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1.
Traffic ; 10(10): 1414-28, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19761539

RESUMEN

Delivery of DNA to the cell nucleus is an essential step in many types of viral infection, transfection, gene transfer by the plant pathogen Agrobacterium tumefaciens and in strategies for gene therapy. Thus, the mechanism by which DNA crosses the nuclear pore complex (NPC) is of great interest. Using nuclei reconstituted in vitro in Xenopus egg extracts, we previously studied DNA passage through the nuclear pores using a single-molecule approach based on optical tweezers. Fluorescently labeled DNA molecules were also seen to accumulate within nuclei. Here we find that this import of DNA relies on a soluble protein receptor of the importin family. To identify this receptor, we used different pathway-specific cargoes in competition studies as well as pathway-specific dominant negative inhibitors derived from the nucleoporin Nup153. We found that inhibition of the receptor transportin suppresses DNA import. In contrast, inhibition of importin beta has little effect on the nuclear accumulation of DNA. The dependence on transportin was fully confirmed in assays using permeabilized HeLa cells and a mammalian cell extract. We conclude that the nuclear import of DNA observed in these different vertebrate systems is largely mediated by the receptor transportin. We further report that histones, a known cargo of transportin, can act as an adaptor for the binding of transportin to DNA.


Asunto(s)
Núcleo Celular/metabolismo , ADN/metabolismo , Carioferinas/fisiología , Transporte Activo de Núcleo Celular , Animales , Citoplasma/metabolismo , ADN/genética , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Carioferinas/metabolismo , Microscopía Electrónica de Rastreo , Poro Nuclear/metabolismo , Óvulo/citología , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/fisiología , Xenopus laevis
2.
Mol Biol Cell ; 14(11): 4387-96, 2003 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-14551248

RESUMEN

Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin beta negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin beta is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin beta down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin beta to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin beta 45-462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin beta-regulation of NPC assembly. Excess full-length importin beta and beta 45-462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin beta NPC block, which maps downstream of GTPgammaS and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 microM RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin beta. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly.


Asunto(s)
Ácido Egtácico/análogos & derivados , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Oocitos/metabolismo , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismo , Animales , Ácido Egtácico/metabolismo , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Fusión de Membrana , Modelos Moleculares , Mutación , Xenopus/metabolismo
3.
Mol Cell ; 11(4): 853-64, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12718872

RESUMEN

The vertebrate nuclear pore complex, 30 times the size of a ribosome, assembles from a library of soluble subunits and two membrane proteins. Using immunodepletion of Xenopus nuclear reconstitution extracts, it has previously been possible to assemble nuclei lacking pore subunits tied to protein import, export, or mRNA export. However, these altered pores all still possessed the bulk of pore structure. Here, we immunodeplete a single subunit, the Nup107-160 complex, using antibodies to Nup85 and Nup133, two of its components. The resulting reconstituted nuclei are severely defective for NLS import and DNA replication. Strikingly, they show a profound defect for every tested nucleoporin. Even the integral membrane proteins POM121 and gp210 are absent or unorganized. Scanning electron microscopy reveals pore-free nuclei, while addback of the Nup107-160 complex restores functional pores. We conclude that the Nup107-160 complex is a pivotal determinant for vertebrate nuclear pore complex assembly.


Asunto(s)
Transporte Activo de Núcleo Celular/genética , Replicación del ADN/genética , Proteínas de Complejo Poro Nuclear/deficiencia , Poro Nuclear/metabolismo , Proteínas Nucleares , Subunidades de Proteína/deficiencia , Vertebrados/metabolismo , Animales , Femenino , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Rastreo , Antígenos de Histocompatibilidad Menor , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Oocitos/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Subunidades de Proteína/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Xenopus , Xenopus laevis
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