Nuclear protein import is decreased by engineered mutants of nuclear transport factor 2 (NTF2) that do not bind GDP-Ran

Wd, Clarkson; Ah, Corbett; Bm, Paschal; Hm, Kent; Aj, McCoy; Gerace, Larry; Pa, Silver; Stewart, Murray

doi:10.1006/jmbi.1997.1255

Cited by 70 publications

(92 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus Tap may rely more on NPC binding domains other than the Tap-UBA for its interaction with GLFG-containing nucleoporins such as Nup98. Such specificity of transport receptors for repeat motifs has been demonstrated for NTF2, which binds FxFG with micromolar affinity but cannot bind GLFG motifs (Clarkson et al, 1997). A similar case has been described for importin ␤ in a study comparing the affinity of its two NPC-binding sites (Bednenko et al, 2003).…”

Section: The Two Npc-binding Domains Of Tap Interact With Nucleoporinsupporting

confidence: 55%

“…It is estimated that more than 3500 FG repeats are distributed throughout each NPC (Strawn et al, 2004). Recent evidence suggests that transport receptors interact with distinct subsets of FG repeats (Clarkson et al, 1997;Damelin and Silver, 2000;Strawn et al, 2001;Blevins et al, 2003;Strawn et al, 2004). This could provide part of the basis for coordinating bidirectional flow of traffic through the NPC, which is estimated to approach 1000 molecules per second (Ribbeck and Gorlich, 2001).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

Lévesque

Bor

Matzat

et al. 2006

MBoC

View full text Add to dashboard Cite

Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins drive the translocation of receptor-cargo complexes through nuclear pores. Tap, a transport receptor that mediates nuclear export of cellular mRNAs, contains a UBA-like and NTF2-like folds that can associate directly with FG repeats. In addition, two nuclear export sequences (NESs) within the NTF2-like region can also interact with nucleoporins. The Tap-RNA complex was shown to bind to three nucleoporins, Nup98, p62, and RanBP2, and these interactions were enhanced by Nxt1. Mutations in the Tap-UBA region abolished interactions with all three nucleoporins, whereas the effect of point mutations within the NTF2-like domain of Tap known to disrupt Nxt1 binding or nucleoporin binding were nucleoporin dependent. A mutation in any of these Tap domains was sufficient to reduce RNA export but was not sufficient to disrupt Tap interaction with the NPC in vivo or its nucleocytoplasmic shuttling. However, shuttling activity was reduced or abolished by combined mutations within the UBA and either the Nxt1-binding domain or NESs. These data suggest that Tap requires both the UBA-and NTF2-like domains to mediate the export of RNA cargo, but can move through the pores independently of these domains when free of RNA cargo. INTRODUCTIONNuclear transport plays an important role in cell function by selectively segregating macromolecules to the nuclear or cytoplasmic compartments. This biased distribution contributes to the regulation of many proteins including transcription factors, cell cycle regulators, and cell signaling components (Komeili and O'Shea, 2000;Macara, 2001;Carmo-Fonseca, 2002). Regulated nuclear export of mRNA serves as a quality control step to ensure that only properly spliced mRNA is exported to the cytoplasm for translation (Lei and Silver, 2002;Stutz and Izaurralde, 2003). All nucleocytoplasmic traffic must go through large, proteinaceous channels known as nuclear pore complexes (NPCs; Suntharalingam and Wente, 2003). Although small molecules (Ͻ40 kDa) can diffuse freely through the NPC, movement of most proteins and RNAs across nuclear pores requires binding to soluble import or export receptors (Macara, 2001;Weis, 2002;Bednenko et al., 2003;Pemberton and Paschal, 2005). Import and export receptors, in turn, mediate protein and RNA translocation through highly transient interactions with nucleoporins in the NPC.The majority of nuclear transport receptors belong to the karyopherin/importin ␤ family of proteins (Macara, 2001;Bednenko et al., 2003). Crm1, the best-characterized export receptor and a member of the importin ␤ family, mediates the export of proteins, U snRNAs, and 5S RNAs (Fornerod et al., 1997;Mattaj and Englmeier, 1998;Cullen, 2003). The transport receptor believed to be responsible for the bulk of mRNA export in eukaryotes is Tap/NXF1, a factor that is unrelated to the importin ␤ family (Katahira et al., 1999;Cullen, 2003). Tap also mediates nuclear export of certain retroviral mRNAs, such as the Mas...

