RESUMO
Many of the known SUMO substrates are nuclear proteins, which regulate gene expression and chromatin dynamics. Sumoylation, in general, appears to correlate with decreased transcriptional activity, and in many cases modulation of the chromatin template is implicated. Sumoylation of the core histones is associated with transcriptional silencing, and transcription factor sumoylation can decrease gene expression by promoting recruitment of chromatin modifying enzymes. Additionally, sumoylation of transcriptional corepressors and chromatin remodeling enzymes can influence interactions with other transcriptional regulators, and alter their enzymatic activity. In some cases, proteins that are components of transcriptional corepressor complexes have been shown to be SUMO E3 ligases, further emphasizing the integration of sumoylation with the regulation of chromatin remodeling. Despite the evidence suggesting that sumoylation is primarily repressive for access to chromatin, recent analyses suggest that protein sumoylation on the chromatin template may play important roles at highly expressed genes. Elucidating the dynamic interplay of sumoylation with other post-translational modifications of histones and chromatin associated proteins will be key to fully understanding the regulation of access to the chromatin template.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , DNA/metabolismo , Histonas/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Animais , Cromatina/química , DNA/química , Histonas/química , Humanos , Conformação de Ácido Nucleico , Conformação Proteica , Relação Estrutura-Atividade , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In spite of their small size, the cellular morphology, structure, and protein localization of yeast cells can be successfully imaged. A detailed protocol for preparing yeast cells for live-cell imaging is described, including techniques to immobilize yeast for time-lapse microscopy. Protocols for indirect immunofluorescence are outlined, including strategies for fixation, cell wall digestion, and the use of primary and secondary antibodies conjugated to fluorescent moieties. Alternative approaches to these techniques are discussed, highlighting the advantages and disadvantages where possible. Using these protocols, investigation of yeast cell structure and protein localization will continue to yield important insights into yeast cell biology and regulation.
Assuntos
Imunofluorescência/métodos , Microscopia de Fluorescência/métodos , Saccharomyces cerevisiae/citologia , Concanavalina A/química , Técnica Indireta de Fluorescência para Anticorpo/métodos , Proteínas Imobilizadas/química , Imagem Óptica/métodos , Saccharomyces cerevisiae/ultraestrutura , Sefarose/químicaRESUMO
Transcriptional activation is typically associated with increased acetylation of promoter histones. However, this paradigm does not apply to transcriptional activation of all genes. In this study we have characterized a group of genes that are repressed by histone acetylation. These histone hypoacetylation-activated genes (HHAAG) are normally repressed during exponential growth, when the cellular level of acetyl-CoA is high and global histone acetylation is also high. The HHAAG are induced during diauxic shift, when the levels of acetyl-CoA and global histone acetylation decrease. The histone hypoacetylation-induced activation of HHAAG is independent of Msn2/Msn4. The repression of HSP12, one of the HHAAG, is associated with well-defined nucleosomal structure in the promoter region, while histone hypoacetylation-induced activation correlates with delocalization of positioned nucleosomes or with reduced nucleosome occupancy. Correspondingly, unlike the majority of yeast genes, HHAAG are transcriptionally upregulated when expression of histone genes is reduced. Taken together, these results suggest a model in which histone acetylation is required for proper positioning of promoter nucleosomes and repression of HHAAG.
Assuntos
Acetilcoenzima A/fisiologia , Cromatina/fisiologia , Histonas/metabolismo , Ativação Transcricional , Acetilação , Cromatina/química , Proteínas de Choque Térmico/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Histone chaperones function in chromatin assembly and disassembly, suggesting they have important regulatory roles in transcription elongation. The Saccharomyces cerevisiae proteins Nap1 and Vps75 are structurally related, evolutionarily conserved histone chaperones. We showed that Nap1 genetically interacts with several transcription elongation factors and that both Nap1 and Vps75 interact with the RNA polymerase II kinase, CTK1. Loss of NAP1 or VPS75 suppressed cryptic transcription within the open reading frame (ORF) observed when strains are deleted for the kinase CTK1. Loss of the histone acetyltransferase Rtt109 also suppressed ctk1-dependent cryptic transcription. Vps75 regulates Rtt109 function, suggesting that they function together in this process. Histone H3 K9 was found to be the important lysine that is acetylated by Rtt109 during ctk1-dependent cryptic transcription. We showed that both Vps75 and Nap1 regulate the relative level of H3 K9 acetylation in the STE11 ORF. This supports a model in which Nap1, like Vps75, directly regulates Rtt109 activity or regulates the assembly of acetylated chromatin. Although Nap1 and Vps75 share many similarities, due to their distinct interactions with SET2, Nap1 and Vps75 may also play separate roles during transcription elongation. This work sheds further light on the importance of histone chaperones as general regulators of transcription elongation.
