RESUMEN
Protein mannosyltransferases (Pmt proteins) initiate O glycosylation of secreted proteins in fungi. We have characterized PMT6, which encodes the second Pmt protein of the fungal pathogen Candida albicans. The residues of Pmt6p are 21 and 42% identical to those of C. albicans Pmt1p and S. cerevisiae Pmt6p, respectively. Mutants lacking one or two PMT6 alleles grow normally and contain normal Pmt enzymatic activities in cell extracts but show phenotypes including a partial block of hyphal formation (dimorphism) and a supersensitivity to hygromycin B. The morphogenetic defect can be suppressed by overproduction of known components of signaling pathways, including Cek1p, Cph1p, Tpk2p, and Efg1p, suggesting a specific Pmt6p target protein upstream of these components. Mutants lacking both PMT1 and PMT6 are viable and show pmt1 mutant phenotypes and an additional sensitivity to the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The lack of Pmt6p significantly reduces adherence to endothelial cells and overall virulence in a mouse model of systemic infection. The results suggest that Pmt6p regulates a more narrow subclass of proteins in C. albicans than Pmt1p, including secreted proteins responsible for morphogenesis and antifungal sensitivities.
Asunto(s)
Candida albicans/fisiología , Candida albicans/patogenicidad , Manosiltransferasas/genética , Alelos , Animales , Antifúngicos , Candida albicans/citología , Adhesión Celular , Diferenciación Celular , Clonación Molecular , Farmacorresistencia Microbiana , Genes Fúngicos , Ratones , Datos de Secuencia Molecular , Morfogénesis , Mutación , Procesamiento Proteico-Postraduccional , Análisis de Secuencia de ADN , Supresión GenéticaRESUMEN
Protein O-mannosylation is an essential protein modification. It is initiated at the endoplasmic reticulum by a family of dolichyl phosphate-mannose:protein O-mannosyltransferases (Pmts), which is evolutionarily conserved from yeast to humans. Saccharomyces cerevisiae Pmt1p is an integral membrane protein of the endoplasmic reticulum. ScPmt1p forms a complex with ScPmt2p that is required for maximum transferase activity. Recently, we proposed a seven-transmembrane structural model for ScPmt1p. A large, hydrophilic, endoplasmic reticulum-oriented segment is flanked by five amino-terminal and two carboxyl-terminal membrane-spanning domains. Based on this model, a structure-function analysis of ScPmt1p was performed. Deletion mutagenesis identified the N-terminal third of the transferase as being essential for the formation of a functional ScPmt1p-ScPmt2p complex. Deletion of the central hydrophilic loop eliminates mannosyltransferase activity, but not ScPmt1p-ScPmt2p interactions. Alignment of all fully characterized PMT family members revealed that this central loop region contains three highly conserved peptide motifs, which can be considered as signatures of the PMT family. In addition, a number of invariant amino acid residues were identified throughout the entire protein sequence. In order to evaluate the functional significance of these conserved residues site-directed mutagenesis was performed. We show that several amino acid substitutions in the conserved motifs significantly reduce ScPmt1p activity. Further, the invariant residues Arg-64, Glu-78, Arg-138, and Leu-408 are essential for ScPmt1p function. In particular, Arg-138 is crucial for ScPmt1p-ScPmt2p complex formation.
Asunto(s)
Manosiltransferasas/química , Manosiltransferasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Manosiltransferasas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The incorporation of radioactive orthophosphate into the cell walls of Saccharomyces cerevisiae was studied. 33P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N-glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368-3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by DAN1 [also called TIR3]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154-2162, 1997). In ccw12, ccw13, or ccw14 single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the 33P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, the ccw12 mutant shows a decrease in mating efficiency and a defect in agglutination.
