RESUMEN
We have isolated a major protein constituent from a highly enriched fraction of yeast nuclear pore complexes (NPCs). The gene encoding this protein, Nup188p, was cloned, sequenced, and found to be nonessential upon deletion. Nup188p cofractionates with yeast NPCs and gives an immunofluorescent staining pattern typical of nucleoporins. Using immunoelectron microscopy, Nup188p was shown to localize to both the cytoplasmic and nucleoplasmic faces of the NPC core. There, Nup188p interacts with an integral protein of the pore membrane domain, Pom152p, and another abundant nucleoporin, Nic96p. The effects of various mutations in the NUP188 gene on the structure of the nuclear envelope and the function of the NPC were examined. While null mutants of NUP188 appear normal, other mutants allelic to NUP188 exhibit a dominant effect leading to the formation of NPC-associated nuclear envelope herniations and growth inhibition at 37 degrees C. In addition, depletion of the interacting protein Pom152p in cells lacking Nup188p resulted in severe deformations of the nuclear envelope. We suggest that Nup188p is one of a group of proteins that form the octagonal core structure of the NPC and thus functions in the structural organization of the NPC and nuclear envelope.
Asunto(s)
Membrana Nuclear/química , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Genes Fúngicos/genética , Genes Letales , Prueba de Complementación Genética , Glicoproteínas de Membrana/análisis , Datos de Secuencia Molecular , Peso Molecular , Mutación , Membrana Nuclear/ultraestructura , Poro Nuclear , Proteínas Nucleares/química , Proteínas Nucleares/aislamiento & purificación , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Análisis de Secuencia , Análisis de Secuencia de ADN , Levaduras/citología , Levaduras/genéticaRESUMEN
The nuclear import factor p10 was cloned from Saccharomyces cerevisiae and found to be essential. The protein p10 can bind directly to several peptide repeat-containing nucleoporins. It also binds to the guanosine triphosphatase (GTPase) Ran in its guanosine diphosphate (GDP)-bound form and to karyopherin beta. Assembly of the karyopherin heterodimer on immobilized nucleoporin yielded cooperative binding of p10 and Ran-GDP. Addition of GTP to this pentameric complex led to dissociation of karyopherin (chi, presumably via in situ formation of Ran-GTP from Ran-GDP. Thus, p10 appears to coordinate the Ran-dependent association and dissociation reactions underlying nuclear import.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Saccharomyces cerevisiae/metabolismo , Transporte Biológico Activo , Proteínas Portadoras/genética , Clonación Molecular , Proteínas de Unión al GTP/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Datos de Secuencia Molecular , Membrana Nuclear/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , alfa Carioferinas , beta Carioferinas , Proteína de Unión al GTP ranRESUMEN
NSP1 is related to a group of nuclear pore proteins termed 'nucleoporins'. We observe that in thermosensitive nsp1 mutants lacZ fusion proteins which contain the nuclear targeting sequence of Mat alpha 2 or Pho2 are located inside the nucleus at the permissive temperature (23 degrees C), but are mislocalized in the cytoplasm at the restrictive temperature (37 degrees C). No evidence was obtained that the large lacZ reporter protein leaks out from the nucleus. Another nuclear passenger protein consisting of the NLS of ribosomal protein L25 and cytosolic dihydrofolate reductase (DHFR) is also accumulating in the cytoplasm after shifting ts nsp1 cells to 37 degrees C. In the latter case, this could be attributed to an increased leakage of the reporter protein from the nucleus into the cytoplasm. These data suggest that NSP1 mutation affects nuclear transport and nuclear retention, but the effects depend on the used NLS and the reporter protein.
Asunto(s)
Proteínas de Unión al Calcio , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Homeodominio , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Proteínas Fúngicas/genética , Mutación , Membrana Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/ultraestructura , Temperatura , Tetrahidrofolato Deshidrogenasa/metabolismo , Transactivadores/metabolismoRESUMEN
The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.
