A Novel Class of RanGTP Binding Proteins

Görlich, Dirk; Dabrowski, M.; Bischoff, F. Ralf; Kutay, Ulrike; Bork, Peer; Hartmann, Enno; Prehn, Siegfried; Izaurralde, Elisa

doi:10.1083/jcb.138.1.65

Cited by 403 publications

(439 citation statements)

References 80 publications

(168 reference statements)

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“…The similarity is higher in the N-terminal half, which mediates RanGTP binding (20). A similar repetitive pattern with a loose consensus of hydrophobic residues can be identified in all the sequences.…”

Section: -4)mentioning

confidence: 68%

Conformational Variability of Nucleo-cytoplasmic Transport Factors

Fukuhara

Fernández

Ebert

et al. 2004

Journal of Biological Chemistry

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The transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is largely mediated by a single family of transport factors, the karyopherin or importin ␤-like family. Structural and biochemical evidence suggests conformational flexibility of these modular HEAT-repeat proteins is crucial for their regulation. Here we use small angle x-ray scattering to assess the extent of conformational variation within a set of nuclear import and export factors. The study reveals that importin ␤, transportin, and the exportin Xpo-t share a similar S-like superhelical conformation in their unbound state. There are no obvious differences in the overall structures that might generally distinguish nuclear export from nuclear import mediators. Two other members of the family, the exportins Cse1 and Xpo1, possess a significantly more globular conformation, indicating that the extended S-like architecture is not a hallmark of all karyopherins. Binding of RanGTP/cargo to importin ␤, transportin, and Xpo-t triggers distinct conformational responses, suggesting that even closely related karyopherins employ different mechanisms of conformational regulation and that cargo and nuclear pore-interacting surfaces of the different receptors may be unique.Nucleo-cytoplasmic transport factors of the karyopherin ␤ family shuttle through nuclear pore complexes spanning the nuclear envelope and mediate the nuclear import and/or export of specific protein or RNA cargoes (for review, see Refs.

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Section: -4)mentioning

confidence: 68%

Conformational Variability of Nucleo-cytoplasmic Transport Factors

Fukuhara

Fernández

Ebert

et al. 2004

Journal of Biological Chemistry

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“…The following proteins were expressed in E. coli BLR/Rep4 and purified as described in the literature indicated: Xenopus importin ␣ (29), human importin ␤ (30), transportin (21), Xenopus importin 7, and importin 5 (28). Expression and purification of Ran, RanQ69L(GTP), NTF2, L23a 2z and 4z fusion proteins, the IBB-6z fusion protein, and the M9-GST fusion protein was performed as described (13,14).…”

Section: Methodsmentioning

confidence: 99%

“…Because, for example, Imp␤ and Imp7 are able to form heterodimers (28), Imp␤ retained by the histone matrix can be bound by Imp7 and thus may incorrectly indicate the binding of Imp7 to the histone. To assess whether direct binding of Imp␤ and Imp7 occurs in vitro, we performed binding experiments with recombinant Imp␤ and Imp7.…”

Section: Fig 1 the Imp␤-imp7 Heterodimer Is The Only Functional Impmentioning

confidence: 99%

The Requirement of H1 Histones for a Heterodimeric Nuclear Import Receptor

Bäuerle¹,

Doenecke²,

Albig³

2002

Journal of Biological Chemistry

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After synthesis in the cytoplasm, H1 histones are imported into the nucleus through an energy-dependent process that can be mediated by an importin ␤-importin 7 (Imp␤-Imp7) heterodimer. H1 histones contain two structurally different types of nuclear localization signals (NLS). The first type of NLS resides within the unstructured C-terminal domain and is rich in basic amino acids. In contrast, the highly conserved central domain of the H1 histone contains comparatively few basic amino acids but also represents a functional NLS. The competence for the nuclear import of this globular domain seems to be based on its secondary structure. Here, we show that the Imp␤-Imp7 heterodimer is the only receptor for H1 import. Furthermore, we identified the import receptors mediating the in vitro transport of different NLS of the H1 histone. Using the digitoninpermeabilized cell import assay we show that Imp␤ is the most efficient import receptor for the globular domain of H1 histones, whereas the heterodimer of Imp␤ and Imp7 is the functional receptor for the entire Cterminal domain. However, short fragments of the Cterminal domain are imported in vitro by at least four different importins, which resembles the import pathway of ribosomal proteins and core histones. In addition, we show that heterodimerization of Imp␤ with Imp7 is absolutely necessary for their proper function as an import receptor for H1 histones. These findings point to a chaperone-like function of the heterodimeric complex in addition to its function as an import receptor. It appears that the Imp␤-Imp7 heterodimer is specialized for NLS consisting of extended basic domains.Histones are the protein component of the fundamental structural unit of eukaryotic chromatin, the nucleosome. The nucleosome is composed of a core particle consisting of a histone octamer with 146 bp of DNA bent around its surface and an approximately 50-bp linker DNA connecting two adjacent core particles. This linker DNA is associated with H1 histones; hence they are termed linker histones (1). The four core histones H2A, H2B, H3, and H4 form the histone octamer. Histones are small, very basic proteins. The linker histone H1 is highly enriched in lysine, H2A and H2B are moderately lysinerich, and H3 and H4 are rich in arginine. The size of core histones varies between 11 kDa (H4) and 15 kDa (H3), whereas the H1 histones have a size of ϳ22 kDa. In mammals the class of H1 histones, the linker histones, comprises seven different subtypes, termed H1.1-H1.5, H1°, and H1t (2). Histones are composed of three domains, the very basic, unstructured C-and N-terminal domains and the hydrophobic, central globular domain, which forms the histone fold motif in the case of the core histones (3) and is involved in histone-histone interactions. During the S phase of the cell cycle, a vast amount of newly synthesized histones has to be imported into the nucleus for the formation of nucleosomes on newly replicated DNA. Thus, histones are among the most abundant substrates for nuclear transport during the...