Asunto(s)
Pared Celular/metabolismo , Proteínas Fúngicas/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia , Secuencia de Aminoácidos , Bencenosulfonatos/farmacología , Cromatografía Liquida , Rojo Congo/farmacología , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Glucano Endo-1,3-beta-D-Glucosidasa/metabolismo , Glicoproteínas/aislamiento & purificación , Espectrometría de Masas , Metilación , Mutación , Oligosacáridos/análisis , Oligosacáridos/aislamiento & purificación , Oligosacáridos/metabolismo , Fenotipo , Fosfatos/metabolismo , Fosforilación , Polisacáridos/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de SecuenciaRESUMEN
The identification of the evolutionarily conserved family of dolichyl-phosphate-D-mannose:protein O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential role in a number of physiologically important processes. Strikingly, all members of the Pmt protein family share almost identical hydropathy profiles; a central hydrophilic domain is flanked by amino- and carboxyl-terminal sequences containing several putative transmembrane helices. This pattern is of particular interest because it diverges from structural models of all glycosyltransferases characterized so far. Here, we examine the transmembrane topology of Pmt1p, an integral membrane protein of the endoplasmic reticulum, from Saccharomyces cerevisiae. Structural predictions were directly tested by site-directed mutagenesis of endogenous N-glycosylation sites, by fusing a topology-sensitive monitor protein domain to carboxyl-terminal truncated versions of the Pmt1 protein and, in addition, by N-glycosylation scanning. Based on our results we propose a seven-transmembrane helical model for the yeast Pmt1p mannosyltransferase. The Pmt1p amino terminus faces the cytoplasm, whereas the carboxyl terminus faces the lumen of the endoplasmic reticulum. A large hydrophilic segment that is oriented toward the lumen of the endoplasmic reticulum is flanked by five amino-terminal and two carboxyl-terminal membrane spanning domains. We could demonstrate that this central loop is essential for the function of Pmt1p.
Asunto(s)
Manosiltransferasas/química , Saccharomyces cerevisiae/enzimología , División Celular/genética , Retículo Endoplásmico/enzimología , Evolución Molecular , Proteínas Fúngicas/química , Glicosilación , Manosiltransferasas/genética , Proteínas de la Membrana/química , Mutagénesis Sitio-Dirigida , Mutación/genética , Fenotipo , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Protein O-mannosylation, originally observed in fungi, starts at the endoplasmic reticulum with the transfer of mannose from dolichyl activated mannose to seryl or threonyl residues of secretory proteins. This reaction is catalyzed by a family of protein O-mannosyltransferases (PMTs), which were first characterized in Saccharomyces cerevisiae. The identification of this evolutionarily conserved PMT gene family has led to the finding that protein O-mannosylation plays an essential role in a number of physiologically important processes. Focusing on the PMT gene family, we discuss here the main aspects of the biogenesis of O-linked carbohydrate chains in S. cerevisiae, Candida albicans, and other fungi. We summarize recent work utilizing pmt mutants that demonstrates the impact of protein O-mannosylation on protein secretion, on maintenance of cell wall integrity, and on budding. Further, the occurrence of PMT orthologs in higher eukaryotes such as Arabidopsis, Drosophila and mammals is reported and discussed.
Asunto(s)
Manosiltransferasas/metabolismo , Proteoglicanos/biosíntesis , Saccharomyces cerevisiae/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Membranas Intracelulares/enzimología , Manosiltransferasas/química , Manosiltransferasas/genética , Oxígeno/química , Proteoglicanos/química , Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
Protein mannosylation by Pmt proteins initiates O-glycosylation in fungi. We have identified the PMT1 gene and analyzed the function of Pmt1p in the fungal human pathogen Candida albicans. Mutants defective in PMT1 alleles lacked Pmt in vitro enzymatic activity, showed reduced growth rates, and tended to form cellular aggregates. In addition, multiple specific deficiencies not known in Saccharomyces cerevisiae (including defective hyphal morphogenesis; supersensitivity to the antifungal agents hygromycin B, G418, clotrimazole, and calcofluor white; and reduced adherence to Caco-2 epithelial cells) were observed in pmt1 mutants. PMT1 deficiency also led to faster electrophoretic mobility of the Als1p cell wall protein and to elevated extracellular activities of chitinase. Homozygous pmt1 mutants were avirulent in a mouse model of systemic infection, while heterozygous PMT1/pmt1 strains showed reduced virulence. The results indicate that protein O-mannosylation by Pmt proteins occurs in different fungal species, where PMT1 deficiency can lead to defects in multiple cellular functions.
