RESUMEN
Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al. J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism. Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified. Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway. We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions.
Asunto(s)
Amidohidrolasas/metabolismo , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas de Plantas/metabolismo , Ubiquitina/metabolismo , Amidohidrolasas/genética , Secuencia de Aminoácidos , Arabidopsis , Evolución Molecular , Datos de Secuencia Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Proteínas de Plantas/genética , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Peptide:N-glycanase (PNGase) cleaves oligosaccharide chains from glycopeptides and glycoproteins. Recently the deduced amino acid sequence of a cytoplasmic PNGase has been identified in various eukaryotes ranging from yeast to mammals, suggesting that deglycosylation may play a central role in some catabolic process. Several lines of evidence indicate that the cytoplasmic enzyme is involved in the quality control system for newly synthesized glycoproteins. Two-hybrid library screening by using mouse PNGase as the target yielded several PNGase-interacting proteins that previously had been implicated in proteasome-dependent protein degradation: mHR23B, ubiquitin, a regulatory subunit of the 19S proteasome, as well as a protein containing an ubiquitin regulatory motif (UBX) and an ubiquitin-associated motif (UBA). These findings by using the two-hybrid system were further confirmed either by in vitro binding assays or size fractionation assays. These results suggest that PNGase may be required for efficient proteasome-mediated degradation of misfolded glycoproteins in mammalian cells.
Asunto(s)
Amidohidrolasas/metabolismo , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas/metabolismo , Células 3T3 , Amidohidrolasas/química , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , Citoplasma/enzimología , Escherichia coli , Biblioteca de Genes , Mamíferos , Ratones , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Complejo de la Endopetidasa Proteasomal , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Transfección , Ubiquitina/metabolismoRESUMEN
In the sea urchin embryo, inhibition of collagen processing and deposition affects both gastrulation and embryonic skeleton (spicule) formation. It has been found that cell-free extracts of gastrula-stage embryos of Strongylocentrotus purpuratus contain a procollagen C-terminal proteinase (PCP) activity. A rationally designed non-peptidic organic hydroxamate, which is a potent and specific inhibitor of human recombinant PCP (FG-HL1), inhibited both the sea urchin PCP as well as purified chick embryo tendon PCP. In the sea urchin embryo, FG-HL1 inhibited gastrulation and blocked spicule elongation, but not spicule nucleation. A related compound with a terminal carboxylate rather than a hydroxamate (FG-HL2) did not inhibit either chick PCP or sea urchin PCP activity in a procollagen-cleavage assay. However, FG-HL2 did block spicule elongation without affecting spicule nucleation or gastrulation. Neither compound was toxic, because their effects were reversible on removal. It was shown that the inhibition of gastrulation and spicule elongation were independent of tissue specification events, because both the endoderm specific marker Endo1 and the primary mesenchyme cell specific marker SM50 were expressed in embryos treated with FG-HL1 and FG-HL2. These results suggest that disruption of the fibrillar collagen deposition in the blastocoele blocks the cell movements of gastrulation and may disrupt the positional information contained within the extracellular matrix, which is necessary for spicule formation.
Asunto(s)
Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Gástrula/fisiología , Metaloendopeptidasas/antagonistas & inhibidores , Péptidos/farmacología , Inhibidores de Proteasas/farmacología , Erizos de Mar/embriología , Animales , Proteína Morfogenética Ósea 1 , Proteínas Morfogenéticas Óseas/metabolismo , Embrión de Pollo , Colágeno/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Inmunohistoquímica , Metaloendopeptidasas/metabolismo , Erizos de Mar/efectos de los fármacos , Erizos de Mar/metabolismoRESUMEN
Renaturation of two enzymes lacking disulfide bonds, citrate synthase (CS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and another protein containing disulfide bonds, lysozyme (LZM), were studied in order to dissect the possible chaperone function from the isomerase function of yeast protein disulfide isomerase (PDI). Our findings suggest no independent chaperone activity of yeast PDI with respect to the two enzymes lacking disulfide bonds, GAPDH and CS, since neither of these enzymes required PDI for renaturation. In contrast, a high level of renaturation of LZM was observed in the presence of PDI. Renaturation of LZM involved formation and rearrangement of disulfide bonds. Additional studies using LZM as a substrate were done to examine the role of cysteine residues in the two active sites of PDI. Studies with a series of cysteine to serine mutants and truncation mutants of yeast PDI revealed that the two active sites of PDI were not equal in activity. An intramolecular disulfide bond in at least one active site of PDI was required for the oxidation of reduced LZM. The first cysteine in each active site was necessary for disulfide bond rearrangement, i.e., isomerization, in LZM, while the second cysteine was not.
