RESUMO
Congenital disorders of glycosylation (CDG) are caused by enzymatic defects of the formation or processing of lipid-linked oligosaccharides and glycoproteins. Since the majority of proteins is glycosylated, a defect in a singular CDG enzyme leads to a multisytemic disease with secondary malfunction of thousands of proteins. CDG-Ij (DPAGT1-CDG) is caused by a defect of the human DPAGT1 (UDP-GlcNAc: Dolichol Phosphate N-Acetylglucosamine-1-Phosphotransferase), catalyzing the first step of N-linked glycosylation. So far the clinical phenotype of only one CDG-Ij patient has been described. The patient showed severe muscular hypotonia, intractable seizures, developmental delay, mental retardation, microcephaly and exotropia. Molecular studies of this patient revealed the heterozygous mutation c.660A>G (Y170C; paternal) in combination with an uncharacterized splicing defect (maternal). Two further mutations, c.890A>T (I297F) and c.162-8G>A as a splicing defect were detected when analyzing DPAGT1 in two affected siblings of a second family. We report two new patients with the novel homozygous mutation, c.341C>G (A114 G), causing a severe clinical phenotype, characterized by hyperexcitability, intractable seizures, bilateral cataracts, progressive microcephaly and muscular hypotonia. Both our patients died within their first year of life. With the discovery of this novel mutation and a detailed clinical description we extend the clinical features of CDG-Ij in order to improve early detection of this disease.
Assuntos
Defeitos Congênitos da Glicosilação/enzimologia , Defeitos Congênitos da Glicosilação/genética , Mutação/genética , Doenças Raras/enzimologia , Doenças Raras/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Adulto , Sequência de Aminoácidos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Glicosilação , Homozigoto , Humanos , Imunoprecipitação , Recém-Nascido , Lipopolissacarídeos/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Pele/citologia , Pele/enzimologiaRESUMO
Factors for efficient synthesis of mRNA in vitro and its subsequent translation in cell free lysates from reticulocyte and wheat germ were studied using yeast invertase as a probe. Among various transcription systems tested, containing either SP6, T5, T7 or a bacterial synthetic consensus promoter, the T7 system was superior both from a quantitative and qualitative point of view. Transcription with SP6 polymerase, but not with the other enzymes, resulted in premature transcript termination, which is ascribed to a sensitivity of the SP6 polymerase towards a hairpin loop structure in the invertase coding region. In-frame fusion of the critical DNA sequence to a different gene promoted premature transcription termination of the resulting chimeric template, which in its original form is transcribed correctly. Transcripts with additional sequences 5' upstream of the natural translation start revealed a diminished protein synthesis presumably due to the presence of out of frame ATG codons. In contrast, no influence on translation was found when additional sequences at the 3' end were present or when the stop codon was missing. Capping of transcripts was essential for translation in wheat germ lysates, whereas protein synthesis in reticulocytes was only reduced in the absence of a cap. The influence of polyadenylation on translation was studied using transcripts with engineered poly(A) tracts of different size. Increasing poly(A) chain length abolished translation in vitro in both translation systems. Inhibition was poly(A)-specific and is discussed as interference of the poly(A) sequences with a crucial component(s) of the protein synthesis machinery.
Assuntos
Glicosídeo Hidrolases/genética , Biossíntese de Proteínas , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , Bacteriófagos/enzimologia , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Vetores Genéticos , Plasmídeos , Poli A/metabolismo , Regiões Promotoras Genéticas , Análogos de Capuz de RNA/metabolismo , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Fagos T/genética , beta-FrutofuranosidaseRESUMO
S. cerevisiae contains many mannose-rich glycoproteins that possess N- and O-linked carbohydrate chains, and both types may even occur on one and the same protein. The steps in the synthesis of asparagine-linked chains begin with assembly and transfer of the lipid-linked precursor to protein in a way common to all eucaryotes. Subsequent modifications lead to mannosyl extensions of various lengths, but complex type carbohydrate structures are not formed. Oligosaccharides O-linked to serine/threonine consist exclusively of mannose in S. cerevisiae. The mannose residue attached directly to the protein is transferred from Dol-P-Man in a unique way, which has been observed so far for fungal cells only. The cellular localization of the glycosylation reactions is summarized and the problem of transmembrane translocation of the sugar precursors at the ER and the Golgi is discussed. Some aspects of secretory (sec) and asparagine linked glycosylation (alg) mutants have been covered, and the various hypotheses related to the possible functions of this costly protein modification process are discussed. The article may also be helpful for those, who want to exploit the yeast's protein synthesizing machinery by genetically manipulating the cells.
