Biophysical Characterization of the Recombinant Importin-α from Neurospora crassa

Takeda, Agnes Alessandra Sekijima; Freitas, Fernanda Zanolli; Magro, Ângelo J.; Bernardes, Natália Elisa; Fernandes, Carlos A. H.; Gonçalves, Rodrigo Duarte; Bertolini, Maria Célia; Fontes, M.R.M.

doi:10.2174/092986613804096829

Cited by 10 publications

(8 citation statements)

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“…Dim-3 was mapped to the right arm of Linkage Group V, and high-throughput sequencing identified two point mutations in the open reading frame of gene NCU01249, E396K and R469H; both were confirmed by Sanger sequencing. NCU01249 encodes NUP-6 (NIH GenBank accession EAA31416.1), which is predicted to form a structure similar to yeast Importin α [ 21 ], a protein that is highly conserved in eukaryotes [ 19 ]. Neurospora NUP-6 includes an N-terminal ∼80 amino acid Importin β-binding domain (IBB), followed by ten ∼40 amino acid ARM repeats ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation

et al. 2015

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Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In Neurospora crassa, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, dim-3 (defective in methylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in dim-3 results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in dim-3 strains. Interactions between DCDC members also appear normal, but the nup-6dim-3 allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6dim-3 itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in dim-3, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport.

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Section: Resultsmentioning

confidence: 99%

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation

et al. 2015

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“…The gene (NCU01249) encoding importin-α was cloned into the pET28a expression vector, and the recombinant protein was expressed as a truncated protein consisting of residues 75–529 fused to a 6-His tag at N-terminus, as previously described [37]. The protein was expressed in the Escherichia coli host strain RosettaTM (DE3) pLysS (Novagen) and purified by affinity chromatography [37].…”

Section: Methodsmentioning

confidence: 99%

“…The protein was expressed in the Escherichia coli host strain RosettaTM (DE3) pLysS (Novagen) and purified by affinity chromatography [37]. The protein was eluted with a 0.15–3.0 M imidazole linear gradient, followed by dialysis in buffer (20 mM Tris-HCl, pH 8.0 and 100 mM NaCl) and stored at cryogenic temperatures.…”

Section: Methodsmentioning

confidence: 99%

Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor

et al. 2016

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Environmental pH induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. Neurospora crassa shares all six components of the Aspergillus nidulans pH signaling pathway, and we investigate here their regulation during an alkaline pH stress response. We show that the N. crassa pal mutant strains, with the exception of Δpal-9, which is the A. nidulans palI homolog, exhibit low conidiation and are unable to grow at alkaline pH. Moreover, they accumulate the pigment melanin, most likely via regulation of the tyrosinase gene by the pH signaling components. The PAC-3 transcription factor binds to the tyrosinase promoter and negatively regulates its gene expression. PAC-3 also binds to all pal gene promoters, regulating their expression at normal growth pH and/or alkaline pH, which indicates a feedback regulation of PAC-3 in the pal gene expression. In addition, PAC-3 binds to the pac-3 promoter only at alkaline pH, most likely influencing the pac-3 expression at this pH suggesting that the activation of PAC-3 in N. crassa results from proteolytic processing and gene expression regulation by the pH signaling components. In N. crassa, PAC-3 is proteolytically processed in a single cleavage step predominately at alkaline pH; however, low levels of the processed protein can be observed at normal growth pH. We also demonstrate that PAC-3 preferentially localizes in the nucleus at alkaline pH stress and that the translocation may require the N. crassa importin-α since the PAC-3 nuclear localization signal (NLS) has a strong in vitro affinity with importin-α. The data presented here show that the pH signaling pathway in N. crassa shares all the components with the A. nidulans and S. cerevisiae pathways; however, it exhibits some properties not previously described in either organism.

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“…The recombinant Imp α protein from N. crassa (NcImp α ) was expressed as a truncated hexa-His fusion protein, consisting of residues 75–529, using the Escherichia coli host strain RosettaTM (DE3) pLysS (Novagen), as previously described [ 17 ]. The protein was purified using nickel affinity chromatography and eluted in a 0.15–3.0 M imidazole linear gradient, followed by dialysis.…”

Section: Methodsmentioning

confidence: 99%

Structure of Importin-α from a Filamentous Fungus in Complex with a Classical Nuclear Localization Signal

et al. 2015

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Neurospora crassa is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. To advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. Importin-α (Imp-α) is the receptor for cargo proteins that contain specific nuclear localization signals (NLSs) that play a key role in the classical nuclear import pathway. Structures of Imp-α from different organisms (yeast, rice, mouse, and human) have been determined, revealing that this receptor possesses a conserved structural scaffold. However, recent studies have demonstrated that the Impα mechanism of action may vary significantly for different organisms or for different isoforms from the same organism. Therefore, structural, functional, and biophysical characterization of different Impα proteins is necessary to understand the selectivity of nuclear transport. Here, we determined the first crystal structure of an Impα from a filamentous fungus which is also the highest resolution Impα structure already solved to date (1.75 Å). In addition, we performed calorimetric analysis to determine the affinity and thermodynamic parameters of the interaction between Imp-α and the classical SV40 NLS peptide. The comparison of these data with previous studies on Impα proteins led us to demonstrate that N. crassa Imp-α possess specific features that are distinct from mammalian Imp-α but exhibit important similarities to rice Imp-α, particularly at the minor NLS binding site.

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Biophysical Characterization of the Recombinant Importin-α from Neurospora crassa

Cited by 10 publications

References 0 publications

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation

Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor

Structure of Importin-α from a Filamentous Fungus in Complex with a Classical Nuclear Localization Signal

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