Asunto(s)
Proteína Disulfuro Isomerasas/metabolismo , Sitios de Unión , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/metabolismo , Activación Enzimática , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Muramidasa/química , Muramidasa/metabolismo , Mutación , Oxidación-Reducción , Desnaturalización Proteica , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Pliegue de Proteína , Especificidad por Sustrato , LevadurasRESUMEN
A glycoprotein of the Xenopus vitelline envelope, gp 69/64, which mediates sperm binding, is closely related to the components of ZPA family, such as the mouse zona pellucida ZP2. To test the generality of these findings, we studied Discoglossus pictus, a species evolutionary distant from Xenopus and identified as a protein of 63 kDa in the vitelline envelope. Preliminary studies suggest that this protein may bind sperm at fertilization. We found that the 63-kDa protein is glycosylated and contains both N- and O-linked chains. We have cloned the cDNA encoding the Discoglossus protein of 63 kDa (Dp ZP2) by screening a Discoglossus cDNA library using Xenopus gp 69/64 cDNA as a probe. Analysis of the deduced sequence of Discoglossus protein revealed 48% identity with Xenopus gp 69/64 and 37-40% identity with mouse ZP2. The sequence conservation included a ZP domain, a potential furin cleavage site and a putative transmembrane domain. The N-terminus region of Dp ZP2 was 40% identical to the corresponding region of Xenopus gp 69/64 which has been shown to be essential for sperm binding to the VE. Although, as of yet, there is no evidence for sperm binding at the Dp ZP2 N-terminus, it is interesting that in this region three potential O-glycosylation sites are conserved in both species, in contrast to N-glycosylation sites. It was found that the Dp ZP2 mRNA is expressed in stage 1 oocytes and in the follicle cells surrounding the oocyte. Similarly, in Xenopus oocytes, the gp 69/64m RNA, was found in the oocytes, as well as in the somatic cells. Mol. Reprod. Dev. 59:133-143, 2001.
Asunto(s)
Anuros/metabolismo , Anuros/fisiología , Proteínas del Huevo/química , Proteínas del Huevo/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Oocitos/metabolismo , Membrana Vitelina/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Anuros/genética , Secuencia de Bases , Clonación Molecular , Proteínas del Huevo/metabolismo , Femenino , Expresión Génica , Glicosilación , Humanos , Inmunohistoquímica , Hibridación in Situ , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Oocitos/citología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Alineación de Secuencia , Interacciones Espermatozoide-Óvulo , Membrana Vitelina/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Glicoproteínas de la Zona PelúcidaRESUMEN
The process of sperm-egg binding is one of the barriers to cross-fertilization between related sea urchin species. A 350 kDa glycoprotein in the egg vitelline layer of Strongylocentrotus purpuratus has been shown to be a sperm-binding protein (SBP). Sulfated O-linked oligosaccharide chains on the 350 kDa glycoprotein, as well as domains of the polypeptide chain, serve as ligands for this binding process. The hypothesis that species-specific sperm-egg binding is attributed to the interaction between the sperm and the 350 kDa glycoprotein was tested using S. purpuratus and S. franciscanus. It was found that both species had a 350 kDa glycoprotein on the egg surface that cross-reacted immunologically using antibodies prepared against a recombinant form of the SBP. Because earlier studies had implicated the carbohydrate chains of the 350 kDa glycoprotein of S purpuratus in sperm binding, differences in carbohydrate chains on the 350 kDa glycoproteins of these species were examined. It was found that among the lectins tested only wheat germ agglutinin and Sambucus nigra agglutinin showed a significant difference in reactivity to the 350 kDa glycoproteins between species. Finally, using a bead-binding assay, it was shown that the isolated 350 kDa glycoproteins exhibited species-specific sperm-binding activity.
Asunto(s)
Proteínas de la Membrana/metabolismo , Erizos de Mar/fisiología , Interacciones Espermatozoide-Óvulo/fisiología , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Fertilización/fisiología , Immunoblotting , Inmunohistoquímica , Lectinas/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Oocitos/química , Oocitos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Espermatozoides/metabolismoRESUMEN
In addition to a role in DNA repair events in yeast, several lines of evidence indicate that the Rad23 protein (Rad23p) may regulate the activity of the 26 S proteasome. We report evidence that a de-N-glycosylating enzyme, Png1p, may be involved in the proteasomal degradation pathway via its binding to Rad23p. Interaction of Rad23p and Png1p was first detected by two-hybrid screening, and this interaction in vivo was confirmed by biochemical analyses. The Png1p-Rad23p complex was shown to be distinct from the well established DNA repair complex, Rad4p-Rad23p. We propose a model in which Rad23p functions as an escort protein to link the 26 S proteasome with proteins such as Rad4p or Png1p to regulate their cellular activities.