Assuntos
Proteínas Fúngicas/metabolismo , Glicoproteínas de Membrana , Saccharomyces cerevisiae/metabolismo , Asparagina/fisiologia , Metabolismo dos Carboidratos , Membrana Celular/metabolismo , Combinação de Medicamentos/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Glicoproteínas/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
Yeast membranes incorporate radioactivity from GDP[14C]mannose into various glycolipids. These can be separated by thin layer chromatography into at least seven components. The major component has been identified previously as dolichyl monophosphate mannose. Only one additional component is not sensitive to mild alkaline saponification, but is hydrolyzed instead under mild acidic conditions. This latter glycolipid has all the characteristics of a polyprenyl diphosphate oligosaccharide with a sugar moiety of more than 12 hexose units. It runs like dolichyl diphosphate derivatives on a DEAE column and evidence is presented that the lipid moiety is a polyprenol. When radioactive Dol-PP-di-N-acetylchitobiose is incubated with yeast membranes in the presence of non-radioactive GDPmannose a small amount of a larger lipid oligosaccharides is formed besides the previously-described Dol-PP-(GlcNAc)2 mannose. This oligosaccharide has all the properties of the glycolipid described above. Its formation is greatly increased when Triton is omitted from the incubation. Radioactivity of the polyprenyl diphosphate [14C]oligosaccharide is transferred to ethanol-insoluble material, most likely endogenous membrane glycoproteins.
Assuntos
Oligossacarídeos de Poli-Isoprenil Fosfato/metabolismo , Açúcares de Poli-Isoprenil Fosfato/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Dolicol Monofosfato Manose/metabolismo , Guanosina Difosfato Manose/metabolismo , Monossacarídeos de Poli-Isoprenil Fosfato/metabolismoRESUMO
Incubation of a membrane fraction from Saccharomyces cerevisiae with UDP-N-acetyl [14C]glucosamine catalyzes the transfer of N-acetylglucosamine to an endogenous lipid fraction as well as a methanol-insoluble polymer. The glycolipid was shown to separate into three compounds by thin-layer chromatography. The biosynthesis of two of them could clearly be stimuated by the addition of dolichol monophosphate to the incubation mixture. Evidence is presented that the substances are dolichol pyrophosphate derivatives: dolichol pyrophosphate N-acetylglucosamine and dolichol pyrophosphate di-N-acetylchitobiose. The formation of the chitobiose-containing lipid was increased by reincubation of the glycolipid with non-radioactive UDP-N-acetylglucosamine. The same particulate preparation transferred mannose from GDPmannose to dolichol pyrophosphate di-N-acetylchitobiose, giving rise to a lipid-bound oligosaccharide. Molecular weight determination of the oligosaccharide moiety gave a value of 780, which is consistent with a tetrasaccharide containing two mannose subunits attached to di-N-acetylchitobiose. The methanol-insoluble radioactive product obtained in the presence of UDP-N-acetyl[14C]glucosamine was transformed by pronase treatment to a large extent into dialyzable material. It is suggested that the glycolipids described serve as intermediates in the glycosylation of yeast mannoproteins.
