RESUMEN
Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three DeltaF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Omega.cm(2). In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1beta, TNF-alpha, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development.
Asunto(s)
Bronquios/citología , Técnicas de Cultivo de Célula/métodos , Fibrosis Quística/patología , Células Epiteliales/citología , Mucosa Respiratoria/citología , Adolescente , Adulto , Línea Celular Transformada , Senescencia Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Cámaras de Difusión de Cultivos , Células Epiteliales/metabolismo , Femenino , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Virus 40 de los Simios , Telomerasa/genéticaRESUMEN
Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Yeast mutants, defective at various steps of the secretory pathway, have been used to dissect the mechanisms of biosynthetic processing and intracellular transport of several proteins. To exploit these yeast mutants, we have employed an expression system in which the CFTR gene is driven by the promoter of a structurally related yeast ABC protein, Pdr5p. Pulse-chase experiments revealed a turnover rate similar to that of nascent CFTR in mammalian cells. Immunofluorescence microscopy showed that most CFTR colocalized with the endoplasmic reticulum (ER) marker protein Kar2p and not with a vacuolar marker. Degradation was not influenced by the vacuolar protease mutants Pep4p and Prb1p but was sensitive to the proteasome inhibitor lactacystin beta-lactone. Blocking ER-to-Golgi transit with the sec18-1 mutant had little influence on turnover indicating that it occurred primarily in the ER compartment. Degradation was slowed in cells deficient in the ER degradation protein Der3p as well as the ubiquitin-conjugating enzymes Ubc6p and Ubc7p. Finally a mutation (sec61-2) in the translocon protein Sec61p that prevents retrotranslocation across the ER membrane also blocked degradation. These results indicate that whereas approximately 75% of nascent wild-type CFTR is degraded at the ER of mammalian cells virtually all of the protein meets this fate on heterologous expression in Saccharomyces cerevisiae.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Saccharomyces cerevisiae/metabolismo , Cisteína Endopeptidasas/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Técnica del Anticuerpo Fluorescente , Glicosilación , Proteínas Fluorescentes Verdes , Humanos , Immunoblotting , Proteínas Luminiscentes/metabolismo , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Complejos Multienzimáticos/metabolismo , Mutación , Pruebas de Precipitina , Complejo de la Endopetidasa Proteasomal , Canales de Translocación SEC , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , TransfecciónRESUMEN
Some disease-associated truncations within the 100-residue domain C-terminal of the second nucleotide-binding domain destabilize the mature protein (Haardt, M., Benharouga, M., Lechardeur, D., Kartner, N., and Lukacs, G. L. (1999) J. Biol. Chem. 274, 21873-21877). We now have identified three short oligopeptide regions in the C-terminal domain which impact cystic fibrosis transmembrane conductance regulator (CFTR) maturation and stability in different ways. A highly conserved hydrophobic patch (region I) formed by residues 1413-1416 (FLVI) was found to be crucial for the stability of the mature protein. Nascent chain stability was severely decreased by shortening the protein by 81 amino acids (1400X). This accelerated degradation was sensitive to proteasome inhibitors but not influenced by brefeldin A, indicating that it occurred at the endoplasmic reticulum. The five residues at positions 1400 to 1404 (region II) normally maintain nascent CFTR stability in a positional rather than a sequence-specific manner. A third modulating region (III) constituted by residues 1390 to 1394 destabilizes the protein. Hence the nascent form regains stability on further truncation back to residues 1390 or 1380, permitting some degree of maturation and a low level of cyclic AMP-stimulated chloride channel activity at the cell surface. Thus while not absolutely essential, the C-terminal domain strongly modulates the biogenesis and maturation of CFTR.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/fisiología , Cricetinae , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Cells of the yeast Saccharomyces cerevisiae choose bud sites in a manner that is dependent upon cell type: a and alpha cells select axial sites; a/alpha cells utilize bipolar sites. Mutants specifically defective in axial budding were isolated from an alpha strain using pseudohyphal growth as an assay. We found that a and alpha mutants defective in the previously identified PMT4 gene exhibit unipolar, rather than axial budding: mother cells choose axial bud sites, but daughter cells do not. PMT4 encodes a protein mannosyl transferase (pmt) required for O-linked glycosylation of some secretory and cell surface proteins (Immervoll, T., M. Gentzsch, and W. Tanner. 1995. Yeast. 11:1345-1351). We demonstrate that Axl2/Bud10p, which is required for the axial budding pattern, is an O-linked glycoprotein and is incompletely glycosylated, unstable, and mislocalized in cells lacking PMT4. Overexpression of AXL2 can partially restore proper bud-site selection to pmt4 mutants. These data indicate that Axl2/Bud10p is glycosylated by Pmt4p and that O-linked glycosylation increases Axl2/ Bud10p activity in daughter cells, apparently by enhancing its stability and promoting its localization to the plasma membrane.
