The Nuclear Localization Signal of Lymphoid Enhancer Factor-1 Is Recognized by Two Differentially Expressed Srp1-Nuclear Localization Sequence Receptor Proteins

Abstract

Proteins are directed to the nucleus by their nuclear localization sequences (NLSs) in a multistep process. The first step, which is to dock the NLS-containing protein to the nuclear pore, is carried out in part by a recently identified NLS receptor named Srp1/importin-␣. Using the high mobility group (HMG) DNA binding domain of human lymphoid enhancer factor-1 (hLEF-1) as bait in a yeast two-hybrid screen, we have identified two different mouse Srp1 proteins (pendulin/importin-␣ and mSrp1) that each bind to a 9-amino acid sequence in hLEF-1 called the B box. We show that the B box of hLEF-1, a region essential for high affinity DNA binding, is also necessary and sufficient for nuclear localization, lending support to the model that NLSs can function both in nuclear transport and DNA binding. Pendulin and mSrp1 are the mouse homologues of hRch1/hSrp1␣/ importin-␣ and hSrp1/karyopherin ␣/NPI-1, respectively, and show considerable sequence divergence from each other. We find a surprising and significant difference in the expression pattern of pendulin and mSrp1 mRNA, suggesting that these two Srp1 proteins are distinguishable in function as well as sequence.Analysis of the nuclear localization of transcription regulatory factors has largely focused on defining the amino acid sequences within transcription factor proteins that direct nuclear entry. A universal consensus sequence that signals nuclear localization (NLS) 1 has not been found; nevertheless short regions, rich in basic residues are a feature common to most identified NLSs (1, 2). It is also common for the NLS of transcription factors to be in regions that overlap or are near the DNA binding domain, prompting speculation that (a) nuclear occupancy by a protein is due to both nuclear import and retention by DNA binding, (b) NLS sequences contribute to DNA binding, or (c) there is coordinate regulation of the two functions due to their proximity (3). It is known that the NLS directs transport through nuclear pores via a receptor-mediated, multistep process (4 -6). Recently, a cytosolic NLS receptor protein in Xenopus named importin-␣ was found to carry out the first step along with the Xenopus importin-␤ subunit (also known as p97 or karyopherin ␤) by docking NLS-containing proteins at the nuclear pore (7). Xenopus importin-␣ is homologous to yeast Srp1p, an essential gene first identified as a suppressor of RNA polymerase I mutations in Saccharomyces cerevisiae (8). Immunofluorescence studies localized ySrp1p mainly to the nuclear pore, and genetic and biochemical evidence established a tight association of ySrp1p with known protein components of the pore (8, 9). More recently, work with human Srp1␣ (hSrp1␣; also known as hRch1) and its mouse homolog pendulin/importin-␣, and hSrp1/karyopherin ␣/NPI-1, a different human Srp1 protein, has confirmed in vivo and in vitro that Srp1 proteins and p97/karyopherin ␤ function as cytosolic NLS receptors that usher NLS-containing proteins to the cytoplasmic side of the nuclear pore, where additional GTP-requiring fac...

Drosophila Importin α1 Performs Paralog-Specific Functions Essential For Gametogenesis

“…An exception is the spermatogenesis defect of Da2 mutant flies that is rescued by any of the three paralogs (Mason et al 2002). It is established that importin a1's each have both shared and distinct cargo repertoires (Michaud and Goldfarb 1993;Prieve et al 1996;Miyamoto et al 1997;Prieve et al 1998;Kohler et al 1999;Talcott and Moore 2000;Fagerlund et al 2002;Quensel et al 2004;Lange et al 2007). The simplest explanations for the paralog-specific phenotypes associated with Da1, Da2, and Da3 mutants invoke deficiencies in the nuclear import of their distinct NLS cargoes.…”

“…Although importin a1, a2, and a3 proteins are coexpressed in many adult tissues, they exhibit complex temporal and spatial expression patterns during development (see Kamei et al 1999;Hogarth et al 2006). Animal importin a1's, a2's, and a3's all mediate the import of classical NLS-containing cargoes and, in addition, each paralog is specialized to bind and mediate the import of distinct repertoires of NLS cargoes (for example, see Michaud and Goldfarb 1993;Prieve et al 1996;Miyamoto et al 1997;Prieve et al 1998;Kohler et al 1999;Talcott and Moore 2000;Fagerlund et al 2002;Quensel et al 2004;Lange et al 2007). Importin a's may also mediate the import of a few cargoes independent of b1 (Hubner et al 1999;Miyamoto et al 2002;Kotera et al 2005).…”

A New β-Catenin-dependent Activation Domain in T Cell Factor

“…Immunofluorescence-Immunofluorescence detection of TCF-1E proteins in transiently transfected COS cells was performed as described previously with the following modifications (24). Cells were transfected by electroporation and plated on glass coverslips.…”

Thrombin Causes a Marked Delay in Skeletal Myogenesis That Correlates with the Delayed Expression of Myogenin and p21

“…Immunofluoresence and Immunoblotting-Immunofluoresence analysis was performed on differentiating myoblasts grown in paranox chamber slides (Nunc Corp.) as described by Prieve et al (21). Antiskeletal myosin was used at a 1:500 dilution.…”