Assuntos
Metabolismo dos Lipídeos , Oligossacarídeos/biossíntese , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Dolicóis/metabolismo , Glicolipídeos/biossíntese , Peso Molecular , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
N-Glycosylation of eukaryotic secretory and membrane-bound proteins is an essential and highly conserved protein modification. The key step of this pathway is the en bloc transfer of the high mannose core oligosaccharide Glc3Man9GlcNAc2 from the lipid carrier dolichyl phosphate to selected Asn-X-Ser/Thr sequences of nascent polypeptide chains during their translocation across the endoplasmic reticulum membrane. The reaction is catalysed by the enzyme oligosaccharyltransferase (OST). Recent biochemical and molecular genetic studies in yeast have yielded novel insights into this enzyme with multiple tasks. Nine proteins have been shown to be OST components. These are assembled into a heterooligomeric membrane-bound complex and are required for optimal expression of OST activity in vivo in wild type cells. In accord with the evolutionary conservation of core N-glycosylation, there are significant homologies between the protein sequences of OST subunits from yeast and higher eukaryotes, and OST complexes from different sources show a similar organisation as well.
Assuntos
Hexosiltransferases , Proteínas de Membrana , Saccharomyces cerevisiae/enzimologia , Transferases/metabolismo , Sítios de Ligação , Clonagem Molecular , Fosfatos de Dolicol/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Oligossacarídeos/metabolismo , Peptídeos/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Transferases/química , Transferases/genéticaRESUMO
A membrane fraction from Saccharomyces cerevisiae as well as a mannosyltransferase purified therefrom was shown to catalyze the transfer of mannose from GDPmannose to retinyl phosphate. The product formed has chromatographic and chemical properties characteristic for retinylphosphate mannose. The enzyme requires divalent cations. Mg2+ is more effective than Mn2+ with an optimum concentration around 25 mM. Amphomycin at a concentration of 0.1 mg/ml inhibits the reaction to 50%. Glycosyl transfer was specific for mannose residues from GDPmannose and did not occur with dolichylphosphate mannose nor with UDP galactose; UDPglucose is a poor donor. Formation of retinylphosphate mannose is inhibited by dolichyl phosphate. This observation as well as similarities between retinylphosphate mannose and dolichylphosphate mannose synthesis in respect to ion requirement, inhibition by amphomycin are suggestive that both reactions are catalyzed by one and the same enzyme. In experiments studying the glycosyl donor specificity in the assembly of lipid-linked oligosaccharide intermediates involved in N-glycosylation of proteins, it could be demonstrated that retinylphosphate mannose can replace dolichylphosphate mannose in the final steps of mannosylation.
Assuntos
Metabolismo dos Lipídeos , Oligossacarídeos/biossíntese , Monossacarídeos de Poli-Isoprenil Fosfato/biossíntese , Açúcares de Poli-Isoprenil Fosfato/biossíntese , Saccharomyces cerevisiae/metabolismo , Catálise , Diterpenos , Fosfatos de Dolicol/farmacologia , Guanosina Difosfato Manose/metabolismo , Monossacarídeos de Poli-Isoprenil Fosfato/fisiologia , Especificidade por SubstratoRESUMO
N-Oligosaccharyltransferase catalyzes the N-glycosylation of asparagine residues of nascent polypeptide chains in the endoplasmic reticulum, a pathway highly conserved in all eukaryotes. An enzymatically active complex was isolated from microsomal membranes from Saccharomyces cerevisiae, which is composed of four proteins: Wbp1p and Swp1p (previously found to be encoded by two essential genes necessary for N-glycosylation in vivo and in vitro) and two additional proteins with a molecular mass of 60/62 kDa and 34 kDa. The 60/62 component represents differentially glycosylated forms of a protein that has sequence homology to ribophorin I. Wbp1p and Swp1p reveal homology to mammalian OST 48 and ribophorin II, respectively. Ribophorin I and II and OST 48 were recently shown to be constituents of the mammalian transferase from dog pancreas. The data reveal a high conservation of the organization of this enzyme activity.