Assuntos
Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Proteínas Quinases/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Montagem e Desmontagem da Cromatina , Histona Acetiltransferases/genética , Chaperonas de Histonas , Histonas/genética , MAP Quinase Quinase Quinases/metabolismo , Metiltransferases/metabolismo , Chaperonas Moleculares/genética , Proteína 1 de Modelagem do Nucleossomo/genética , Fases de Leitura Aberta , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.
Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Chaperonas de Histonas/fisiologia , Histonas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Humanos , Proteína 1 de Modelagem do Nucleossomo/fisiologiaRESUMO
Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.
RESUMO
Modification of histones is critical for the regulation of all chromatin-templated processes. Yeast Rtt109 is a histone acetyltransferase (HAT) that acetylates H3 lysines 9, 27 and 56. Rtt109 associates with and is stabilized by Nap1 family histone chaperone Vps75. Our data suggest Vps75 and Nap1 have some overlapping functions despite their different cellular localization and histone binding specificity. We determined that Vps75 contains a classical nuclear localization signal and is imported by Kap60-Kap95. Rtt109 nuclear localization depends on Vps75, and nuclear localization of the Vps75-Rtt109 complex is not critical for Rtt109-dependent functions, suggesting Rtt109 may be able to acetylate nascent histones before nuclear import. To date, the effects of VPS75 deletion on Rtt109 function had not been separated from the resulting Rtt109 degradation; thus, we used an Rtt109 mutant lacking the Vps75-interaction domain that is stable without Vps75. Our data show that in addition to promoting Rtt109 stability, Vps75 binding is necessary for Rtt109 acetylation of the H3 tail. Direct interaction of Vps75 with H3 likely allows Rtt109 access to the histone tail. Furthermore, our genetic interaction data support the idea of Rtt109-independent functions of Vps75. In summary, our data suggest that Vps75 influences chromatin structure by regulating histone modification and through its histone chaperone functions.
Assuntos
Núcleo Celular/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilação , Transporte Ativo do Núcleo Celular/fisiologia , Histona Acetiltransferases/genética , Histonas/genética , Chaperonas Moleculares/genética , Sinais de Localização Nuclear , Proteína 1 de Modelagem do Nucleossomo/genética , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
We analyzed the nuclear import and regulation of the yeast histone variant Htz1 (H2A.Z), and the role of histone chaperones Nap1 and Chz1 in this process. Copurification suggested that Htz1 and H2B dimerized in the cytoplasm prior to import. Like H2B, Htz1 contained a nuclear localization signal (NLS) in its N-terminus that is recognized by multiple karyopherins (also called importins), indicating multiple transport pathways into the nucleus. However, Kap114 and Kap123 appeared to play the major role in Htz1 import. We also identified a role for Nap1 in the import of Htz1/H2B heterodimers, and Nap1 formed a RanGTP-insensitive import complex with Htz1/H2B and Kap114. Nap1 was necessary for maintaining a soluble pool of Htz1, indicating that its chaperone function may be important for the dynamic exchange of histones within nucleosomes. In contrast, Chz1 was imported by a distinct import pathway, and Chz1 did not appear to interact with Htz1 in the cytoplasm. Genetic analysis indicated that NAP1 has a function in the absence of HTZ1 that is not shared with CHZ1. This provides further evidence that the histone chaperones Nap1 and Chz1 have separate Htz1-dependent and -independent functions.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Citosol/metabolismo , Carioferinas/metabolismo , Transdução de SinaisRESUMO
Budding yeast Saccharoymyces cerevisiae is a powerful model system for analyzing eukaryotic cell cycle regulation. Yeast cell cycle analysis is typically performed by visual analysis or flow cytometry, and both have limitations in the scope and accuracy of data obtained. This study demonstrates how multispectral imaging flow cytometry (MIFC) provides precise quantitation of cell cycle distribution and morphological phenotypes of yeast cells in flow. Cell cycle analysis of wild-type yeast, nap1Delta, and yeast overexpressing NAP1, was performed visually, by flow cytometry and by MIFC. Quantitative morphological analysis employed measurements of cellular length, thickness, and aspect ratio in an algorithm to calculate a novel feature, bud length. MIFC demonstrated reliable quantification of the yeast cell cycle compared to morphological and flow cytometric analyses. By employing this technique, we observed both the G2/M delay and elongated buds previously described in the nap1Delta strain. Using MIFC, we demonstrate that overexpression of NAP1 causes elongated buds yet only a minor disruption in the cell cycle. The different effects of NAP1 expression level on cell cycle and morphology suggests that these phenotypes are independent. Unlike conventional yeast flow cytometry, MIFC generates complete cell cycle profiles and concurrently offers multiple parameters for morphological analysis.