Asunto(s)
Candida albicans/enzimología , Proteínas Fúngicas/metabolismo , Manosa/metabolismo , Manosiltransferasas/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Candida albicans/crecimiento & desarrollo , Candida albicans/patogenicidad , Cartilla de ADN , Prueba de Complementación Genética , Humanos , Masculino , Manosiltransferasas/química , Manosiltransferasas/genética , Ratones , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas , VirulenciaRESUMEN
In wild-type budding yeast strains, the proteins encoded by SIR3, SIR4 and RAP1 co-localize with telomeric DNA in a limited number of foci in interphase nuclei. Immunostaining of Sir2p shows that in addition to a punctate staining that coincides with Rap1 foci, Sir2p localizes to a subdomain of the nucleolus. The presence of Sir2p at both the spacer of the rDNA repeat and at telomeres is confirmed by formaldehyde cross-linking and immunoprecipitation with anti-Sir2p antibodies. In strains lacking Sir4p, Sir3p becomes concentrated in the nucleolus, by a pathway requiring SIR2 and UTH4, a gene that regulates life span in yeast. The unexpected nucleolar localization of Sir2p and Sir3p correlates with observed effects of sir mutations on rDNA stability and yeast longevity, defining a new site of action for silent information regulatory factors.
Asunto(s)
Nucléolo Celular/ultraestructura , ADN Ribosómico , Proteínas de Unión al ADN/aislamiento & purificación , Histona Desacetilasas , Saccharomyces cerevisiae/ultraestructura , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Telómero , Transactivadores/aislamiento & purificación , Anticuerpos Antifúngicos , Especificidad de Anticuerpos , Compartimento Celular , Nucléolo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Proteínas Fúngicas/aislamiento & purificación , Proteínas de Unión al GTP/inmunología , Proteínas de Unión al GTP/aislamiento & purificación , Modelos Biológicos , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Saccharomyces cerevisiae/genética , Sirtuina 2 , Sirtuinas , Transactivadores/genética , Transactivadores/inmunología , Proteínas de Unión al GTP rapRESUMEN
Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect. SIR3 overproduction can extend the silenced domain. We examine here the nature of these multiprotein complexes. SIR2 and SIR4 were immunoprecipitated from whole-cell extracts. In addition, using formaldehyde cross-linking we have mapped SIR2, SIR4, and RAP1 along telomeric chromatin before and after SIR3 overexpression. Our data demonstrate that SIR2 and SIR4 interact in a protein complex and that SIR2, SIR3, SIR4, and RAP1 map to the same sites along telomeric heterochromatin in wild-type cells. However, when overexpressed, SIR3 spreads along the chromosome and its interactions are dominant to those of SIR4 and especially SIR2, whose detection is decreased in extended heterochromatin. RAP1 binding at the core region is unaffected by SIR3 overproduction and RAP1 shows no evidence of spreading. Thus, we propose that the structure of core telomeric heterochromatin differs from that extended by SIR3.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Heterocromatina/metabolismo , Histona Desacetilasas , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Telómero/metabolismo , Transactivadores/metabolismo , Western Blotting , Extractos Celulares/química , Reactivos de Enlaces Cruzados/metabolismo , Proteínas de Unión al ADN/inmunología , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/inmunología , Proteínas de Unión al GTP/inmunología , Proteínas de Unión al GTP/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Histonas/inmunología , Histonas/metabolismo , Modelos Genéticos , Mutación/genética , Pruebas de Precipitina , Unión Proteica , Sirtuina 2 , Sirtuinas , Telómero/genética , Transactivadores/inmunología , Proteínas de Unión al GTP rapRESUMEN
Telomeric genes and the HM loci in saccharomyces cerevisiae are transcriptionally repressed and adopt a heterochromatin-like structure. The trans-acting factors RAP1, SIR3 and SIR4 are required for telomeric and HM silencing, and are thought to be chromosomal, but how they contribute to histone-dependent repression of adjacent chromatin is unclear. SIR3 suppresses silencing defects in histones, is limiting for silencing adjacent to telomeres, and interacts with the H3 and H4 amino termini in vitro. Here we show that SIR3 co-immunoprecipitates SIR4, RAP1 and histones from cellular extracts, suggesting the presence of large chromatin-associated protein complexes. Crosslinking experiments show that SIR3 is present at HMRa, HMLalpha and telomeres in vivo, and that is spreads from telomeric regions into adjacent chromatin when overexpressed. Thus SIR3 is a structural component of yeast heterochromatin, repressing adjacent genes as it spreads along the chromosome.