Assuntos
Hexosiltransferases , Saccharomyces cerevisiae/enzimologia , Transferases/isolamento & purificação , Sequência de Aminoácidos , Animais , Asparagina/metabolismo , Cães , Glicosilação , Membranas Intracelulares/enzimologia , Proteínas de Membrana/química , Microssomos/enzimologia , Microssomos/ultraestrutura , Dados de Sequência Molecular , Pâncreas/enzimologia , Homologia de Sequência , Transferases/química , Transferases/metabolismoRESUMO
Outer chain glycosylation in Saccharomyces cerevisiae leads to heterogeneous and immunogenic asparagine-linked saccharide chains containing more than 50 mannose residues on secreted glycoproteins. Using a [3H]mannose suicide selection procedure a collection of N-glycosylation defective mutants (designated ngd) was isolated. One mutant, ngd29, was found to have a defect in the initiation of the outer chain and displayed a temperature growth sensitivity at 37 degrees C allowing the isolation of the corresponding gene by complementation. Cloning, sequencing and disruption of NGD29 showed that it is a non lethal gene and identical to OCH1. It complemented both the glycosylation and growth defect. Membranes isolated from an ngd29 disruptant or an ngd29mnn1 double mutant were no longer able, in contrast to membranes from wild type cells, to transfer mannose from GDPmannose to Man8GlcNAc2, the in vivo acceptor for building up the outer chain. Heterologous expression of glucose oxidase from Aspergillus niger in an ngd29mnn1 double mutant produced a secreted uniform glycoprotein with exclusively Man8GlcNAc2 structure that in wild type yeast is heavily hyperglycosylated. The data indicate that this mutant strain is a suitable host for the expression of recombinant glycoproteins from different origin in S. cerevisiae to obtain mammalian oligomannosidic type N-linked carbohydrate chains.
Assuntos
Proteínas Fúngicas/biossíntese , Genes Fúngicos , Glicoproteínas de Membrana/biossíntese , Mutação , Oligossacarídeos/biossíntese , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Alelos , Animais , Asparagina , Sequência de Carboidratos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose Oxidase/biossíntese , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/biossíntese , Glicosilação , Mamíferos , Manosiltransferases/biossíntese , Manosiltransferases/genética , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oligossacarídeos/química , Fases de Leitura Aberta , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , beta-FrutofuranosidaseRESUMO
The PMR1 gene of Saccharomyces cerevisiae is thought to encode a putative Ca(2+)-ATPase [1]. Membranes isolated from wild-type cells and from pmr1 null mutant of S. cerevisiae were fractionated on sucrose density gradients. In the pmr1 mutant we found a decrease in activity of the P-type ATPase and of ATP-dependent, protonophore-insensitive Ca2+ transport in light membranes, that comigrate with the Golgi marker GDPase. We conclude that the product of the PMR1 gene (Pmr1p) is indeed a Ca(2+)-ATPase of the Golgi and Golgi-like membranes. Surprisingly, the pmr1 null mutation abolished Ca(2+)-ATPase activity in Golgi and/or Golgi-like membranes only to 50% under conditions where they are separated from vacuolar membranes. This indicates that an additional Ca(2+)-ATPase is localized in Golgi and/or Golgi-like membranes. Moreover, a third Ca(2+)-ATPase is found in the ER and ER-like membranes. The data are consistent with the assumption that these Ca(2+)-ATPases are encoded by gene(s) different from PMR1. Disruption of PMR1 Ca(2+)-ATPase causes significant redistribution of enzyme activities and of total protein in compartments of the secretory pathway. A decrease in activity is observed for three integral membrane proteins: NADPH cytochrome c reductase, dolichyl phosphate mannose synthase, and Ca(2+)-ATPase, and also for total protein in Golgi, Golgi-like compartments and in vacuoles, whereas a corresponding increase of these activities is observed in endoplasmic reticulum and endoplasmic reticulum-like membranes. We assume that Ca(2+)-ATPases and sufficient Ca2+ gradients across the organellar membranes are important for the correct sorting of proteins to the various compartments of the secretory apparatus.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Proteínas Fúngicas/metabolismo , Complexo de Golgi/enzimologia , Saccharomyces cerevisiae/enzimologia , Transporte Biológico , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , Fracionamento Celular , Membrana Celular/enzimologia , Retículo Endoplasmático/enzimologia , Inibidores Enzimáticos/farmacologia , Transporte de Íons , Manosiltransferases/metabolismo , Mutação , NADPH-Ferri-Hemoproteína Redutase/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Pirofosfatases/metabolismo , Vacúolos/enzimologia , Vanadatos/farmacologiaAssuntos
