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

Abstract

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...

Splicing Factor hSlu7 Contains a Unique Functional Domain Required to Retain the Protein within the Nucleus

“…Additional mutations between the ␣ and ␤ NLS regions, without disrupting the putative zinc-knuckle conserved amino acids, were introduced and shown to have no effect on the cellular distribution of hSlu7 (Figure 4, V and VP; and EDE, at amino acid position 238 -240). We note that the spacer region of the NLS indeed was shown to tolerate point mutations without affecting localization (Robbins et al, 1991;Cokol et al, 2000;Romanelli and Morandi, 2002), which strengthens the functional specificity of the four identified NLS regions. Interestingly, fusing the NLS region only to the GFP protein increased its nuclear localization by 12% (Figure 4, B and C, GFP-NLS), even though GFP is a small enough protein to freely diffuse in and out of the nucleus (Xiao et al, 2003).…”

“…We assume that we did not affect the overlapping NLS, because only the basic amino acids (R/K) control the NLS's function (also see Robbins et al, 1991;Cokol et al, 2000;Romanelli and Morandi, 2002). Before mutational studies, and to predict the mutational affect on the three-dimensional structure of hSlu7's zinc-knuckle domain, we performed the following procedure.…”

Sequence Elements in Both Subunits of the DNA Fragmentation Factor Are Essential for Its Nuclear Transport

“…Bipartite cNLSs consist of two basic clusters connected by a 10-to 12-amino acid spacer following the consensus 2(K/R)-X 10 -12 -3(K/R) (22,25,28). In certain cases the spacer might exceed a length of up to 32 amino acids (53). Simultaneous interaction of the two basic stretches with the importin ␣ binding sites characterizes a bipartite cNLS (27).…”

The importance of RNA binding proteins in preproinsulin mRNA stability

“…PTB contains a nuclear export signal (NES) located to the first 25 residues of the N-terminal region, with amino acids 11-16 being the most important (Li and Yen, 2002). The first 60 amino acids of the N-terminal part of both PTB and PTBP2 also contain a nuclear localization sequence (NLS) (Perez et al, 1997;Romanelli and Morandi, 2002). The PTBP2 NLS sequence has been reported to bind to the import receptor importin- (Romanelli and Morandi, 2002), indicating that PTB and PTBP2 entry into the nucleus follows the classic nuclear import pathway.…”

“…The first 60 amino acids of the N-terminal part of both PTB and PTBP2 also contain a nuclear localization sequence (NLS) (Perez et al, 1997;Romanelli and Morandi, 2002). The PTBP2 NLS sequence has been reported to bind to the import receptor importin- (Romanelli and Morandi, 2002), indicating that PTB and PTBP2 entry into the nucleus follows the classic nuclear import pathway. It has also been shown that the export of PTB, unlike other hnRNPs, is uncoupled from RNA export.…”