Functional Analysis of the Hydrophobic Patch on Nuclear Transport Factor 2 Involved in Interactions with the Nuclear Porein Vivo

Abstract

Nuclear transport factor 2 (NTF2) is a small homodimeric protein that interacts simultaneously with both RanGDP and FxFG nucleoporins. The interaction between NTF2 and Ran is essential for the import of Ran into the nucleus. Here we use mutational analysis to dissect the in vivo role of the interaction between NTF2 and nucleoporins. We identify a series of surface residues that form a hydrophobic patch on NTF2, which when mutated disrupt the NTF2-nucleoporin interaction. Analysis of these mutants in vivo demonstrates that the strength of this interaction can be significantly reduced without affecting cell viability. However, cells cease to be viable if the interaction between NTF2 and nucleoporins is abolished completely, indicating that this interaction is essential for the function of NTF2 in vivo. In addition, we have isolated a dominant negative mutant of NTF2, N77Y, which has increased affinity for nucleoporins. Overexpression of the N77Y protein blocks nuclear protein import and concentrates Ran at the nuclear rim. These data support a mechanism in which NTF2 interacts transiently with FxFG nucleoporins to translocate through the pore and import RanGDP into the nucleus.Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs), 1 large proteinaceous channels that perforate the nuclear membrane. Protein cargoes to be imported into the nucleus contain internal sequences, termed nuclear localization signals (NLS), that target them to the nucleus (1-3). However, cargo does not interact directly with the NPC but is transported bound to soluble transport receptors of the importin-␤ family of proteins (4, 5). The recent purification and analysis of the yeast NPC indicates that the majority of the ϳ40 proteins that make up the NPC, collectively termed nucleoporins, are localized symmetrically throughout the pore complex (6). A subset of these proteins contains a phenylalanine-glycine (FG) repeat motif. These proteins appear to line the nuclear pore channel and can be subdivided further into either GLFG repeat-containing proteins or FxFG proteins (6,7). Although the precise mechanism by which the transport receptor-cargo complex translocates through the NPC is largely unknown, importin-␤ family transport receptors have been shown to interact with both the GLFG repeat and the FxFG repeat nucleoporins (8 -16) suggesting that these proteins are involved directly in the translocation process.The small GTPase Ran is central to nucleocytoplasmic transport. Although Ran is localized throughout the cell, ϳ80% is concentrated within the nucleus (17). The Ran guanine nucleotide exchange factor is tethered to DNA in the nucleus (18,19), suggesting that the majority of nuclear Ran is GTP-bound. On the other hand, RanGAP (Ran GTPase-activating protein) is predominantly cytoplasmic (20 -22), suggesting that the majority of cytoplasmic Ran is GDP-bound. Thus, the nucleotide bound state of Ran may act as a cellular marker that allows the transport machinery to distinguish between the nuclear and cytoplasmic compartme...

Identification and Characterization of the Arabidopsis Orthologs of Nuclear Transport Factor 2, the Nuclear Import Factor of Ran

“…Although the C-terminal similarity between the two Arabidopsis NTF2 orthologs and yeast NTF2 is striking, this is unlikely the reason for the lacking function of AtNTL in yeast, as human NTF2 can functionally replace yeast NTF2 (Corbett and Silver, 1996) but also has limited sequence similarity at the very C terminus. While it is currently not known why NTL is not functional as an NTF2, one notable difference is a reduction in the number of bulky hydrophobic amino acids compared to AtNTF2a and AtNTF2b, which are involved in interaction with the xFxFG repeats and Ran (Stewart et al, 1998a;Quimby et al, 2001).…”

“…In yeast, overexpression of wild-type NTF2 is slightly dominant negative (Quimby et al, 2001). To investigate the role of AtNTF2a in plant nuclear import, we tested the effect of AtNTF2a overexpression on the localization of a nuclear protein.…”

Nuclear Localization Signal Receptor Affinity Correlates with in Vivo Localization in Saccharomyces cerevisiae

“…Using a galactose-inducible multicopy expression vector, we tested the effects of overexpressing both the SV40-GFP-GFP and the BPSV40-GFP-GFP reporters on growth of wild-type yeast cells. As controls, we included the empty vector and a known dominant negative variant of the protein Ntf2 (39). As shown in Fig.…”

“…Overexpression of NLS Cargo-Wild-type cells (ACY192) or srp1-55 cells (ACY642) were transformed with galactoseinducible plasmids encoding GFP (pAC1350), SV40 NLS (ESPKKKRKVE)-GFP (pAC1352), bipartite SV40 NLS (KRTADGSEFESPKKKRKVE)-GFP (pAC1353), or as a control a known dominant negative mutant of yeast Ntf2 (N77Y Ntf2, pAC253) (14,26,39). Single transformants were grown in liquid culture to saturation, serially diluted (1:10), and spotted on minimal media plates containing 2% galactose or, as a control, 2% glucose.…”

The nuclear pore complex: structure and function

“…FG nucleoporins are located throughout the NPC (Rout et al, 2000;Fahrenkrog et al, 2002;Walther et al, 2002;Paulillo et al, 2005;Schwarz-Herion et al, 2007;Chatel and Fahrenkrog, 2012) and they are mediating the interaction to nuclear transport receptors. Crystal structures have shown that the interaction between FG repeats and transport receptors mainly involves the Phe residues of the FG repeats, together with the flanking Gly residues that provide conformational flexibility, and hydrophobic residues of the receptor (Quimby et al, 2001;Fribourg et al, 2001;Bayliss et al, 2002a;Bayliss et al, 2002b;Liu and Stewart, 2005;Vognsen et al, 2013). Karyopherins possess several FG-binding sites and their translocation through the NPC is accomplished due to multiple and rapid binding events to the FG-nucleoporins (Rexach and Blobel, 1995;Kutay et al, 1997;Bayliss et al, 2000;Allen et al, 2001;Gilchrist et al, 2002;Tetenbaum-Novatt et al, 2012).…”