Asunto(s)
Proteínas Fúngicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Telómero/metabolismo , Transactivadores/metabolismo , Cromosomas Fúngicos/metabolismo , Reactivos de Enlaces Cruzados , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Histonas/genética , Histonas/metabolismo , Mutación , Pruebas de Precipitina , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rapRESUMEN
The deletion of the protein mannosyltransferase 1 gene (PMT1) of Saccharomyces cerevisiae results in viable cells. O-Mannosylation of proteins is reduced to about half of the value in comparison to wild-type cells. In order to distinguish between the the PMT1 gene product (= Pmt1p) and residual transferase activity, an in vitro assay to measure Dol-P-Man:protein mannosyltransferase activity in cells deleted for PMT1 has been developed. The transferase activity of these cells exhibits a pH optimum of 6.5 as compared to pH 7.5 for Pmt1p. The Km value of the residual enzyme activity for the hexapeptide YNPTSV is 7 times higher than that of Pmt1p and shows a clear preference for the seryl residue. Differences in substrate affinities as well as in seryl/threonyl depend on the specific sequence of the peptides used in the enzyme assay. The new enzyme activity shows a significantly lower thermal stability as compared to Pmt1p.
Asunto(s)
Isoenzimas/metabolismo , Manosiltransferasas/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Membrana Celular/enzimología , Eliminación de Gen , Genes Fúngicos , Cinética , Manosiltransferasas/genética , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Especificidad por Sustrato , TermodinámicaRESUMEN
The silent mating loci and chromosomal regions adjacent to telomeres of S. cerevisiae have features similar to heterochromatin of more complex eukaryotes. Transcriptional repression at these sites depends on the silent information regulators SIR3 and SIR4 as well as histones H3 and H4. We show here that the SIR3 and SIR4 proteins interact with specific silencing domains of the H3 and H4 N-termini in vitro. Certain mutations in these factors, which affect their silencing functions in vivo, also disrupt their interactions in vitro. Immunofluorescence studies with antibodies against RAP1 and SIR3 demonstrate that the H3 and H4 N-termini are required for the association of SIR3 with telomeric chromatin and the perinuclear positioning of yeast telomeres. Based on these interactions, we propose a model for heterochromatin-mediated transcriptional silencing in yeast, which may serve as a paradigm for other eukaryotic organisms as well.
Asunto(s)
Proteínas Fúngicas/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Transactivadores/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Compartimento Celular , Núcleo Celular/ultraestructura , Análisis Mutacional de ADN , Técnica del Anticuerpo Fluorescente , Regulación Fúngica de la Expresión Génica , Heterocromatina/ultraestructura , Histonas/genética , Factor de Apareamiento , Modelos Moleculares , Datos de Secuencia Molecular , Biosíntesis de Péptidos , Péptidos/genética , Relación Estructura-Actividad , Telómero/ultraestructuraRESUMEN
The yeast cell wall as a good antifungal target is discussed in general. More specifically the reaction, catalyzed by Dol-P-Man: protein O-D-mannosyltransferase is proposed as a new potential target. Six genes responsible for this endoplasmic reticulum-localized reaction have been cloned and characterized so far. Triple disruptions of these genes are either lethal or the corresponding cells have to be osmotically stabilized to survive. No inhibitors of this reaction are as yet known.
Asunto(s)
Antifúngicos/farmacología , Glicoproteínas/biosíntesis , Saccharomyces cerevisiae/metabolismo , Pared Celular/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
Yeast chromosomes may lack the linker histone H1 (normally required to compact 10 nm beads-on-a-string fiber into the 30 nm fiber) and there is no cytological evidence for higher order fiber structure but they do contain regions which correspond to euchromatin and heterochromatin of higher eukaryotes. Both euchromatin and heterochromatin contain nucleosomal particles (composed of two molecules each of H2A, H2B, H3 and H4), however histones have been shown to regulate genes in these regions in quite different ways. The mechanisms by which such regulation occurs are the topic of this paper.
