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1.
Mol Cell Biol ; 27(18): 6569-79, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17636033

RESUMEN

mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA binding proteins. Poly(U) binding protein 1 (Pub1) is a cytoplasmic Saccharomyces cerevisiae mRNA binding protein that stabilizes transcripts containing AU-rich elements (AREs) or stabilizer elements (STEs). In a yeast two-hybrid screen, we identified nuclear poly(A) binding protein 2 (Nab2) as being a Pub1-interacting protein. Nab2 is an essential nucleocytoplasmic shuttling mRNA binding protein that regulates poly(A) tail length and mRNA export. The interaction between Pub1 and Nab2 was confirmed by copurification and in vitro binding assays. The interaction is mediated by the Nab2 zinc finger domain. Analysis of the functional link between these proteins reveals that Nab2, like Pub1, can modulate the stability of specific mRNA transcripts. The half-life of the RPS16B transcript, an ARE-like sequence-containing Pub1 target, is decreased in both nab2-1 and nab2-67 mutants. In contrast, GCN4, an STE-containing Pub1 target, is not affected. Similar results were obtained for other ARE- and STE-containing Pub1 target transcripts. Further analysis reveals that the ARE-like sequence is necessary for Nab2-mediated transcript stabilization. These results suggest that Nab2 functions together with Pub1 to modulate mRNA stability and strengthen a model where nuclear events are coupled to the control of mRNA turnover in the cytoplasm.


Asunto(s)
Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Hibridación Fluorescente in Situ , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/aislamiento & purificación , Proteínas de Unión a Poli(A)/aislamiento & purificación , Unión Proteica , Estructura Terciaria de Proteína , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Técnicas del Sistema de Dos Híbridos
2.
Trends Cell Biol ; 14(9): 505-14, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15350979

RESUMEN

The importin alpha/beta heterodimer targets hundreds of proteins to the nuclear-pore complex (NPC) and facilitates their translocation across the nuclear envelope. Importin alpha binds to classical nuclear localization signal (cNLS)-containing proteins and links them to importin beta, the karyopherin that ferries the ternary complex through the NPC. A second karyopherin, the exportin CAS, recycles importin alpha back to the cytoplasm. In this article, we discuss control mechanisms that importin alpha exerts over the assembly and disassembly of the ternary complex and we describe how new groups of importin alpha genes arose during the evolution of metazoan animals to function in development and differentiation. We also describe activities of importin alpha that seem to be distinct from its housekeeping functions in nuclear transport.


Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/química , Animales , Caenorhabditis elegans , Diferenciación Celular , Citoplasma/metabolismo , Dimerización , Drosophila , Humanos , Carioferinas/química , Modelos Moleculares , Filogenia , Conformación Proteica , beta Carioferinas/metabolismo
3.
J Mol Biol ; 376(4): 1048-59, 2008 Feb 29.
Artículo en Inglés | MEDLINE | ID: mdl-18190927

RESUMEN

Nuclear abundant poly(A) RNA-binding protein 2 (Nab2) is an essential yeast heterogeneous nuclear ribonucleoprotein that modulates both mRNA nuclear export and poly(A) tail length. The N-terminal domain of Nab2 (residues 1-97) mediates interactions with both the C-terminal globular domain of the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), and the mRNA export factor, Gfd1. The solution and crystal structures of the Nab2 N-terminal domain show a primarily helical fold that is analogous to the PWI fold found in several other RNA-binding proteins. In contrast to other PWI-containing proteins, we find no evidence that the Nab2 N-terminal domain binds to nucleic acids. Instead, this domain appears to mediate protein:protein interactions that facilitate the nuclear export of mRNA. The Nab2 N-terminal domain has a distinctive hydrophobic patch centered on Phe73, consistent with this region of the surface being a protein:protein interaction site. Engineered mutations within this hydrophobic patch attenuate the interaction with the Mlp1 C-terminal domain but do not alter the interaction with Gfd1, indicating that this patch forms a crucial component of the interface between Nab2 and Mlp1.


Asunto(s)
Proteínas Nucleares/química , Proteínas de Transporte Nucleocitoplasmático/química , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sitios de Unión , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fenilalanina , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Soluciones
4.
J Biol Chem ; 282(5): 3042-9, 2007 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-17158105

RESUMEN

Recent work has demonstrated that some actively transcribed genes closely associate with nuclear pore complexes (NPC) at the nuclear periphery. The Saccharomyces cerevisiae Mlp1 and Mlp2 proteins are components of the inner nuclear basket of the nuclear pore that mediate interactions with these active genes. To investigate the physical link between the NPC and active loci, we identified proteins that interact with the carboxyl-terminal globular domain of Mlp1 by tandem affinity purification coupled with mass spectrometry. This analysis led to the identification of several components of the Spt-Ada-Gcn5-acetyltransferase (SAGA) histone acetyltransferase complex, Gcn5, Ada2, and Spt7. We utilized co-immunoprecipitation and in vitro binding assays to confirm the interaction between the Mlp proteins and SAGA components. Chromatin immunoprecipitation experiments revealed that Mlp1 and SAGA components associate with the same region of the GAL promoters. Critically, this Mlp-promoter interaction depends on the integrity of the SAGA complex. These results identify a physical association between SAGA and the NPC, and support previous results that relied upon visualization of GAL loci at the nuclear periphery by microscopy (Cabal, G. G. Genovesio, A., Rodriguez-Navarro, S., Zimmer, C., Gadal, O., Lesne, A., Buc, H., Feuerbach-Fournier, F., Olivo-Marin, J.-C., Hurt, E. C., and Nehrbass, U. (2006) Nature 441, 770-773). We propose that a physical interaction between nuclear pore components and the SAGA complex can link the actively transcribed GAL genes to the nuclear pore.


