Karyopherin β2 mediates nuclear import of a mRNA binding protein

Bonifaci, Neris; Moroianu, Junona; Radu, Aurelian; Blobel, Günter

doi:10.1073/pnas.94.10.5055

Cited by 154 publications

(148 citation statements)

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“…Kap ␤2B has 84% amino acid identity and 92% homology with Kap ␤2A, which was shown to function in nuclear import of heterogeneous ribonucleoproteins (31)(32)(33). The nonidentical residues are scattered all along the sequences, and the longest segment of contiguous nonidentical residues has a length of only nine residues.…”

Section: Discussionmentioning

confidence: 99%

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Karyopherin β2B participates in mRNA export from the nucleus

Shamsher

Ploski

Radu

2002

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

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Transport of macromolecules between the cell nucleus and cytoplasm occurs through the nuclear pores and is mediated by soluble carriers known as karyopherins (Kaps), transportins, importins, or exportins. We report that Kap ␤2B (transportin-2) forms complexes with the mRNA export factor TAP in the presence of RanGTP, as shown by coimmunoprecipitation from HeLa cells. The interaction strictly depends on the presence of RanGTP. In digitoninpermeabilized cells, Kap ␤2B mediates TAP-GFP export from the nuclei in the presence of RanGTP. A TAP mutant that does not coimmunoprecipitate with Kap ␤2B is also not exported by Kap ␤2B. In the permeabilized cells assay, TAP is also exported independently of Kap ␤2B by direct interaction with nucleoporins, in agreement with previous reports. The export rate is, however, significantly lower than the Kap ␤2B-mediated pathway. Both Kap ␤2B and TAP are present and enriched in the poly(A) ؉ RNA complexes isolated from HeLa cell nuclear lysates. Poly(A) ؉ RNA strongly accumulates in the nuclei of HeLa cells treated with Kap ␤2B short interfering RNA, indicating that Kap ␤2B is involved in the export of at least a large proportion of the mRNA species. The export of ␤-actin and GAPDH mRNA is also inhibited, whereas 28S RNA is not affected. The data support the conclusion that Kap ␤2B participates directly in the export of a large proportion of cellular mRNAs, and TAP connects Kap ␤2B to the mRNAs to be exported.T rafficking of proteins and nucleic acids in or out of the cell nucleus occurs through the nuclear pores (1-5). Macromolecules below a certain size limit (40-60 kDa for proteins) can diffuse through the pores, whereas species above the size limit (and some smaller species) are transported by a complex system of soluble carriers generally known as karyopherins (Kaps), transportins, importins, or exportins (6-10). The carriers bind their cargoes either in the cytoplasm or in the nucleoplasm, dock them to components of the nuclear pore complexes, and assist their passage across the nuclear envelope. Most Kaps belong to the Kap ␤ family and are characterized by the ability to bind the small GTPase Ran that regulates the interaction of Kaps with their cargoes (11,12). Ran is present in the nucleus in the GTP-bound form and has opposite action on the import versus the export complexes. Ran-GTP induces release of the cargoes from the Kaps that mediated their import and facilitates binding of the export Kaps to the cargoes that are subsequently exported.In mRNA export several conserved factors are required in both metazoans and yeast: Mex67 (the orthologue of the mammalian TAP͞NXF1), Mtr2, Yra1, Sub2, Dbp5, Gle1, Gle2, members of the IP6 pathway, pre-mRNA-processing factors, and the metazoan counterparts (12-23). Among these proteins TAP plays a prominent role by mediating the translocation step across the nuclear pore of cellular and viral mRNAs by direct interaction with nucleoporins (24-30).Contrary to most nucleocytoplasmic export pathways characterized to date, no Kap ␤ family me...

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Section: Discussionmentioning

confidence: 99%

“…Kap ␤2A was purified as described (33). Full-length Kap ␤2B amplified by PCR from human brain cDNA (CLON-TECH) was sequenced (GenBank accession no.…”

Section: Methodsmentioning

confidence: 99%

Karyopherin β2B participates in mRNA export from the nucleus

Shamsher

Ploski

Radu

2002

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

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“…Karyopherin ␣ recognizes the nuclear localization signals (NLS) and nuclear export signals (NES) of substrates that are being transported, but requires karyopherin ␤1 to dock on to nucleoporins. 17,[26][27][28] Once docked, the small GTPase Ran and p10 (NTF2) facilitate entry into or out of the nucleus.…”

Section: Figurementioning

confidence: 99%

NUP98 gene fusions in hematologic malignancies

Lam

Aplan

2001

Leukemia

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Acute leukemia is associated with a wide spectrum of recurrent, non-random chromosomal translocations. Molecular analysis of the genes involved in these translocations has led to a better understanding of both the causes of chromosomal rearrangements as well as the mechanisms of leukemic transformation. Recently, a number of laboratories have cloned translocations involving the NUP98 gene on chromosome 11p15.5, from patients with acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), and T cell acute lymphoblastic leukemia (T-ALL). To date, at least eight different chromosomal rearrangements involving NUP98 have been identified. The resultant chimeric transcripts encode fusion proteins that juxtapose the N-terminal GLFG repeats of NUP98 to the C-terminus of the partner gene. Of note, several of these translocations have been found in patients with therapy-related acute myelogenous leukemia (t-AML) or myelodysplastic syndrome (t-MDS), suggesting that genotoxic chemotherapeutic agents may play an important role in generating chromosomal rearrangements involving NUP98. Leukemia (2001) 15, 1689-1695.