Assuntos
Ciclo Celular , Citometria de Fluxo/métodos , Citometria por Imagem/métodos , Saccharomyces cerevisiae/citologia , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Chromatin remodeling is central to the regulation of transcription elongation. We demonstrate that the conserved Saccharomyces cerevisiae histone chaperone Nap1 associates with chromatin. We show that Nap1 regulates transcription of PHO5, and the increase in transcript level and the higher phosphatase activity plateau observed for Deltanap1 cells suggest that the net function of Nap1 is to facilitate nucleosome reassembly during transcription elongation. To further our understanding of histone chaperones in transcription elongation, we identified factors that regulate the function of Nap1 in this process. One factor investigated is an essential mRNA export and TREX complex component, Yra1. Nap1 interacts directly with Yra1 and genetically with other TREX complex components and the mRNA export factor Mex67. Additionally, we show that the recruitment of Nap1 to the coding region of actively transcribed genes is Yra1 dependent and that its recruitment to promoters is TREX complex independent. These observations suggest that Nap1 functions provide a new connection between transcription elongation, chromatin assembly, and messenger RNP complex biogenesis.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Montagem e Desmontagem da Cromatina/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Proteínas Nucleares/fisiologia , Transporte de RNA/fisiologia , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Transcrição Gênica/fisiologia , Fosfatase Ácida , Proteínas de Ciclo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Regulação Fúngica da Expressão Gênica/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Proteína 1 de Modelagem do Nucleossomo , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Fases de Leitura Aberta , Mapeamento de Interação de Proteínas , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Transporte de RNA/genética , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica/genéticaRESUMO
In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.
Assuntos
Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/enzimologia , Transporte Ativo do Núcleo Celular , Ciclo Celular , Núcleo Celular/metabolismo , Ciclina B/metabolismo , Histonas/metabolismo , Proteína 1 de Modelagem do Nucleossomo , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Core histones H3 and H4 are rapidly imported into the nucleus by members of the karyopherin (Kap)/importin family. We showed that H3 and H4 interact with Kap123p, histone acetyltransferase-B complex (HAT-B), and Asf1p in cytosol. In vivo analysis indicated that Kap123p is required for H3-mediated import, whereas H4 utilizes multiple Kaps including Kap123p. The evolutionary conservation of H3 and H4 cytoplasmic acetylation led us to analyze the role of acetylation in nuclear transport. We determined that lysine 14 is critical for H3 NLS function in vivo and demonstrated that mutation of H3 lysine 14 to the acetylation-mimic glutamine decreased association with Kap123p in vitro. Several lysines in the H4 NLS are important for its function. We showed that mutation of key lysines to glutamine resulted in a greater import defect than mutation to arginine, suggesting that positive charge promotes NLS function. Lastly we determined that six of ten N-terminal acetylation sites in H3 and H4 can be mutated to arginine, indicating that deposition acetylation is not absolutely necessary in vivo. However, the growth defect of these mutants suggests that acetylation does play an important role in import. These findings suggest a model where cytosolic histones bind import karyopherins prior to acetylation. Other factors are recruited to this complex such as HAT-B and Asf1p; these factors in turn promote acetylation. Acetylation may be important for modulating the interaction with transport factors and may play a role in the release of histones from karyopherins in the nucleus.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Transporte Ativo do Núcleo Celular/fisiologia , Substituição de Aminoácidos , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Citoplasma/genética , Citoplasma/metabolismo , Histona Acetiltransferases/genética , Histonas/genética , Carioferinas/genética , Carioferinas/metabolismo , Chaperonas Moleculares , Mutação de Sentido Incorreto , Processamento de Proteína Pós-Traducional/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , beta CarioferinasAssuntos