Asunto(s)
Polaridad Celular , Proteínas Fúngicas/metabolismo , Manosiltransferasas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Amidohidrolasas/metabolismo , División Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Polaridad Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Proteínas Fúngicas/análisis , Proteínas Fúngicas/genética , Expresión Génica , Genes Fúngicos/genética , Genes Fúngicos/fisiología , Glicosilación/efectos de los fármacos , Aparato de Golgi/metabolismo , Manosiltransferasas/genética , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Modelos Biológicos , Peso Molecular , Mutación , Oligosacáridos/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Fenotipo , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tunicamicina/farmacologíaRESUMEN
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
Intact Saccharomyces cerevisiae cells were biotinylated with the non-permeable sulfosuccinimidyl-6-(biotinamido) hexanoate reagent. Twenty specifically labelled cell wall proteins would be extracted and visualized on SDS gels via streptavidin/horseradish peroxidase. Nine cell wall proteins were released by SDS extraction under reducing conditions and were designated Scw1-9p for (soluble cell wall proteins); five proteins were released from SDS-extracted cell walls by laminarinase (Ccw1-5p for covalently linked cell wall proteins) and six with mild (30 mM-NaOH, 4 degrees C, 14 h) alkali treatment (Ccw6-11p). N-terminal sequences of the Ccw proteins 6, 7, 8 and 11 showed that these cell wall proteins are members of the PIR gene family (predicted proteins with internal repeats), CCW6 being identical to PIR1 and CCW8 to PIR3. Single gene disruptions of all four genes did not yield a phenotype. In the CCW11 disruption the Ccw11p as well as the laminarinase-extracted Ccw5 protein was missing. The new cell wall proteins are O-mannosylated, contain a Kex2 processing site, but no C-terminal GPI anchor sequence.
Asunto(s)
Biotina/metabolismo , Proteínas Fúngicas/metabolismo , Manosa/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Pared Celular/química , Proteínas Fúngicas/análisis , Glicosilación , Datos de Secuencia MolecularRESUMEN
S. cerevisiae contains at least six genes (PMT1-6) for dolicholphosphate-D-mannose: protein-O-D-mannosyltransferases. The in vivo mannosylation of seven O-mannosylated yeast proteins has been analyzed in a number of pmt mutants. The results clearly indicate that the various protein O-mannosyltransferases have different specificities for protein substrates. Five of the proteins tested (chitinase, a-agglutinin, Kre9p, Bar1p, Pir2p/hsp 150) are mainly underglycosylated in pmt1 and pmt2 mutants, whereby qualitative differences exist among the various proteins. Two of the O-mannosylated proteins (Ggp1p and Kex2p) are not at all affected in pmt1 and pmt2 mutants but are clearly underglycosylated when PMT4 is mutated. Although the PMT4 gene product is shown to be responsible for O-mannosylating a Ser-rich region of Ggp1p in vivo, a penta-seryl-peptide is not an in vitro substrate for this transferase. A PMT3 mutation does affect O-mannosylation of chitinase only in the genetic background of a pmt1pmt2 double mutation, indicating that PMT1 and PMT2 can compensate for a deleted PMT3 gene.
Asunto(s)
Proteínas Fúngicas/metabolismo , Manosiltransferasas/metabolismo , Saccharomyces cerevisiae/enzimología , Quitinasas/química , Quitinasas/metabolismo , Dimerización , Eliminación de Gen , Glicosilación , Manosa/metabolismo , Manosiltransferasas/química , Manosiltransferasas/genética , Mutación , Especificidad por SustratoRESUMEN
The transfer of mannose to seryl and threonyl residues of secretory proteins is catalyzed by a family of protein mannosyltransferases coded for by seven genes (PMT1-7). Mannose dolichylphosphate is the sugar donor of the reaction, which is localized at the endoplasmic reticulum. By gene disruption and crosses all single, double and triple mutants of genes PMT1-4 were constructed. Two of the double and three of the triple mutants were not able to grow under normal conditions; three of these mutants could grow, however, when osmotically stabilized. The various mutants were extensively characterized concerning growth, morphology and their sensitivity to killer toxin K1, caffeine and calcofluor white. O-Mannosylation of gp115/Gas1p was affected only in pmt4 mutants, whereas glycosylation of chitinase was mainly affected in pmt1 and pmt2 mutants. The results show that protein O-glycosylation is essential for cell wall rigidity and cell integrity and that this protein modification, therefore, is vital for Saccharomyces cerevisiae.