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“…Importins a consist of two structural and functional domains, a short basic N-terminal importin b binding (IBB) domain, and a large NLS-binding domain comprising armadillo (Arm) repeats [17][18][19]. Crystal structures of karyopherins a complexes with NLS peptide have revealed the determinants of specificity for the binding of NLS sequences [20][21][22].A number of NLS sequences that do not conform to the classical NLS consensus motif have also been identified, such as the M9 sequence, present in the hnRNP A1, which is recognized by transportin (karyopherin-b2), rich in glycine rather than basic residues [23][24][25]. A unique signal, called KNS, which allows nuclear transport via a mechanism independent of soluble factors has also been described in the hnRNP type K [26].…”

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“…A number of NLS sequences that do not conform to the classical NLS consensus motif have also been identified, such as the M9 sequence, present in the hnRNP A1, which is recognized by transportin (karyopherin-b2), rich in glycine rather than basic residues [23][24][25]. A unique signal, called KNS, which allows nuclear transport via a mechanism independent of soluble factors has also been described in the hnRNP type K [26].…”

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Importin α binds to an unusual bipartite nuclear localization signal in the heterogeneous ribonucleoprotein type I

Romanelli

Morandi

2002

European Journal of Biochemistry

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The heterogeneous nuclear ribonucleoprotein (hnRNP) type I, a modulator of alternative splicing, localizes in the nucleoplasm of mammalian cells and in a discrete perinucleolar structure. HnRNP I contains a novel type of bipartite nuclear localization signal (NLS) at the N-terminus of the protein that we have previously named nuclear determinant localization type I (NLD-I). Recently, a neural counterpart of hnRNP I has been identified that contains a putative NLS with two strings of basic amino acids separated by a spacer of 30 residues. In the present study we show that the neural hnRNP I NLS is necessary and sufficient for nuclear localization and represents a variant of the novel bipartite NLS present in the NLD-I domain. Furthermore, we demonstrate that the NLD-I is transported into the nucleus by cytoplasmic factor(s) with active transport modality. Binding assays using recombinant importin a show an interaction with NLD-I similar to that of SV40 large T antigen NLS. Deletion analysis indicates that both stretches of basic residues are necessary for binding to importin a. The above experimental results lead to the conclusion that importin a acts as cytoplasmic receptor for proteins characterized by a bipartite NLS signal that extends up to 37 residues.Keywords: heterogeneous ribonucleoprotein-I; polypyrimidine tract-binding protein; PTB; nuclear localization signal; importin a.Transport of proteins and RNA into and out of the nucleus occurs through nuclear pore complexes (NPCs), which are plugged through the double membrane of the nuclear envelope [1,2]. Small molecules and ions may pass the NPC passively, while macromolecules larger than 40-45 kDa are actively transported through the NPC. The active nuclear import and export of proteins is mediated by specific aminoacid sequences that are referred as nuclear localization signals (NLSs) [3,4] and nuclear export signal (NESs) [5]. At least two different types of ÔclassicalÕ NLSs have been defined: a short stretch of basic amino acids, exemplified by the SV40 large T antigen NLS (T-ag PKKKRKV) [6] and a bipartite NLS composed of two stretches of basic amino acids separated by a spacer of 10-12 amino acids, exemplified by nucleoplasmin (KRPAATKKAGQAKKKK). The two sets of basic residues of bipartite-type NLS are required for sufficient nuclear localization, while the spacer is mutant-tolerant in sequence [7]. NLSs are usually recognized by the heterodimeric import receptor complex comprising importins a and b, also named karyopherins [8,9]. Importins a contain the NLS-binding site and importins b are responsible for the docking of the importin-substrate complex to the cytoplasmic side of the NPC and its subsequent translocation through the pore. Transfer through the pore of importin-NLS protein complex requires two additional soluble proteins, RanGTPase and nuclear transport factor-2 (NTF2) [1]. Once inside the nucleus, Ran-GTP binding to importin b causes the dissociation of the import complex and release of the cargo [10,11]. The directionality of the nuclear...

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Karyopherin β2 mediates nuclear import of a mRNA binding protein

Cited by 154 publications

References 31 publications

Karyopherin β2B participates in mRNA export from the nucleus

Karyopherin β2B participates in mRNA export from the nucleus

NUP98 gene fusions in hematologic malignancies

Importin α binds to an unusual bipartite nuclear localization signal in the heterogeneous ribonucleoprotein type I

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