Asunto(s)
Membrana Nuclear/metabolismo , Saccharomyces/metabolismo , Evolución Biológica , Transporte BiológicoRESUMEN
NSP1 is a nuclear pore protein (nucleoporin) essential for cell growth. To identify the components that functionally interact with NSP1 in the living cell, we developed a genetic screen for mutants that are lethal in a genetic background of mutated, but not wild type NSP1. Fourteen synthetic lethal mutants were obtained, belonging to at least four different complementation groups. The genes of two complementation groups, NSP116 and NSP49, were cloned. Like the previously described nucleoporins, these genes encode proteins with many repeat sequences. NSP116 and NSP49, however, contain a new repetitive sequence motif 'GLFG', which classifies them as a subclass of nucleoporins. NSP116 and NSP49, tagged with the IgG binding domain of protein A and expressed in yeast, are located at the nuclear envelope. These data provide in vivo evidence that distinct subclasses of nucleoporins physically interact or share overlapping function in nuclear pore complexes.
Asunto(s)
Proteínas de Unión al Calcio , Proteínas Fúngicas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos , Proteínas Fúngicas/genética , Prueba de Complementación Genética , Glicoproteínas de Membrana/clasificación , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Membrana Nuclear/metabolismo , Proteínas Nucleares/clasificación , Proteínas Nucleares/genética , Plásmidos , Mapeo Restrictivo , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Proteína Estafilocócica A/metabolismoRESUMEN
The functional regulation of chromatin is closely related to its spatial organization within the nucleus. In yeast, perinuclear chromatin domains constitute areas of transcriptional repression. These 'silent' domains are defined by the presence of perinuclear telomere clusters. The only protein found to be involved in the peripheral localization of telomeres is Yku70/Yku80. This conserved heterodimer can bind telomeres and functions in both repair of DNA double-strand breaks and telomere maintenance. These findings, however, do not address the underlying structural basis of perinuclear silent domains. Here we show that nuclear-pore-complex extensions formed by the conserved TPR homologues Mlp1 and Mlp2 are responsible for the structural and functional organization of perinuclear chromatin. Loss of MLP2 results in a severe deficiency in the repair of double-strand breaks. Furthermore, double deletion of MLP1 and MLP2 disrupts the clustering of perinuclear telomeres and releases telomeric gene repression. These effects are probably mediated through the interaction with Yku70. Mlp2 physically tethers Yku70 to the nuclear periphery, thus forming a link between chromatin and the nuclear envelope. We show, moreover, that this structural link is docked to nuclear-pore complexes through a cleavable nucleoporin, Nup145. We propose that, through these interactions, nuclear-pore complexes organize a nuclear subdomain that is intimately involved in the regulation of chromatin metabolism.
Asunto(s)
Antígenos Nucleares , Cromatina/fisiología , ADN Helicasas , Membrana Nuclear/fisiología , Proteínas de Saccharomyces cerevisiae , Telómero/fisiología , Cromatina/metabolismo , Cromatina/ultraestructura , Daño del ADN , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Autoantígeno Ku , Mutación , Membrana Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/fisiología , Pruebas de Precipitina , Proteínas de Unión al ARN , Telómero/ultraestructura , LevadurasRESUMEN
NSP1 is located at the nuclear periphery in yeast and is essential for cell growth. Employing immunoelectron microscopy on yeast cells, we show that NSP1 is located at the nuclear pores. The molecular analysis of the NSP1 protein points to a two domain model: a nonessential domain (the first 603 amino acids) composed of repetitive sequences common to other nuclear proteins and an essential, carboxy-terminal domain (residues 604-823) mediating the vital function of NSP1. The NSP1 carboxy-terminal domain, which shows a heptad repeat organization, affected the correct location of two nuclear proteins: site-specific amino acid substitutions within a predicted alpha-helical structure of this domain caused a temperature-sensitive growth arrest at 37 degrees C and the appearance of NSP1 and NOP1, a nucleolar protein, in the cytosol.
Asunto(s)
Proteínas de Unión al Calcio , Proteínas Fúngicas/genética , Membrana Nuclear/ultraestructura , Proteínas Nucleares , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/ultraestructura , Alcohol Deshidrogenasa/genética , Secuencia de Aminoácidos , Cromosomas Fúngicos , Diploidia , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/aislamiento & purificación , Genes Fúngicos , Haploidia , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación , Membrana Nuclear/análisis , Proteínas de Complejo Poro Nuclear , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Conformación Proteica , Proteínas Recombinantes de Fusión/aislamiento & purificación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Esporas Fúngicas/fisiología , Supresión Genética , Temperatura , beta-Galactosidasa/aislamiento & purificaciónRESUMEN
Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.