Proteínas Fúngicas/biossíntese , Glicoproteínas/biossíntese , Hexosiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Boroidretos , Radioisótopos de Carbono , Cromatografia em Gel , Cromatografia por Troca Iônica , Cromatografia em Papel , Cromatografia em Camada Fina , Eletroforese em Papel , Glicosídeo Hidrolases , Concentração de Íons de Hidrogênio , Cinética , Manganês/farmacologia , Manose , Conformação Molecular , Relação Estrutura-Atividade , Fatores de TempoAssuntos
Detergentes/farmacologia , Hexosiltransferases/antagonistas & inibidores , Fosfatos de Poli-Isoprenil/farmacologia , Saccharomyces cerevisiae/enzimologia , Membrana Celular/enzimologia , Fosfatos de Dolicol/farmacologia , Cinética , Relação Estrutura-Atividade , Uridina Difosfato N-AcetilglicosaminaRESUMO
A new membrane preparation from Saccharomyces cerevisiae was developed, which effectively catalyzes the synthesis of large oligosaccharide-lipids from GDP-Man and UDP-Glc allowing a detailed study of their formation and size. The oligosaccharide from an incubation with GDP-Man could be separated by gel filtration chromatography into several species consisting of two N-acetylglucosamine (GlcNAc) residues at the reducing end and differing by one mannos unit; the major compound formed has the composition (Man)9(GlcNAc)2. Upon incubation with UDP-Glc, three oligosaccharides corresponding to the size of (Glc)1-3(Man)9(GlcNAc)2 are formed. Thus, the oligosaccharides generated in vitro by the yeast membranes appear to be identical in size with the oligosaccharides found in animal systems. In addition the results indicate that dolichyl phosphate mannoe (DolP-Man) is the immediate donor in assembling the oligosaccharide moiety from (Man)5(GlcNAc)2 to (Man)9(GlcNAc)2. All three glucose residues are transferred from DolP-Glc. Experiments with isolated [Glc-14C]oligosaccharide-lipid as substrate demonstrated that the oligosaccharide chain is transferred to an endogenous membrane protein acceptor. Moreover, transfer is followed by an enzymic removal of glucose residues, due to a glucosidase activity associated with the membranes. Glucose release from the free [Glc-14C]oligosaccharide is less effective than from protein-bound oligosaccharide. Glycosylation was also observed using [Man-14C]oligosaccharide-lipid or DolPP-(GlcNAc)2 as donor. However, transfer in the presence of glucose seems to be more rapid. The mannose-containing oligosaccharide, released from the lipid, was shown to function as a substrate for further chain elongation reactions utilizing GDP-Man but not DolPP-Man as donor. It is suggested that the immediate precursor in the synthesis of the heterogeneous core region, (Man)12-17(GlcNAc)2, of yeast mannoproteins is a glucose-containing lipid-oligosaccharide with the composition (Glc)3(Man)9(GlcNAc)2, i.e. only part of what has been defined as inner core is built up on the lipid carrier. After transfer to protein the oligosaccharide is modified by excision of the glucose residues, followed subsequently by further elongation from GDP-Man to give the size of th oligosaccharide chains found in native mannoproteins.
Assuntos
Diterpenos/metabolismo , Dolicóis/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas de Membrana , Oligossacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Dolicol Monofosfato Manose/metabolismo , Guanosina Difosfato Manose/metabolismo , Cinética , Uridina Difosfato Glucose/metabolismoRESUMO
The translation and translocation of two yeast glycoproteins, invertase and carboxypeptidase Y, were studied in a heterologous cell-free translation system from reticulocytes supplemented with dog pancreas microsomes. Using in vitro synthesized mRNA transcripts, encoding complete or truncated invertase forms, the influence of polypeptide size and ribosome dependence was studied. It was found that C-terminal truncated fragments of 25 kDa, i.e. a size larger than the average size of a domain structure, are translocated and processed post-translationally with a similar efficiency to the cotranslational events. Post-translational import decreases with increasing peptide chain, mature polypeptide (60 kDa) being no longer translocated. Post-translational competence is only maintained as long as the peptide remains associated with ribosomes. Translocation of invertase depends on the presence of the leader peptide and requires energy independent of protein synthesis. Size dependence of post-translational import could also be demonstrated for carboxypeptidase Y.