Asunto(s)
Poro Nuclear/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transactivadores/metabolismo , Transcripción Genética , Sitios de Unión , Cromatina/genética , Cromatina/fisiología , Genes Fúngicos , Cinética , Proteínas Nucleares/metabolismo , Plásmidos , Proteínas de Unión al ARN , Proteínas Recombinantes de Fusión/metabolismo
5.
J Biol Chem ; 281(33): 23545-56, 2006 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-16785238

RESUMEN

Nuclear localization signals (NLSs) target proteins into the nucleus through mediating interactions with nuclear import receptors. Here, we perform a quantitative analysis of the correlation between NLS receptor affinity and the steady-state distribution of NLS-bearing cargo proteins between the cytoplasm and the nucleus of live yeast, which reflects the relative import rates of various NLS sequences. We find that there is a complicated, but monotonic quantitative relationship between the affinity of an NLS for the import receptor, importin alpha, and the steady-state accumulation of the cargo in the nucleus. This analysis takes into consideration the impact of protein size. In addition, the hypothetical upper limit to an NLS affinity for the receptors is explored through genetic approaches. Overall, our results indicate that there is a correlation between the binding affinity of an NLS cargo for the NLS receptor, importin alpha, and the import rate for this cargo. This correlation, however, is not maintained for cargoes that bind to the NLS receptor with very weak or very strong affinity.


Asunto(s)
Señales de Localización Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Señales de Exportación Nuclear/fisiología , Unión Proteica/fisiología , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , alfa Carioferinas/química
6.
J Biol Chem ; 278(8): 5854-63, 2003 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-12486120

RESUMEN

Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/karyopherin alpha and beta. The importin alpha subunit recognizes classical NLS sequences, and the importin beta subunit directs the complex to the nuclear pore. Recent work shows that the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding in the absence of importin beta in vitro. To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin alpha protein. These mutants dissect the two functions of the N-terminal IBB domain, importin beta binding and auto-inhibition. One of these importin alpha mutations, A3, decreases auto-inhibitory function without impacting binding to importin beta or the importin alpha export receptor, Cse1p. We used this mutant to show that the auto-inhibitory function is essential in vivo and to provide evidence that this auto-inhibitory-defective importin alpha remains bound to NLS-cargo within the nucleus. We propose a model where the auto-inhibitory activity of importin alpha is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of importin alpha to the cytoplasm.


Asunto(s)
Saccharomyces cerevisiae/fisiología , alfa Carioferinas/fisiología , beta Carioferinas/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Núcleo Celular/fisiología , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Subunidades de Proteína/fisiología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , alfa Carioferinas/antagonistas & inhibidores
7.
EMBO J ; 22(20): 5358-69, 2003 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-14532109

RESUMEN

The yeast nucleoporin Nup2p is associated primarily with the nuclear basket of nuclear pore complexes and is required for efficient importin-alpha:beta-mediated nuclear protein import as well as efficient nuclear export of Kap60p/importin-alpha. Residues 1-51 of Nup2p bind tightly to Kap60p and are required for Nup2p function in vivo. We have determined the 2.6 A resolution crystal structure of a complex between this region of Nup2p and the armadillo repeat domain of Kap60p. Nup2p binds along the inner concave groove of Kap60p, but its interaction interface is different from that employed for nuclear localization signal (NLS) recognition although there is some overlap between them. Nup2p binds Kap60p more strongly than NLSs and accelerates release of NLSs from Kap60p. Nup2p itself is released from Kap60p by Cse1p:RanGTP only in the presence of the importin-beta binding (IBB) domain of Kap60p. These data indicate that Nup2p increases the overall rate of nuclear trafficking by coordinating nuclear import termination and importin recycling as a concerted process.


Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Carioferinas/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/genética , Fragmentos de Péptidos/química , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
8.
J Immunol ; 173(1): 410-9, 2004 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-15210800

RESUMEN

Comprised of RFX5, RFXAP, and RFX-B/ANK, the regulatory factor X (RFX) complex is an obligate transcription factor required for the expression of MHC class II genes. RFX functions by binding to the conserved X1 box sequence located upstream of all MHC class II genes. Using a mutagenesis scheme and a yeast heterologous reporter system, the mechanism by which the RFX complex is transported into the nucleus was examined. The results have identified specific nuclear localization signals (NLS) in both RFX5 and RFXAP that direct the nuclear translocation and expression of MHC class II genes. Additionally, a nuclear export signal was identified in the N terminus of RFXAP. RFX-B was poorly localized to the nucleus, and no specific NLS was identified. Whereas RFX5 could import an RFXAP NLS mutant into the nucleus, it had no effect on the import of RFX-B. The results suggest that although RFX5 and RFXAP could assemble before nuclear import, RFX-B association with the complex does not take place until after the subunits enter the nucleus. The identification of nuclear import and export sites on RFX molecules provides potential targets to modulate MHC class II expression.


Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas de Unión al ADN/metabolismo , Antígenos de Histocompatibilidad Clase II/análisis , Señales de Localización Nuclear , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Proteínas de Unión al ADN/química , Antígenos de Histocompatibilidad Clase II/fisiología , Humanos , Datos de Secuencia Molecular , Transporte de Proteínas , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/química
9.
J Biol Chem ; 279(20): 20613-21, 2004 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-14998990

RESUMEN

Many important regulatory proteins, including cell cycle regulators and transcription factors, contain a phosphorylation site within or adjacent to a classic nuclear localization signal (NLS) sequence. Previous studies show that the nuclear localization of these cargoes can be regulated by phosphorylation at these sites. It was hypothesized that this phosphorylation regulates the nuclear import of NLS cargo proteins by modulating the interaction of the cargo with the classic nuclear transport receptor, importin alpha. In this study, we utilize in vitro solution binding assays and in vivo analyses to directly test this model. We demonstrate that mimicking phosphorylation at a site adjacent to an NLS decreases the binding affinity of the NLS for importin alpha. This decrease in cargo affinity for importin alpha correlates with a decrease in nuclear accumulation in vivo. Through these analyses, we show that the cell cycle-dependent nuclear import of the Saccharomyces cerevisiae transcription factor Swi6p correlates with a phosphorylation-dependent change in affinity for importin alpha. Furthermore, we present data using the SV40 NLS to suggest that this form of regulation can be utilized to artificially modulate the nuclear import of a cargo, which is usually constitutively targeted to the nucleus. This work defines one molecular mechanism for regulating nuclear import by the classic NLS-mediated transport pathway.


Asunto(s)
Señales de Localización Nuclear/metabolismo , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Ciclo Celular , Homeostasis , Datos de Secuencia Molecular , Señales de Localización Nuclear/química , Fragmentos de Péptidos/química , Fosforilación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo
10.
J Biol Chem ; 278(43): 41947-53, 2003 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-12917403

RESUMEN

Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.


Asunto(s)
Señales de Localización Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae , alfa Carioferinas/metabolismo , Alelos , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico/genética , Mutación , Unión Proteica/genética , Temperatura , alfa Carioferinas/genética
11.
J Biol Chem ; 278(24): 21361-9, 2003 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-12672802

RESUMEN

Protein cargoes that contain a classic nuclear localization signal (NLS) are transported into the nucleus through binding to a heterodimeric receptor comprised of importin/karyopherin alpha and beta. An evolutionarily conserved auto-inhibitory sequence within the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding to the NLS binding pocket on importin alpha. In this study, we have used site-directed mutagenesis coupled with in vitro binding assays and in vivo analyses to investigate the intramolecular interaction of the N-terminal IBB domain and the NLS binding pocket of Saccharomyces cerevisiae importin alpha, Srp1p. We find that mutations within the IBB domain that decrease the binding affinity of the auto-inhibitory sequence for the NLS binding pocket impact importin alpha function in vivo. In addition, the severity of the in vivo phenotype is directly correlated to the reduction of auto-inhibition measured in vitro, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function. We exploit a conditional auto-inhibitory mutant, srp1-55, to study the in vivo functional overlap between the N-terminal IBB domain of importin alpha and other factors implicated in NLS-cargo release, Cse1p and Nup2p. We propose that the N-terminal IBB domain of importin alpha and Cse1p function together in NLS-cargo release, whereas Nup2p contributes to cargo release/importin alpha recycling through a distinct mechanism.


Asunto(s)
Proteínas Fúngicas/química , Proteínas de Choque Térmico/química , Proteínas de Saccharomyces cerevisiae , Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes , Proteínas de Choque Térmico/metabolismo , Immunoblotting , Carioferinas/química , Cinética , Proteínas Luminiscentes/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/química , Saccharomyces cerevisiae/metabolismo , Temperatura
12.
EMBO J ; 21(21): 5833-42, 2002 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-12411501

RESUMEN

A classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin alpha/beta complex. In this study, we found that importin alpha migrated into the nucleus without the addition of importin beta, Ran or any other soluble factors in an in vitro transport assay. A mutant importin alpha lacking the importin beta-binding domain efficiently entered the nucleus. Competition experiments showed that this import pathway for importin alpha is distinct from that of importin beta. These results indicate that importin alpha alone can enter the nucleus via a novel pathway in an importin beta- and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin alpha differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin alpha was affected by the addition of ATP, but not ATPgammaS. These results suggest that the nuclear import machinery for importin alpha at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis.


Asunto(s)
Núcleo Celular/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismo , Animales , Bovinos , Citosol/metabolismo , Proteínas Fluorescentes Verdes , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólisis , Proteínas Luminiscentes/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo
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