Transporte Ativo do Núcleo Celular , Animais , Ciclo Celular , Georgia , Humanos , Técnicas In Vitro , Carioferinas/metabolismo , Laminas/metabolismo , Modelos Biológicos , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Fenômenos Fisiológicos ViraisRESUMO
Nuclear import and export is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (referred to as importins and exportins). The yeast karyopherin Kap114p has previously been shown to import histones H2A and H2B, Nap1p, and a component of the preinitiation complex (PIC), TBP. Using a proteomic approach, we have identified several potentially new cargoes for Kap114p. These cargoes include another PIC component, the general transcription factor IIB or Sua7p, which interacted directly with Kap114p. Consistent with its role as a Sua7p import factor, deletion of KAP114 led to specific mislocalization of Sua7p to the cytoplasm. An interaction between Sua7p and TBP was also detected in cytosol, raising the possibility that both Sua7p and TBP can be coimported by Kap114p. We have also shown that Kap114p possesses multiple overlapping binding sites for its partners, Sua7p, Nap1p, and H2A and H2B, as well as RanGTP and nucleoporins. In addition, we have assembled an in vitro complex containing Sua7p, Nap1p, and histones H2A and H2B, suggesting that this Kap may import several proteins simultaneously. The import of more than one cargo at a time would increase the efficiency of each import cycle and may allow the regulation of coimported cargoes.
Assuntos
Transporte Ativo do Núcleo Celular , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIB/química , Sítios de Ligação , Transporte Biológico , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Citoplasma/metabolismo , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/química , Deleção de Genes , Genes Reporter , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Histonas/química , Microscopia , Modelos Biológicos , Proteínas Nucleares/química , Proteína 1 de Modelagem do Nucleossomo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/química , Espectrometria de Massas por Ionização por Electrospray , beta Carioferinas , Proteína ran de Ligação ao GTP/químicaRESUMO
Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.
Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Carioferinas/fisiologia , Transporte Proteico/fisiologia , Transporte de RNA/fisiologia , Animais , Humanos , Proteínas de Neoplasias/metabolismo , Sinais de Localização Nuclear/fisiologia , Poro Nuclear/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Receptores de Esteroides/metabolismo , Transdução de Sinais , Proteína ran de Ligação ao GTP/fisiologiaRESUMO
The nuclear import of histones is a prerequisite for the downstream deposition of histones to form chromatin. However, the coordinate regulation of these processes remains poorly understood. Here we demonstrate that Kap114p, the primary karyopherin/importin responsible for the nuclear import of histones H2A and H2B, modulates the deposition of histones H2A and H2B by the histone chaperone Nap1p. We show that a complex comprising Kap114p, histones H2A and H2B, and Nap1p is present in the nucleus and that the presence of this complex is specifically promoted by Nap1p. This places Kap114p in a position to modulate Nap1p function, and we demonstrate by the use of two different assay systems that Kap114p inhibits Nap1p-mediated chromatin assembly. The inhibition of H2A and H2B deposition by Kap114p results in the concomitant inhibition of RCC1 loading onto chromatin. Biochemical evidence suggests that the mechanism by which Kap114p modulates histone deposition primarily involves direct histone binding, while the interaction between Kap114p and Nap1p plays a secondary role. Furthermore, we found that the inhibition of histone deposition by Kap114p is partially reversed by RanGTP. Our results indicate a novel mechanism by which cells can regulate histone deposition and establish a coordinate link between histone nuclear import and chromatin assembly.