Assuntos
Carboxipeptidases/genética , Retículo Endoplasmático/metabolismo , Glicosídeo Hidrolases/genética , Processamento de Proteína Pós-Traducional , Catepsina A , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/metabolismo , Peso Molecular , Plasmídeos , Ribossomos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , beta-FrutofuranosidaseRESUMO
The plant growth-hormone auxin (indole-3-acetic acid, IAA) is involved in regulating such diverse processes as cell elongation, cell division and differentiation. The sequence of events leading to the various phenomena is still poorly understood. Both changes in extra- and intracellular pH (refs 1-4) and selective transcription are known to be induced by auxin. Evidence for auxin receptors at the plasmalemma membrane has been reported, but the signal transduction pathway is not known, for this nor for other plant hormones. In animal cells, hydrolysis of inositolphospholipids is a major mechanism for transmembrane signalling in response to external stimuli such as hormones, growth factors, neurotransmitters, antigens or light (reviewed in refs 8-11). Here we report that auxin can generate transient changes in inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol bisphosphate (InsP2) within minutes in Catharanthus roseus cells arrested in G1. These changes are accompanied by a redistribution within the polyphosphoinositide fraction. As the physiological response to auxin addition is to relieve the arrest in G1, we suggest that these effects are an element in the signal transduction of this plant hormone.
Assuntos
Ácidos Indolacéticos/farmacologia , Fosfatidilinositóis/metabolismo , Ácido 2,4-Diclorofenoxiacético/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/metabolismo , Células VegetaisRESUMO
We have cloned and sequenced a wound-inducible cDNA clone designated WIP1 (for wound-induced protein) from maize coleoptiles. It was isolated by differential screening of a cDNA library prepared from excised maize coleoptile segments. The deduced amino acid sequence predicts a secretory, cysteine-rich protein of 102 residues with a calculated molecular mass of 11 kDa and a typical N-terminal signal sequence. The protein has about 30% identity with various Bowman-Birk type proteinase inhibitors. Most interestingly, it is novel in that it is double-headed with exclusive specificity for chymotrypsin. WIP1 is strongly wound-induced in contrast to other members of the Bowman-Birk proteinase inhibitor family, which occur in seeds and are regulated during development. The response is fast, similar to defence-induced genes, and measurable as early as 30 min after wounding. Induction can also be evoked in the intact coleoptiles and the signal is systematically transmitted in the coleoptile to adjacent regions of the wounded area. Isolation and analysis of the corresponding genomic clone reveals that WIP1 contains an intron of 90 nucleotides.
Assuntos
Regulação da Expressão Gênica , Proteínas de Plantas/genética , Inibidores de Serina Proteinase , Inibidor da Tripsina de Soja de Bowman-Birk/genética , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA , Dados de Sequência Molecular , Proteínas de Plantas/química , Homologia de Sequência de Aminoácidos , Inibidor da Tripsina de Soja de Bowman-Birk/química , Zea mays/citologia , Zea mays/crescimento & desenvolvimentoRESUMO
In an effort to understand factors that control glycosylation of proteins and processing of carbohydrate chains, invertase from Saccharomyces cerevisiae was expressed in a heterologous system. Microinjection of invertase-specific in vitro transcripts into oocytes from Xenopus laevis resulted in synthesis, glycosylation and secretion of enzymatically active invertase. It was found that although the number of carbohydrate chains acquired is the same as in yeast, the carbohydrate processing is different. This is consistent with the notion that the usage of a glycosylation site is determined by the protein part, whereas subsequent processing occurs in a host-dependent manner. Both, high-mannose and complex type glycans, most likely tri- and tetra-antennary structures, were synthesized in oocytes. The data obtained suggests that in this system the core chains of yeast invertase remain high-mannose type, whereas the more extensively processed polymannose chains are modified to complex oligosaccharides. In the presence of the glycosylation inhibitor, tunicamycin, and the glucosidase processing inhibitor, methyldeoxynojirimycin, secretion of invertase is significantly decreased, whereas in the presence of the mannosidase inhibitor, deoxymannojirimycin, no influence of secretion is seen. This may suggest that glycosylation of invertase is important for early secretion events. Expression of invertase lacking the leader sequence results in loss of glycosylation and secretion in oocytes. This indicates that yeast signals for secretion are functional in this higher eukaryote.