Asunto(s)
Genes Fúngicos , Manosiltransferasas/genética , Manosiltransferasas/metabolismo , Familia de Multigenes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glicosilación , Datos de Secuencia Molecular , Mutación , Fenotipo , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The protein O-mannosyltransferases Pmt1p and Pmt2p are catalyzing the O-glycosylation of serine and threonine residues in the endoplasmic reticulum of yeast. Deletion of each of these proteins by disruption of the corresponding gene leads to a dramatic decrease of mannosyltransferase activity in vitro. With an anti-Pmt1p immunoaffinity column a complex of Pmt1p and a second protein was purified; this protein turned out to be Pmt2p. Overexpression of Pmt1p or Pmt2p, respectively, does not increase mannosyltransferase activity in vitro. Overexpression of both mannosyltransferases together, however, raises in vitro activity threefold. These data indicate that Pmt1p and Pmt2p function as a complex catalyzing protein O-glycosylation in yeast.
Asunto(s)
Proteínas Fúngicas/metabolismo , Isoenzimas/metabolismo , Manosiltransferasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Glicosilación , Manosiltransferasas/genética , Manosiltransferasas/aislamiento & purificación , Datos de Secuencia Molecular , Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
Two genes PMT3 and PMT4 were identified by polymerase chain reaction of genomic DNA using primers derived from regions of high homology between the products of three genes PMT1, PMT2 of Saccharomyces cerevisiae and part of a PMT1 related sequence of Kluyveromyces lactis. Pmt1p and Pmt2p are mannosyltransferases involved in the transfer of a mannosyl residue from dolichyl phosphate-D-mannose (Dol-P-Man) to seryl and threonyl residues in proteins. The products encoded by the PMT3 and PMT4 genes have almost identical hydropathy profiles in comparison to PMT1 and PMT2: a hydrophobic N- and C-terminal third each with multiple potential transmembrane helices and a central hydrophilic part. The predicted Pmt3p contains 753 amino acids, four potential N-glycosylation sites and it is significantly homologous to Pmt1p, Pmt2p and Pmt4p. Pmt4p contains 762 amino acids and two potential N-glycosylation sites. Northern blot analysis showed a single mRNA transcript of PMT3 and PMT4 of 2.8 kb. Thus PMT3 and PMT4 are two new members of the PMT gene family. The pmt4 null mutant the pmt3 pmt4 double null mutant, but not pmt3 null mutant, showed a significant shift of chitinase due to under glycosylation of the enzyme. The triple disruption pmt2 pmt3 pmt4 and the quadruple disruption result in a lethal phenotype.
Asunto(s)
Genes Fúngicos/genética , Manosiltransferasas/genética , Familia de Multigenes/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Quitinasas/metabolismo , Clonación Molecular , Glicosilación , Manosiltransferasas/metabolismo , Datos de Secuencia Molecular , Mutación , ARN de Hongos/análisis , ARN Mensajero/análisis , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de AminoácidoRESUMEN
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 PMT2 gene from Saccharomyces cerevisiae was identified as FUN25, a transcribed open reading frame on the left arm of chromosome I (Ouellette, B. F. F., Clark, M. W. C., Keng, T., Storms, R. G., Zhong, W., Zeng, B., Fortin, N., Delaney, S., Barton, A., Kaback, D.B., and Bussey, H. (1993) Genome 36, 32-42). The product encoded by the PMT2 gene shows significant similarity with the dolichyl phosphate-D-mannose:protein O-D-mannosyltransferase, Pmt1p (EC 2.4.1.109), which is required for initiating the assembly of O-linked oligosaccharides in S. cerevisiae (Strahl-Bolsinger, S., Immervoll, T., Deutzmann, R., and Tanner, W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8164-8168). The PMT2 gene encodes a new protein O-D-mannosyltransferase. Yeast cells carrying a PMT2 disruption show a diminished in vitro and in vivo O-mannosylation activity and resemble mutants with a nonfunctional PMT1 gene. Strains bearing a pmt1 pmt2 double disruption show a severe growth defect but retain residual O-mannosylation activity indicating the presence of at least one more protein-O-mannosyltransferase.
Asunto(s)
Proteínas Fúngicas/genética , Manosiltransferasas/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Cromosomas Fúngicos , Proteínas Fúngicas/metabolismo , Ligamiento Genético , Glicosilación , Manosiltransferasas/metabolismo , Datos de Secuencia Molecular , Oligosacáridos/química , Mutación Puntual , Homología de Secuencia de AminoácidoRESUMEN
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.