RESUMO
In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.
Assuntos
Dano ao DNA , RNA Polimerase II/metabolismo , Reparo do DNA , Células HEK293 , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Ubiquitinação , Raios UltravioletaRESUMO
DNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a "mechanism of last resort" employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transcrição Gênica , Sequência de Aminoácidos , Proteínas Cromossômicas não Histona/química , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Alinhamento de Sequência , Estresse Fisiológico , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Different approaches have been described to study RNAPII poly-ubiquitylation and degradation, each method with its own advantages and caveats. Here, we describe optimised strategies for detecting ubiquitylated RNAPII and studying its degradation, but these protocols are suitable for studying other ubiquitylated proteins as well.
Assuntos
RNA Polimerase II/análise , RNA Polimerase II/metabolismo , Ubiquitinação , Animais , Dano ao DNA , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/genética , Transcrição Gênica , Raios Ultravioleta , Leveduras/enzimologia , Leveduras/genética , Leveduras/metabolismoRESUMO
During its journey across a gene, RNA polymerase II has to contend with a number of obstacles to its progression, including nucleosomes, DNA-binding proteins, DNA damage, and sequences that are intrinsically difficult to transcribe. Not surprisingly, a large number of elongation factors have evolved to ensure that transcription stalling or arrest does not occur. If, however, the polymerase cannot be restarted, it becomes poly-ubiquitylated and degraded by the proteasome. This process is highly regulated, ensuring that only RNAPII molecules that cannot otherwise be salvaged are degraded. In this review, we describe the mechanisms and factors responsible for the last resort mechanism of transcriptional elongation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
Assuntos
Instabilidade Genômica/genética , Proteólise , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética/fisiologia , Ubiquitinação/fisiologia , Animais , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Humanos , Modelos Biológicos , RNA Polimerase II/química , RNA Polimerase II/fisiologia , Ubiquitinação/genéticaRESUMO
The proteasome degrades proteins modified by polyubiquitylation, so correctly controlled ubiquitylation is crucial to avoid unscheduled proteolysis of essential proteins. The mechanism regulating proteolysis of RNAPII has been controversial since two distinct ubiquitin ligases (E3s), Rsp5 (and its human homologue NEDD4) and Elongin-Cullin complex, have both been shown to be required for its DNA-damage-induced polyubiquitylation. Here we show that these E3s work sequentially in a two-step mechanism. First, Rsp5 adds mono-ubiquitin, or sometimes a ubiquitin chain linked via ubiquitin lysine 63 that does not trigger proteolysis. When produced, the K63 chain can be trimmed to mono-ubiquitylation by an Rsp5-associated ubiquitin protease, Ubp2. Based on this mono-ubiquitin moiety on RNAPII, an Elc1/Cul3 complex then produces a ubiquitin chain linked via lysine 48, which can trigger proteolysis. Likewise, for correct polyubiquitylation of human RNAPII, NEDD4 cooperates with the ElonginA/B/C-Cullin 5 complex. These data indicate that RNAPII polyubiquitylation requires cooperation between distinct, sequentially acting ubiquitin ligases, and raise the intriguing possibility that other members of the large and functionally diverse family of NEDD4-like ubiquitin ligases also require the assistance of a second E3 when targeting proteins for degradation.
Assuntos
Poliubiquitina/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Humanos , Lisina/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Hibridização in Situ Fluorescente , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/isolamento & purificação , Proteínas de Ligação a Poli(A)/isolamento & purificação , Ligação Proteica , Estrutura Terciária de Proteína , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/isolamento & purificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/química , Animais , Caenorhabditis elegans , Diferenciação Celular , Citoplasma/metabolismo , Dimerização , Drosophila , Humanos , Carioferinas/química , Modelos Moleculares , Filogenia , Conformação Proteica , beta Carioferinas/metabolismoRESUMO
Upon DNA damage, eukaryotic cells activate a conserved signal transduction cascade known as the DNA damage checkpoint (DDC). We investigated the influence of DDC kinases on nucleotide excision repair (NER) in Saccharomyces cerevisiae and found that repair of both strands of an active gene is affected by Mec1 but not by the downstream checkpoint kinases, Rad53 and Chk1. Repair of the nontranscribed strand (by global genome repair) requires new protein synthesis, possibly reflecting the involvement of Mec1 in the activation of repair genes. In contrast, repair of the transcribed strand by transcription-coupled NER (TC-NER) occurs in the absence of new protein synthesis, and DNA damage results in Mec1-dependent but Rad53-, Chk1-, Tel1-, and Dun1-independent phosphorylation of the TC-NER factor Rad26, a member of the Swi/Snf group of ATP-dependent translocases and yeast homologue of Cockayne syndrome B. Mutation of the Rad26 phosphorylation site results in a decrease in the rate of TC-NER, pointing to direct activation of Rad26 by Mec1 kinase. These findings establish a direct role for Mec1 kinase in transcription-coupled repair, at least partly via phosphorylation of Rad26, the main transcription-repair coupling factor.
Assuntos
Adenosina Trifosfatases/metabolismo , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Dano ao DNA , Mutação , Fosforilação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Proteínas Nucleares/química , Proteínas de Transporte Nucleocitoplasmático/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sítios de Ligação , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fenilalanina , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , SoluçõesRESUMO
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.
Assuntos
Poro Nuclear/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Transcrição Gênica , Sítios de Ligação , Cromatina/genética , Cromatina/fisiologia , Genes Fúngicos , Cinética , Proteínas Nucleares/metabolismo , Plasmídeos , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Sinais de Localização Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Dados de Sequência Molecular , Sinais de Exportação Nuclear/fisiologia , Ligação Proteica/fisiologia , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , alfa Carioferinas/químicaRESUMO
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.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Carioferinas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Clonagem Molecular , Cristalografia por Raios X , Dados de Sequência Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fragmentos de Peptídeos/química , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Saccharomyces cerevisiae/fisiologia , alfa Carioferinas/fisiologia , beta Carioferinas/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Núcleo Celular/fisiologia , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Subunidades Proteicas/fisiologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , alfa Carioferinas/antagonistas & inibidoresRESUMO
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.
Assuntos
Transporte Ativo do Núcleo Celular , Proteínas de Ligação a DNA/metabolismo , Antígenos de Histocompatibilidade Classe II/análise , Sinais de Localização Nuclear , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Proteínas de Ligação a DNA/química , Antígenos de Histocompatibilidade Classe II/fisiologia , Humanos , Dados de Sequência Molecular , Transporte Proteico , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/químicaRESUMO
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.
Assuntos
Sinais de Localização Nuclear/metabolismo , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Ciclo Celular , Homeostase , Dados de Sequência Molecular , Sinais de Localização Nuclear/química , Fragmentos de Peptídeos/química , Fosforilação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Sinais de Localização Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae , alfa Carioferinas/metabolismo , Alelos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico/genética , Mutação , Ligação Proteica/genética , Temperatura , alfa Carioferinas/genéticaRESUMO
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.
Assuntos
Proteínas Fúngicas/química , Proteínas de Choque Térmico/química , Proteínas de Saccharomyces cerevisiae , Proteínas Fúngicas/metabolismo , Proteínas de Fluorescência Verde , Proteínas de Choque Térmico/metabolismo , Immunoblotting , Carioferinas/química , Cinética , Proteínas Luminescentes/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMO
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.