Asunto(s)
Amidohidrolasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Amidohidrolasas/química , Amidohidrolasas/genética , Amidohidrolasas/aislamiento & purificación , Secuencia de Bases , Cromatografía en Gel , Clonación Molecular , Citosol/enzimología , Cartilla de ADN , ADN de Hongos/genética , ADN de Hongos/efectos de la radiación , Proteínas de Unión al ADN/química , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas Fúngicas/química , Glicosilación , Modelos Biológicos , Datos de Secuencia Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Plásmidos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Rayos UltravioletaRESUMEN
A simple microadsorption technique is described to remove detergent additives from oligosaccharide samples before their mass spectrometric analysis. The described methodology has been validated with submicrogram quantities of contaminated glycoproteins. This procedure is applicable to investigating minute quantities of glycans in both the positive- and negative-ion mode of matrix-assisted laser desorption/ionization mass spectrometry.
Asunto(s)
Oligosacáridos/análisis , Oligosacáridos/química , Animales , Humanos , Espectrometría de Masas/métodos , Resinas de Plantas , Sensibilidad y EspecificidadRESUMEN
Oligosaccharyltransferase (OT) in Saccharomyces cerevisiae is an enzyme complex consisting of 8 transmembrane proteins located in the endoplasmic reticulum (ER). Studies on potential protein-protein interactions in OT using a two-hybrid library screen revealed that protein kinase C (Pkc1p) interacted with the lumenal domains of several OT subunits. Additional genetic experiments revealed that overexpression of two OT subunits rescued the growth defect caused by overexpression of a Pkc1 active site mutant, implying that there are specific genetic interactions between PKC1 and OT. These in vivo findings were complemented by in vitro studies that showed that several of the OT subunits bound to a fusion protein consisting of glutathione S-transferase linked via its C-terminus to Pkc1p. Assays of OT activity, in which glycosylation of a simple acceptor peptide was assayed in microsomes from wild-type and a pkc1 null revealed a 50% reduction in activity in the microsomes from the null strain. In contrast, strains containing null mutations of two other genes known to be downstream of Pkc1p in the PKC1-MAP kinase pathway had a level of OT activity comparable to that of wild-type cells. These in vivo and in vitro experiments suggest that in yeast cells Pkc1p may be involved in regulation of the N-glycosylation of proteins.
Asunto(s)
Retículo Endoplásmico/metabolismo , Hexosiltransferasas , Proteínas de la Membrana/metabolismo , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Transferasas/metabolismo , Asparagina/metabolismo , Pared Celular/metabolismo , Proteínas Fúngicas/metabolismo , Glicosilación , Sistema de Señalización de MAP Quinasas , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Transferasas/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
It has been proposed that cytoplasmic peptide:N-glycanase (PNGase) may be involved in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not correctly fold in the ER. However, a lack of information about the structure of the enzyme has limited our ability to obtain insight into its precise biological function. A PNGase-defective mutant (png1-1) was identified by screening a collection of mutagenized strains for the absence of PNGase activity in cell extracts. The PNG1 gene was mapped to the left arm of chromosome XVI by genetic approaches and its open reading frame was identified. PNG1 encodes a soluble protein that, when expressed in Escherichia coli, exhibited PNGase activity. PNG1 may be required for efficient proteasome-mediated degradation of a misfolded glycoprotein. Subcellular localization studies indicate that Png1p is present in the nucleus as well as the cytosol. Sequencing of expressed sequence tag clones revealed that Png1p is highly conserved in a wide variety of eukaryotes including mammals, suggesting that the enzyme has an important function.