Asunto(s)
Cromatina/fisiología , Regulación Fúngica de la Expresión Génica/genética , Heterocromatina/fisiología , Histonas/fisiología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Eucromatina , Datos de Secuencia MolecularRESUMEN
The integral endoplasmic reticulum membrane protein catalyzing the initial reaction of protein O-glycosylation in Saccharomyces cerevisiae has been purified to homogeneity. The 92-kDa N-glycosylated protein transfers mannose residues from dolichyl phosphate-D-mannose to specific serine/threonine residues of proteins entering the secretory pathway. This type of mannosyl transfer reaction has so far been observed only in fungal cells. Oligonucleotides derived from peptide sequences of the transferase were used to screen a genomic yeast library. A clone was isolated which contains an open reading frame of 2451 bp corresponding to an mRNA transcript of 3 kb. The predicted protein consists of 817 amino acids including three potential N-glycosylation sites. The hydropathy plot indicates a tripartite structure of the protein: an amino-terminal third and a carboxyl-terminal third, both with multiple potential transmembrane helices, and a central hydrophilic part. Expression of the clone in Escherichia coli resulted in mannosyltransferase activity. Gene disruption led to a complete loss of in vitro mannosyltransferase activity from dolichyl phosphate-D-mannose to a peptide used as acceptor in the enzymatic assay. In vivo it was observed, however, that protein O-mannosylation in the disruptant had decreased only to about 40-50%, indicating the existence of an additional transferase which had not been measured by the in vitro enzyme assay.
Asunto(s)
Genes Fúngicos , Manosiltransferasas/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Retículo Endoplásmico/enzimología , Escherichia coli/genética , Expresión Génica , Glicosilación , Manosiltransferasas/aislamiento & purificación , Manosiltransferasas/metabolismo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , Conformación Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Especificidad por SustratoRESUMEN
An unknown open reading frame from Saccharomyces cerevisiae was identified and sequenced. The predicted amino acid sequence shows high homology to the DEAD-box family of proteins. Gene disruption revealed that the gene is not essential for yeast but necessary for normal cell growth.
Asunto(s)
Genes Fúngicos/genética , ARN Nucleotidiltransferasas/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Genoma Fúngico , Datos de Secuencia Molecular , Familia de Multigenes/genética , Mutagénesis Insercional , ARN Helicasas , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is O-glycosylated at residues Ser9 and Thr10 during secretion by yeast and COS-1 cells [Ernst, J.F., Mermod, J.-J. and Richman, L.I. (1992) Eur. J. Biochem. 203, 663-667]. Two types of octapeptides encompassing residues 4-11 (peptide 4-11) and variants thereof, or residues 8-15 (peptide 8-15) of hGM-CSF were tested as substrates for in vitro O-glycosylation using dolichyl-phosphate- D-mannose: protein O-D-mannosyltransferase (Man-transferase) of the yeast Saccharomyces cerevisiae, or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) of rat liver cells. Peptide 8-15 was found to be O-glycosylated at residues Ser9 and Thr10 by GalNAc-transferase and, with reduced efficiency, also by Man-transferase. Peptide 4-11 was a good substrate for yeast Man-transferase, leading to mannosylation of only Thr10, whereas it was very poorly O-glycosylated at positions Ser5 and Ser7 by GalNAc-transferase. The observed differences in peptide-acceptor activities indicate that the site of O-glycosylation depends on similar, but not identical protein structural features in yeast and mammalian cells.
Asunto(s)
Glicosiltransferasas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Hígado/enzimología , Oligopéptidos/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Animales , Glicosilación , Humanos , Masculino , Manosiltransferasas/metabolismo , Datos de Secuencia Molecular , Ratas , Ratas EndogámicasRESUMEN
The enzyme dolichyl-phosphate-D-mannose:protein O-D-mannosyltransferase has been solubilized from Saccharomyces cerevisiae membranes and its mannosyltransferase activity demonstrated using short peptides. The specific activity of the protein was enriched 130-fold before it was further purified by native and SDS gel chromatography. A 92-kDa band correlated well with the enzyme activity; an antibody raised against this protein precipitated the mannosyltransferase. The 92-kDa band was hydrolysed to 84 kDa after treatment with endoglycosidase F, indicating that the protein is a glycoprotein which may contain four carbohydrate chains. The purified mannosyltransferase is distinctly influenced in transfer specificity by amino acids next to serine and threonine within the acceptor peptides. Thus acidic amino acids strongly inhibit acceptor activity as do glycine and proline residues as amino-terminal and carboxy-terminal neighbours, respectively.