Assuntos
Histonas/metabolismo , Carioferinas/fisiologia , Proteínas Nucleares/fisiologia , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Animais , Proteínas de Ciclo Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Histonas/antagonistas & inibidores , Masculino , Modelos Biológicos , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Proteína 1 de Modelagem do Nucleossomo , Mutação Puntual/genética , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína/fisiologia , Proteínas/análise , Proteínas/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo , Espermatozoides/química , Xenopus laevis , beta Carioferinas , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/fisiologiaRESUMO
Autonomously replicating sequence-binding factor 1 (Abf1p) is a site-specific DNA binding protein in Saccharomyces cerevisiae that functions to regulate multiple nuclear events including DNA replication, transcriptional activation, and gene silencing. Previous work indicates that the multiple functions of Abf1p are conferred by the carboxy-terminus of the protein, which can be further dissected into two important clusters of amino acid residues (CS1 and CS2). Here we present genetic and cell biological evidence for a critical role of CS1 in proper nuclear localization of Abf1p. Mutations in CS1 cause severe defects in cell growth, nuclear translocation, and Abf1p-mediated gene regulation, which can be rescued by a heterologous nuclear localization sequence (NLS). In addition, the CS1-domain can mediate the import of a CS1-GFP fusion protein. Importantly, the CS1-mediated nuclear import depends on the Ran guanine nucleotide exchange factor Prp20p. Interestingly, a single amino acid change in CS1 (K625I) also causes the protein to be exported out of the nucleus via the Crm1p-dependent pathway. The temperature-sensitive growth phenotype of this particular mutant can be overcome by overexpression of Kap121p/Pse1p, a well-established nuclear transport receptor. Biochemical studies indicate that Pse1p binds to a region of Abf1p upstream of CS1 in a RanGTP-sensitive manner, suggesting that Abf1p has a second distinct NLS and can be imported into the nucleus by several overlapping pathways. We propose that the link between Abf1p and the nuclear transport machinery may also be important for partitioning multiple Abf1p-mediated nuclear processes.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
The karyopherin beta (or importin beta) family comprises soluble transport factors that mediate the movement of proteins and RNAs between the nucleus and cytoplasm. Recent studies have extended the role of karyopherins to regulating assembly of the nuclear pore complex (NPC), assembly of the nuclear envelope, mitosis and replication. New data also address how karyopherins specifically recognize and transport many distinct cargoes and traverse the NPC. These data raise the possibility that, although there might be a universal mechanism for nuclear transport, specific interactions between karyopherins and components of the NPC might function to regulate differentially the ability of the different karyopherins to cross the NPC.
Assuntos
Núcleo Celular/fisiologia , Carioferinas/fisiologia , Poro Nuclear/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/química , Humanos , Carioferinas/química , Poro Nuclear/químicaRESUMO
Import of core histones into the nucleus is a prerequisite for their deposition onto DNA and the assembly of chromatin. Here we demonstrate that nucleosome assembly protein 1 (Nap1p), a protein previously implicated in the deposition of histones H2A and H2B, is also involved in the transport of these two histones. We demonstrate that Nap1p can bind directly to Kap114p, the primary karyopherin/importin responsible for the nuclear import of H2A and H2B. Nap1p also serves as a bridge between Kap114p and the histone nuclear localization sequence (NLS). Nap1p acts cooperatively to increase the affinity of Kap114p for these NLSs. Nuclear accumulation of histone NLS-green fluorescent protein (GFP) reporters was decreased in deltanap1 cells. Furthermore, we demonstrate that Nap1p promotes the association of the H2A and H2B NLSs specifically with the karyopherin Kap114p. Localization studies demonstrate that Nap1p is a nucleocytoplasmic shuttling protein, and genetic experiments suggest that its shuttling is important for maintaining chromatin structure in vivo. We propose a model in which Nap1p links the nuclear transport of H2A and H2B to chromatin assembly.
Assuntos
Núcleo Celular/metabolismo , Histonas/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular , Técnicas In Vitro , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 de Modelagem do Nucleossomo , Ligação Proteica , Retroelementos/genética , Leveduras/genética , Leveduras/metabolismo , beta CarioferinasRESUMO
The correct assembly of chromatin is necessary for the maintenance of genomic stability in eukaryotic cells. A critical step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. Here we demonstrate that the nuclear import pathway of histones H3 and H4 is mediated by at least two karyopherins/importins, Kap123p and Kap121p. Cytosolic H4 is found associated with Kap123p and H3. Kap121p is also present in the H4-PrA-associated fractions, albeit in lesser amounts than Kap123p, suggesting that this Kap serves as an additional import receptor. We further demonstrate that cytosolic Kap123p is associated with acetylated H3 and H4. H3 and H4 each contain a nuclear localization signal (NLS) in their amino-terminal domains. These amino-terminal domains were found to be essential for the nuclear accumulation of H3 and H4-green fluorescent protein reporters. Each NLS mediated direct binding to Kap123p and Kap121p, and decreased nuclear accumulation of H3 and H4 NLS-green fluorescent protein reporters was observed in specific kap mutant strains. H3 and H4 are the first histones to be assembled onto DNA, and these results show that their import is mediated by at least two import pathways.