Asunto(s)
Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Secuencia Conservada , Amidohidrolasas/análisis , Carboxipeptidasas/genética , Carboxipeptidasas/metabolismo , Catepsina A , Núcleo Celular/enzimología , Mapeo Cromosómico/normas , Cromosomas Fúngicos , Clonación Molecular , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Citosol/enzimología , Eliminación de Gen , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutación/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Complejo de la Endopetidasa Proteasomal , Control de Calidad , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Solubilidad , TemperaturaRESUMEN
Differentially expressed integrins have been shown to be involved in the intricate cell movements that occur during early development. Because the migration and movement of cells have been well characterized in sea urchin embryos, we searched for alpha-integrin subunits in this organism. An alpha integrin subunit, alphaP, was cloned from Strongylocentrotus purpuratus mesenchyme blastula stage mRNA by RT-PCR and RACE and found to exhibit 74-77% sequence similarity to mammalian alpha(5), alpha(8), alpha(IIb), and alpha(v) integrin. The 8-kb transcript was most abundant at the prism stage, although low levels could be detected at all stages by Northern blot analysis and RT-PCR. A polyclonal antibody to this novel integrin was generated against a 100-amino-acid alphaP fragment fused to glutathione S-transferase and shown to recognize a 180-kDa alpha-integrin in the egg and in all stages of embryogenesis studied.
Asunto(s)
Cadenas alfa de Integrinas , Integrinas/análisis , Integrinas/genética , Erizos de Mar/embriología , Erizos de Mar/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Blastocisto/química , Blastocisto/metabolismo , Western Blotting , Clonación Molecular , Embrión no Mamífero/química , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Sueros Inmunes/inmunología , Integrinas/química , Integrinas/inmunología , Datos de Secuencia Molecular , Peso Molecular , Óvulo/química , Óvulo/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Erizos de Mar/química , Erizos de Mar/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT), which catalyzes the transfer of dolichol-linked oligosaccharide chains to nascent polypeptides in the endoplasmic reticulum, consists of nine nonidentical membrane protein subunits. Genetic and biochemical evidence indicated these nine proteins exist in three subcomplexes. Three of the OT subunits (Ost4p, Ost3p, and Stt3p) have been proposed to exist in one subcomplex. To investigate the interaction of these three membrane proteins, initially we carried out a mutational analysis of Ost4p, which is an extraordinarily small membrane protein containing only 36 amino acid residues. This analysis indicated that when single amino acid residues in a region close to the luminal face of the putative transmembrane domain of Ost4p were changed into an ionizable amino acid such as Lys or Asp, growth at 37 degrees C and OT activity measured in vitro were impaired. In addition, using immunoprecipitation techniques and Western blot analysis, we found that with these mutations the interaction between Ost4p, Ost3p, and Stt3p was disrupted. Introduction of Lys or Asp residues at other positions in the putative transmembrane domain or at the N or C terminus of Ost4p had no effect on disrupting subunit interactions or impairing the activity of OT. These findings suggest that a localized region of the putative transmembrane domain of Ost4p mediates in stabilization of the interaction with the two other OT subunits (Ost3p and Stt3p) in a subcomplex in the endoplasmic reticulum membrane.
Asunto(s)
Proteínas Fúngicas/metabolismo , Hexosiltransferasas , Saccharomyces cerevisiae/enzimología , Transferasas/metabolismo , Secuencia de Aminoácidos , Retículo Endoplásmico/enzimología , Immunoblotting , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Fenotipo , Estructura Secundaria de Proteína , Transferasas/genéticaAsunto(s)
Microdominios de Membrana/fisiología , Erizos de Mar/fisiología , Transducción de Señal/fisiología , Interacciones Espermatozoide-Óvulo/fisiología , Animales , Adhesión Celular , Proteínas del Huevo/química , Femenino , Glicoesfingolípidos/análisis , Sustancias Macromoleculares , Masculino , Microdominios de Membrana/química , Estructura Terciaria de Proteína , Espermatozoides/química , Espermatozoides/ultraestructuraRESUMEN
The low density, detergent-insoluble membrane fraction (LD-DIM), where gangliosides are likely to be highly enriched, was prepared from sperm of two sea urchin species, Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus. Immunoblotting showed the presence in the LD-DIM of two receptors for egg ligands, a glycosylphosphatidylinositol (GPI)-anchored protein, and four proteins which may be involved in signal transduction. Co-immunoprecipitation revealed that at least three proteins, the speract receptor, the 63kDa GPI-anchored protein and the alpha subunit of a heterotrimeric Gs protein, are localized in the LD-DIM. This suggests that the LD-DIM fraction may be a membrane microdomain for speract-speract receptor interaction, as well as the subsequent signal transduction pathway involved in induction of sperm respiration, motility and possibly the acrosome reaction.
