic of a glycosylation defect. Results obtained from the acid phosphatase assay and high-performance liquid chromatography analysis of N-linked glycans in Spmnn9Δ and Spanp1Δ mutants suggested that the mannan structure in S. pombe is synthesized sequentially by the α-mannosyltransferases in the order of SpOch1p, SpMnn9p and SpAnp1p. Immunoprecipitation and split YFP analyses demonstrated that SpMnn9p and SpAnp1p form the M-Pol-II like complex. Together, these results provided an improved understanding of the mechanism of mannan synthesis by SpMnn9p and SpAnp1p in S. pombe.
Assuntos
Mananas/biossíntese , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Parede Celular/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Manosiltransferases/metabolismo , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Myxococcus xanthus Nudix hydrolase 2 (Nud2) hydrolyzed oxidized deoxynucleotides, such as 8-oxo-dGTP, 8-oxo-dGDP, 8-OH-dTP, and 2-OH-dATP, and showed the highest specific activity toward 8-oxo-dGTP. Mn2+ was the most effective co-factor for stimulating oxidized deoxynucleotide hydrolase activity. The Km of Nud2 with 8-oxo-dGTP for Mn2+ was 19-fold lower than that for Mg2+, and was 2-fold lower than that with dGTP for Mn2+. The specificity constant (kcat/Km) for 8-oxo-dGTP was 6-fold higher than that for dGTP. Nud2 contains a similar Nudix motif (84AX590GX7REX2EEXGX). Replacement of Ala84 and/or Gly90 in the Nudix motif of Nud2 by Gly or Glu had negligible effects on 8-oxo-dGTP hydrolase activity, suggesting that a strict Nudix motif sequence is not essential for complete hydrolase activity of Nud2.
Assuntos
Myxococcus xanthus/enzimologia , Pirofosfatases/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Enzimas Reparadoras do DNA/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Hidrólise , Cinética , Mutação , Myxococcus xanthus/genética , Oxirredução , Monoéster Fosfórico Hidrolases/metabolismo , Pirofosfatases/isolamento & purificação , Especificidade por Substrato , Nudix HidrolasesRESUMO
The N- and O-linked oligosaccharides from fission yeast Schizosaccharomyces pombe not only contain large amounts of d-mannose (Man) but also contain large amounts of d-galactose (Gal). Although the galactomannans of S. pombe are mainly composed of α1,2- or α1,3-linked Gals, some of the terminal α1,2-linked Gals are found to be linked to pyruvylated ß1,3-linked galactose (PvGal). We have determined the structural characteristics of the N-glycans and O-glycans in three Schizosaccharomyces species (S. japonicus, S. octosporus, and S. cryophilus) using lectin blot, 1H NMR spectroscopy, and size-fractionation high performance liquid chromatography (HPLC), and found that the galactosylation of oligosaccharides was a common feature in fission yeasts. In addition, each of the terminal Galα1,2-, Galß1,3- and non-substituted Man residues exhibited distinct characteristics. A BLAST search of gene databases in Schizosaccharomyces identified genes homologous to pvg1 encoding pyruvyltransferase of S. pombe. These genes, when expressed in an S. pombe pvg1Δ strains, led to the pyruvylation of non-reducing terminal ß-linked Gal, suggesting the biosynthetic pathway of PvGal-containing oligosaccharides is highly conserved in fission yeasts.
Assuntos
Oligossacarídeos/química , Polissacarídeos/química , Schizosaccharomyces/química , Lectinas/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oligossacarídeos/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Autophagy is triggered when organisms sense radical environmental changes, including nutritional starvation. During autophagy, cytoplasmic components, including organelles, are enclosed within autophagosomes and are degraded upon lysosome-vacuole fusion. In this study, we show that processing of GFP-tagged Atg8 can serve as a marker for autophagy in the fission yeast Schizosaccharomyces pombe. Using this marker, 13 Atg homologues were also found to be required for autophagy in fission yeast. In budding yeast, autophagy-deficient mutants are known to be sterile, whereas in fission yeast we found that up to 30 % of autophagy-defective cells with amino acid auxotrophy were able to recover sporulation when an excess of required amino acids was supplied. Furthermore, we found that approximately 15 % of the autophagy-defective cells were also able to sporulate when a prototrophic strain was subjected to nitrogen starvation, which suggested that fission yeast may store sufficient intracellular nitrogen to allow partial sporulation under nitrogen-limiting conditions, although the majority of the nitrogen source is supplied by autophagy. Monitoring of the sporulation process revealed that the process was blocked non-specifically at various stages in the atg1Delta and atg12Delta mutants, possibly due to a shortage of amino acids. Taking advantage of this partial sporulation ability of fission yeast, we sought evidence for the existence of a recycling system for nitrogen sources during starvation.
Assuntos
Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Aminoácidos/metabolismo , Autofagia/genética , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia , Sequência de Bases , DNA Fúngico/genética , Genes Fúngicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Mutação , Nitrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomycetales/genética , Saccharomycetales/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Especificidade da Espécie , Esporos Fúngicos/fisiologiaRESUMO
The KTRalpha1,2-mannosyltransferase gene family of Saccharomyces cerevisiae is responsible not only for outer-chain modifications of N-linked oligosaccharides but also for elongation of O-linked mannose residues. To identify genes involved in the elongation step of O-linked oligosaccharide chains in Schizosaccharomyces pombe, we characterized six genes, omh1(+)-omh6(+), that share significant sequence similarity to the S. cerevisiae KTR family. Six deletion strains were constructed, each carrying a single disrupted omh allele. All strains were viable, indicating that none of the omh genes was essential. Heterologous expression of a chitinase from S. cerevisiae in the omh mutants revealed that O-glycosylation of chitinase had decreased in omh1Delta cells, but not in the other mutants, indicating that the other omh genes do not appear to be required for O-glycan synthesis. Addition of the second alpha1,2-linked mannose residue was blocked in omh1Delta cells. An Omh1-GFP fusion protein was found to be localized in the Golgi apparatus. These results indicate that Omh1p plays a major role in extending alpha1,2-linked mannose in the O-glycan pathway in S. pombe.