Assuntos
Glicoproteínas/biossíntese , Glicosídeo Hidrolases/biossíntese , Oócitos/metabolismo , Polissacarídeos/biossíntese , Saccharomyces cerevisiae/genética , Xenopus laevis/metabolismo , Animais , Feminino , Vetores Genéticos , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/genética , Técnicas In Vitro , Oócitos/microbiologia , Plasmídeos , Polissacarídeos/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , beta-FrutofuranosidaseRESUMO
To understand better the structural requirements of the protein moiety important for N-glycosylation, we have examined the influence of proline residues with respect to their position around the consensus sequence (or sequon) Asn-Xaa-Ser/Thr. In the first part of the paper, experiments are described using a cell-free translation/glycosylation system from reticulocytes supplemented with dog pancreas microsomes to test the ability of potential acceptor peptides to interfere with glycosylation of nascent yeast invertase chains. It was found that peptides, being acceptors for oligosaccharide transferase in vitro, inhibit cotranslational glycosylation, whereas nonacceptors have no effect. Acceptor peptides do not abolish translocation of nascent chains into the endoplasmic reticulum. Results obtained with proline-containing peptides are compatible with the notion that a proline residue in an N-terminal position of a potential glycosylation site does not interfere with glycosylation, whereas in the position Xaa or at the C-terminal of the sequon, proline prevents and does not favour oligosaccharide transfer, respectively. This statement was further substantiated by in vivo studies using site-directed mutagenesis to introduce a proline residue at the C-terminal of a selected glycosylation site of invertase. Expression of this mutation in three different systems, in yeast cells, frog oocytes and by cell-free translation/glycosylation in reticulocytes supplemented with dog pancreas microsomes, leads to an inhibition of glycosylation with both qualitative and quantitative differences. This may indicate that host specific factors also contribute to glycosylation.
Assuntos
Glicosídeo Hidrolases/genética , Mutação , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Cães , Genes , Glicosilação , Microssomos/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Pâncreas/metabolismo , Plasmídeos , Reticulócitos/metabolismo , Saccharomyces cerevisiae/genética , beta-FrutofuranosidaseRESUMO
We have isolated and sequenced a genomic clone from Saccharomyces cerevisiae that shows structural features of a novel retrotransposon, designated Ty4. The element is 6.2 kilobases in length, and its genetic organization of the deduced functional domains is similar to Ty1 and Ty2 and thus different from Ty3. In contrast to hitherto known Ty elements from yeast, Ty4 is flanked by long terminal tau-element repeats instead of delta or sigma sequences. Ty4 contains two overlapping open reading frames. The first open reading frame, TYA4, is 1230 base pairs long and encodes a protein with a motif found in the nucleic acid-binding gag-protein of retroviruses. The second 4395-base pair open reading frame, TYB4, encodes a polyprotein that has domains with significant homology to retroviral protease, integrase, reverse transcriptase, and RNase H, structurally arranged in that order. The deduced amino acid sequence shows the greatest similarity with Ty2 and Ty1. The overall identity of the deduced functional protein domains is 28% with Ty2, 25% with Ty1, 19% with copia from Drosophila, and 18% with Ty3. Examination of genomic DNA from several laboratory strains indicates that Ty4 is present in two to four copies. Ty4 mRNA is of low abundance as compared to other Ty retrotransposons. At the 3' end of Ty4, two "solo" delta-elements, a full length and an overlapping, truncated one, are associated.