Asunto(s)
Detergentes/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Glicoesfingolípidos/metabolismo , Receptores de Superficie Celular/metabolismo , Espermatozoides/química , Animales , Western Blotting , Secuencia de Carbohidratos , Membrana Celular/química , Membrana Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Gangliósidos/inmunología , Gangliósidos/metabolismo , Glicoesfingolípidos/química , Masculino , Datos de Secuencia Molecular , Pruebas de Precipitina , Erizos de Mar , Solubilidad , Espermatozoides/metabolismoRESUMEN
A "quality control" system associated with the endoplasmic reticulum (ER) that discriminates between misfolded proteins and correctly folded proteins is present in a variety of eukaryotic cells, including yeast. Recently, it has been shown that misfolded proteins that are N -glycosylated in the lumen of the ER are transported out of the ER, de-N-glycosylated by a soluble peptide: N -glycanase (PNGase) and degraded by action of the proteasome. It also has been shown that small N -glycosylatable peptides follow a fate similar to that of misfolded proteins, i.e., glycosylation in the lumen of the ER, transport out of the ER, and de- N -glycosylation in the cytosol. These processes of retrograde glycopeptide transport and de- N -glycosylation have been observed in mammalian cells, as well as in yeast cells. However, little is known about the mechanism involved in the movement of glycopeptides from the ER to the cytosol. Here we report a simple method for assaying N -glycosylation/de- N -glycosylation by simple paper chromatographic and electrophoretic techniques using an N -glycosylatable(3)H-labeled tripeptide as a substrate. With this method, we confirmed the cytosolic localization of the de- N -glycosylated peptide, which supports the idea that de- N -glycosylation occurs after the export of the glycopeptide from the lumen of the ER to the cytosol. Further, we found that the variations in the structure of the oligosaccharide chain on the glycopeptide did not cause differences in the export of the glycopeptide. This finding suggests that the mechanism for the export of small glycopeptides may differ from that of misfolded (glyco)proteins.
Asunto(s)
Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Glicopéptidos/metabolismo , Oligosacáridos/química , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/farmacología , Transporte Biológico , Conformación de Carbohidratos , Cromatografía en Papel , Electroforesis en Papel , Inhibidores Enzimáticos/farmacología , Glicopéptidos/química , Glicósido Hidrolasas/antagonistas & inhibidores , GlicosilaciónRESUMEN
The attachment of N-linked oligosaccharide chains to proteins is an important cotranslational process. These chains can, in some cases, serve to stabilize the protein, while in other cases they function as recognition elements. A key enzyme in the N-glycosylation process is oligosaccharyltransferase (OT). In yeast this enzyme, which is found in the endoplasmic reticulum, consists of nine different transmembrane protein subunits. Our general aim is to learn more about the functions of the multiple subunits of yeast OT and their mode of interaction with each other. Using a combination of biochemical and genetic techniques the subunit Ost1p has been shown to recognize Asn-X-Ser/Thr glycosylation sites. The principle tool used in the identification process was a benzophenone-based glycosylation site peptide that was shown to be crosslinked to Ost1p. Our current objective is to identify the domain in the primary structure that is involved in recognition of the glycosylation site sequence. By use of bifunctional crosslinkers, the possible interaction of Ost1p with other subunits of OT will be studied. This work and other studies on the OT subunits are concisely summarized.
Asunto(s)
Retículo Endoplásmico/enzimología , Hexosiltransferasas , Proteínas de la Membrana , Transferasas/metabolismo , Especificidad por SustratoRESUMEN
Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.
Asunto(s)
Cisteína/metabolismo , Retículo Endoplásmico/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteínas/metabolismo , Transporte Biológico/genética , Citosol/metabolismo , Citosol/fisiología , Retículo Endoplásmico/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Microsomas/metabolismo , Chaperonas Moleculares/metabolismo , Mutagénesis Sitio-Dirigida , Péptidos/metabolismo , Unión Proteica/genética , Proteína Disulfuro Isomerasas/biosíntesis , Proteína Disulfuro Isomerasas/genética , Pliegue de Proteína , Proteínas/genética , Canales de Translocación SEC , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Eliminación de Secuencia/genética , Especificidad por Sustrato/genéticaRESUMEN
The low density detergent-insoluble membrane (LD-DIM) fraction was obtained by a sucrose-density gradient centrifugation from sperm of three sea urchin species, Hemicentrotus pulcherrimus, Strongylocentrotus purpuratus, and Anthocidaris crassispina. These LD-DIM preparations were characterized by enriched glycosphingolipids (GSL) including gangliosides and sulfatide (SLF), having more than 50% of the total amount of GSL present in these sperm. Interestingly, a minor component of H. pulcherrimus sperm (HO3S-->8Neu5Acalpha2-->8Neu5Acalpha2-->6Glcbeta1++ +-->Cer) was shown to be even more enriched in the LD-DIM as revealed by using monoclonal antibody (mAb.3G9) speific to this ganglioside. In addition to the GSL, phosphatidyl-serine (PS) and diacylglcerol (DG) were enriched in the LD-DIM. On the other hand, cholesterol (CL) and sphingomyelin (SM) were not so enriched, which contrasted with the LD-DIM from Madin-Darby canine kidney (MDCK) cells, where CL and SM were reported to be abundant. Because mammalian somatic cell-derived DIMs have been proposed to be associated with functional signal transduction, it seems possible that the ganglioside-enriched LD-DIM in sea urchin sperm can participate in binding to eggs and the subsequent egg activation process. To our knowledge this is the chemical characterization of the LD-DIM fraction of a gametic cell.
