Molecular Basis for the Recognition of Snurportin 1 by Importin β

Abstract

The nuclear import of uridine-rich ribonucleoproteins is mediated by the transport adaptor snurportin 1 (SNP1). Similar to importin ␣, SNP1 uses an N-terminal importin ␤ binding (sIBB) domain to recruit the receptor importin ␤ and gain access to the nucleus. In this study, we demonstrate that the sIBB domain has a bipartite nature, which contains two distinct binding determinants for importin ␤. The first determinant spans residues 25-65 and includes the previously identified importin ␣ IBB (␣IBB) region of homology. The second binding determinant encompasses residues 1-24 and resembles region 1011-1035 of the nucleoporin 153 (Nup153). The two binding determinants synergize within the sIBB domain to confer a low nanomolar binding affinity for importin ␤ (K d ϳ 2 nM) in an interaction that, in vitro, is displaced by RanGTP. We propose that in vivo the synergy of Nup153 and nuclear RanGTP promotes translocation of uridine-rich ribonucleoproteins into the nucleus.Nuclear import of proteins and nucleic acids is an active signal-mediated process that requires, in most cases, soluble transport factors and GTP hydrolysis by the GTPase Ran (1-4). Nuclear transport factors are sorted into two categories: importins and exportins (also known as karyopherins), which include 14 members in budding yeast and at least 20 in humans (5, 6). All karyopherins fold into superhelical solenoids, which present an external convex surface involved in nucleoporin binding and a concave internal face that interacts with transport cargos and RanGTP. The two best characterized importins, human importin ␤1 and yeast karyopherin ␤2, have been visualized using crystallographic methods bound to specific import cargos (7-10) and RanGTP (11,12). These structures have provided invaluable information on the structural flexibility of karyopherins and their ability to undergo conformational changes during the import process (13-16).The best understood nuclear import pathway involves cargos bearing a classical SV40-like nuclear localization sequence (PKKKRKV) (17). These cargos are imported into the nucleus by the adaptor importin ␣ and the receptor importin ␤ with the aid of RanGTP. GTP hydrolysis by Ran energizes the import reaction by promoting both release of the import complex from nucleoporins as well as unloading of import cargos into the nucleus (1-3). In addition to classical nuclear localization sequence cargos, uridine-rich ribonucleoproteins (U snRNPs) 2 represent an important class of import cargos. Mature U snRNP particles are assembled in the cytoplasm and imported into the nucleus in at least two distinct pathways, both dependent on importin ␤ (18). In the first pathway, the import signal is in the proteinaceous core of the U snRNP formed by the Sm proteins (18 -21). In the second pathway, the trimethylated guanosine cap of the mature U snRNP is recognized by the adaptor snurportin 1 (SNP1) (18 -21), which, in turn, recruits importin ␤. SNP1 consists of an N-terminal IBB domain (sIBB) similar to that found in importin ␣ (␣IBB) and a larg...

Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)

“…Indeed many of the key residues indentified in Importin-13 as directly binding RanGTP are also present in TNPO3 (51). The C-terminal portions of these karyopherins usually bind their respective cargos (69,(77)(78)(79)(80)(81)(82)(83). However, two exceptions have been reported with Ubc9 and parathyroid hormone-related protein interacting with the N-terminal arches of Importin-13 and Importin-␤, respectively (40,66).…”

The Importin β Binding Domain Modulates the Avidity of Importin β for the Nuclear Pore Complex

“…sIBB(25-3A-65) was constructed by long PCR on the sIBB(25-65) template. GST-IBBs were expressed and purified as described (10). To form homogeneous complexes of importin ␤ bound to different IBB domains, each IBB construct was expressed as a GST fusion, immobilized on glutathione beads (GE Healthcare), and washed extensively in a low salt buffer (20 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM ␤-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride).…”

“…The beads were imaged using a Leika CTR5000 with a 20ϫ air objective. Pulldown assay was carried out as previously described (10).…”

“…SNP1 residues 1-24, referred to as sn , has sequence homology to Nup153 and contains a functional nuclear export signal for SNP1 (14,15), which in vitro binds importin ␤ with a K d value of ϳ30 M (10). In contrast, sIBB contains an ␣IBB-like region of homology that binds importin ␤ with nanomolar affinity (10).…”

“…Similar to importin ␣, SNP1 contains an N-terminal IBB domain (sIBB), whereas the rest of the protein folds into an trimethylated guanosine cap-binding domain that resembles the GTP-binding domain of mRNA-guanylyltransferase but has no significant sequence or structural similarity to importin ␣ (37). The sIBB domain encompasses residues 1-65 (32) and presents a bipartite structure (10). SNP1 residues 1-24, referred to as sn , has sequence homology to Nup153 and contains a functional nuclear export signal for SNP1 (14,15), which in vitro binds importin ␤ with a K d value of ϳ30 M (10).…”