The transport of histones from the cytoplasm to the nucleus of the cell, through the nuclear membrane, is a cellular process that regulates the supply of new histones in the nucleus and is key for DNA replication and transcription. Nuclear import of histones is mediated by proteins of the karyopherin family of nuclear transport receptors. Karyopherins recognize their cargos through linear motifs known as nuclear localization/export sequences or through folded domains in the cargos. Karyopherins interact with nucleoporins, proteins that form the nuclear pore complex, to promote the translocation of their cargos into the nucleus. When binding to histones, karyopherins not only function as nuclear import receptors but also as chaperones, protecting histones from non-specific interactions in the cytoplasm, in the nuclear pore and possibly in the nucleus. Studies have also suggested that karyopherins might participate in histones deposition into nucleosomes. In this review we describe structural and biochemical studies from the last two decades on how karyopherins recognize and transport the core histone proteins H3, H4, H2A and H2B and the linker histone H1 from the cytoplasm to the nucleus, which karyopherin is the major nuclear import receptor for each of these histones, the oligomeric state of histones during nuclear import and the roles of post-translational modifications, histone-chaperones and RanGTP in regulating these nuclear import pathways.
Previously we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus (Padavannil et al. 2019). However, unlike most nuclear import systems where RanGTP dissociates cargoes from their importins, RanGTP binds stably to the Importin-9•H2A-H2B complex and formation of RanGTP•Importin-9•H2A-H2B facilitates H2A-H2B release to the assembling nucleosome (Padavannil et al. 2019). Here we show cryo-EM structures of Importin-9•RanGTP and of its yeast homolog Kap114, including Kap114•RanGTP, Kap114•H2A-H2B, and RanGTP•Kap114•H2A-H2B. In combination with hydrogen-deuterium exchange analysis of Importin-9 complexes and nucleosome assembly assays, we explain how the conserved Kap114/Importin-9 importins bind H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. We show that RanGTP binds to the N-terminal repeats of Kap114/Importin-9 as in Kap114/Importin-9•RanGTP, and H2A-H2B binds via its acidic patch to the Kap114/Importin-9 C-terminal repeats as in Kap114/Importin-9•H2A-H2B. RanGTP-binding in RanGTP•Kap114•H2A-H2B changes Kap114/Importin-9 conformation such that it no longer contacts the surface of H2A-H2B proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome. The reduced affinity of RanGTP for Kap114/Importin-9 when H2A-H2B is bound may ensure release of H2A-H2B only at chromatin.
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.
IMPORTIN-4, the primary nuclear import receptor of core histones H3 and H4, binds the H3–H4 dimer and histone chaperone ASF1 prior to nuclear import. However, how H3–H3–ASF1 is recognized for transport cannot be explained by available crystal structures of IMPORTIN-4–histone tail peptide complexes. Our 3.5-Å IMPORTIN-4–H3–H4–ASF1 cryoelectron microscopy structure reveals the full nuclear import complex and shows a binding mode different from suggested by previous structures. The N-terminal half of IMPORTIN-4 clamps the globular H3–H4 domain and H3 αN helix, while its C-terminal half binds the H3 N-terminal tail weakly; tail contribution to binding energy is negligible. ASF1 binds H3–H4 without contacting IMPORTIN-4. Together, ASF1 and IMPORTIN-4 shield nucleosomal H3–H4 surfaces to chaperone and import it into the nucleus where RanGTP binds IMPORTIN-4, causing large conformational changes to release H3–H4–ASF1. This work explains how full-length H3–H4 binds IMPORTIN-4 in the cytoplasm and how it is released in the nucleus.
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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