Asunto(s)
Lípidos de la Membrana/aislamiento & purificación , Espermatozoides/química , Animales , Secuencia de Carbohidratos , Línea Celular , Cromatografía en Capa Delgada , Detergentes , Perros , Gangliósidos/química , Gangliósidos/aislamiento & purificación , Masculino , Lípidos de la Membrana/química , Datos de Secuencia Molecular , Erizos de Mar , SolubilidadRESUMEN
Other laboratories have established that oligosaccharyl transferase (OST) from Saccharomyces cerevisiae can be purified as a protein complex containing eight different subunits. To identify the OST subunit that recognizes the peptide sites that can be glycosylated, we developed photoaffinity probes containing a photoreactive benzophenone derivative, p-benzoylphenylalanine (Bpa), as part of an 125I-labeled peptide that could be expected to be glycosylated. We found that Asn-Bpa-Thr peptides served as substrates for OST and that photoactivation of these probes in the presence of microsomes abolished the OST activity. Photoactivation of 125I-labeled Asn-Bpa-Thr in the presence of microsomes resulted in specific covalent labeling of a protein doublet of molecular mass 62 and 64 kDa. By carrying out the photoactivation of the probe using microsomes containing epitope-tagged Ost1p, we demonstrated that the 125I-labeled protein was Ost1p. Radiolabeling of this protein was dependent on irradiation at 350 nm. No labeling was detected using a probe containing Ala instead of Thr as the third amino acid residue. We conclude that Ost1p is the subunit of the OST complex that recognizes the peptide sites in the nascent chains that are destined to be glycosylated.
Asunto(s)
Proteínas Fúngicas/metabolismo , Hexosiltransferasas , Proteínas de la Membrana , Oligopéptidos/metabolismo , Saccharomyces cerevisiae/enzimología , Transferasas/metabolismo , Secuencia de Bases , Secuencia de Consenso , Cartilla de ADN , Proteínas Fúngicas/química , Glicosilación , Microsomas/enzimología , Microsomas/efectos de la radiación , Oligopéptidos/química , Fotoquímica , Especificidad por Sustrato , Transferasas/químicaRESUMEN
Little is known about sperm-binding proteins in the egg envelope of nonmammalian vertebrate species. We report here the molecular cloning and characterization of a recently identified sperm receptor (gp69/64) in the Xenopus laevis egg vitelline envelope. Our data indicate that the gp69 and gp64 glycoproteins are two glycoforms of the receptor and have the same number of N-linked oligosaccharide chains but differ in the extent of O-glycosylation. The amino acid sequence of the receptor is closely related to that of the mouse zona pellucida protein ZP2. Most of the sequence conservation, including a ZP domain, a potential furin cleavage site, and a putative transmembrane domain are located in the C-terminal half of the receptor. Proteolytic cleavage of the gp69/64 protein by a cortical granule protease during fertilization removes 27 amino acid residues from the N terminus of gp69/64 and results in loss of sperm binding to the activated eggs. Similarly, we find that treatment of eggs with type I collagenase removes 31 residues from the N terminus of gp69/64 and has the same effect on sperm binding. The isolated and purified N terminus-truncated receptor protein is inactive as an inhibitor of sperm-egg binding. Earlier studies on the effect of Pronase digestion on receptor activity suggest that this N-terminal peptide may contain an O-linked glycan that is involved in the binding process. Based on these results and the findings on the primary structure of the receptor, a pathway for the maturation and secretion of gp69/64, as well as its inactivation following fertilization, is proposed.