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“…Nucleocytoplasmic transport is mediated largely by the superfamily of transport receptors that interact with nuclear pore complexes (NPCs), share an N-terminal RanGTP-binding motif and are related to importin ␤ (8,9). CRM1 binds its cargo in the nucleus, translocates it to the cytoplasm, releases the cargo, and returns back to the nucleus.…”

mentioning

confidence: 99%

“…Within Rev, the region Leu-64 to Arg-80 was protected by CRM1, whereas Rev specifically interacts with residue Asp-716 and the neighborhood of Lys-810 of CRM1 (17). RanGTP binding to CRM1 is expected to be mediated by a region near the N terminus (8,9). A comparison of leucine-rich NESs of several proteins revealed that the Rev NES has a relatively low affinity for CRM1 (18).…”

mentioning

confidence: 99%

A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export

Daelemans

Afonina

Nilsson

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

119

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The HIV-1 Rev protein is an essential regulator of the HIV-1 mRNA expression that promotes the export of unspliced and partially spliced mRNA. The export receptor for the leucine-rich nuclear export signal (NES) of Rev has recently been recognized as CRM1. We identified a low molecular weight compound PKF050-638 as an inhibitor of HIV-1 Rev. This drug inhibits in a dose-dependent fashion Rev-dependent mRNA expression in a cellular assay for Rev function. We show that PKF050-638 is an inhibitor of the CRM1-mediated Rev nuclear export. By using a quantitative in vitro CRM1-NES cargo-binding assay, we could demonstrate that PKF050-638 disrupts CRM1-NES interaction. This mode of action is confirmed in cell culture because the drug reversibly interferes with the colocalization of CRM1 and Rev in the nucleolus of the cell. In addition, we prove that the inhibition is through direct interaction of the compound with Cys-539 of CRM1. These effects are similar to those of the known CRM1 inhibitor leptomycin B and suggest that the inhibitory effect of the compound is caused by binding to CRM1 at a similar site. The compound displayed strict structural requirements for its activity, as its enantiomer was inactive in all assays tested. These results show that we identified a drug that interferes with the CRM1-mediated nuclear export of Rev through inhibition of the CRM1-NES complex formation. The reversibility of its binding to CRM1 and its availability through chemical synthesis could make it useful for studying CRM1-mediated export pathways.T he Rev protein is an essential factor for HIV replication and promotes the export of unspliced or partially spliced mRNA responsible for the production of the viral structural proteins. Rev is an 18-kDa protein that has been shown to shuttle continuously between the nucleus and the cytoplasm (1, 2). Nuclear import of Rev is mediated by its nuclear localization signal (NLS) embedded in the RNA-binding domain that binds a unique RNA stem-loop structure termed the Rev responsive element (RRE). The function of the Rev NLS in the context of Rev is apparently multimerization dependent. Mutants defective in multimerization do not accumulate in the nucleus. Instead, these mutants are localized throughout the cell, although their NLS is completely intact (3, 4). Nuclear export of Rev is mediated by its leucine-rich nuclear export signal (NES) and is known to use the CRM1 export factor to export the viral RNA from the nucleus to the cytoplasm. CRM1 is a nuclear export receptor for proteins carrying the leucine-rich NES (5-7).Nucleocytoplasmic transport is mediated largely by the superfamily of transport receptors that interact with nuclear pore complexes (NPCs), share an N-terminal RanGTP-binding motif and are related to importin ␤ (8, 9). CRM1 binds its cargo in the nucleus, translocates it to the cytoplasm, releases the cargo, and returns back to the nucleus. The RanGTPase cycle is key to promoting the directionality of this transport (reviewed in refs. 10 and 11). Export receptors bind their...

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A Novel Class of RanGTP Binding Proteins

Cited by 403 publications

References 80 publications

Conformational Variability of Nucleo-cytoplasmic Transport Factors

Conformational Variability of Nucleo-cytoplasmic Transport Factors

The Requirement of H1 Histones for a Heterodimeric Nuclear Import Receptor

A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export

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