show abstract

Section: The Two Npc-binding Domains Of Tap Interact With Nucleoporinsupporting

confidence: 55%

mentioning

confidence: 99%

Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

Lévesque

Bor

Matzat

et al. 2006

MBoC

View full text Add to dashboard Cite

show abstract

“…Previous work consistent with this hypothesis has shown that residue Trp-7 of rNTF2 contributes to the interaction between rNTF2 and FxFG nucleoporins (35) and that mutation of Trp-7 of rNTF2 to alanine reduces binding to FxFG nucleoporins (35). Moreover, in crystal structures of both wild-type and mutant rNTF2 (35,45), the hydrophobic patch surrounding Trp-7 is often involved in hydrophobic crystal contacts between adjacent molecules in the crystal lattice (29,45). In particular, close interactions between the aromatic ring of Phe-126 and both Trp-7 and Trp-112 are found in the crystal packing of the W7A-rNTF2 mutant (35).…”

Section: Modeling Of Saccharomyces Cerevisiae Ntf2p Reveals a Surfacementioning

confidence: 67%

“…Consistent with the role of NTF2 in importing Ran into the nucleus, NTF2 has been shown to interact at non-overlapping sites with both Ran and a subset of nuclear pore proteins containing FxFG repeats (27). NTF2 specifically interacts with the GDP-bound form of Ran, and this interaction has been extensively characterized through mutational analysis of both NTF2 and Ran (27)(28)(29)(30)(31) as well as analysis of the NTF2-Ran co-crystal structure (32). These studies indicate that the interaction between NTF2 and Ran is required to concentrate Ran in the nucleus and consequently for protein transport between the nucleus and the cytoplasm (33).…”

mentioning

confidence: 99%

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

Quimby¹,

Leung²,

Bayliss³

et al. 2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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...

show abstract

“…The complex is then translocated through a central gated channel of the NPC into the nuclear interior. Movement of the import complex through the NPC requires Ran and NTF2 (5)(6)(7)(8), but the precise role of these components is unclear. An additional protein, hsp/hsc70, has been shown to be required for import of some substrates (e.g.…”

mentioning

confidence: 99%

Nuclear Import of Adenovirus DNA in Vitro Involves the Nuclear Protein Import Pathway and hsc70

Saphire

Guan

Schirmer

et al. 2000

Journal of Biological Chemistry

Self Cite

122

102

View full text Add to dashboard Cite

Adenovirus, a respiratory virus with a doublestranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process.Proteins are imported into the nucleus through aqueous channels that span the nuclear envelope called nuclear pore complexes (NPCs).1 Whereas ions and molecules less than ϳ20 -40 kDa can diffuse passively through the NPC, larger proteins are transported by saturable pathways that are energy-and signal-dependent. The classical signals that specify nuclear protein import (nuclear localization sequences (NLSs)) are short stretches of amino acids rich in basic residues, although other classes of NLSs have been described recently (1, 2). The transport of NLS-bearing proteins into the nucleus requires the participation of soluble cytosolic factors (3). The advent of a cytosol-dependent in vitro assay for nuclear import (3) has led to the identification of a number of these factors (4), namely importin-␣ (also called the NLS receptor and karyopherin-␣), importin-␤ (also called p97 and karyopherin-␤), Ran (TC4), and NTF2 (also called p10 and pp15). An initial step in the import of proteins containing classical NLSs occurs in the cytoplasm, where the substrate binds to the receptor importin-␣. Subsequently, this complex docks at the cytoplasmic face of the NPC together with importin-␤. The complex is then translocated through a central gated channel of the NPC into the nuclear interior. Movement of the import complex through the NPC requires Ran and NTF2 (5-8), but the precise role of these components is unclear. An additional protein, hsp/hsc70, has been shown to be required for import of some substrates (e.g. the large T-antigen protein (9)), but not of others (e.g. the glucocorticoid receptor (10)).Most viruses that replicate in the nucleus must use the NPC to introduce their genome into the nucleoplasm. Although it is known that the gated channel of the NPC can expand up to ϳ25 nm in diameter to allow signal-mediated transport (11), this is still considerably smaller than the diameters of many ...

show abstract

Nuclear protein import is decreased by engineered mutants of nuclear transport factor 2 (NTF2) that do not bind GDP-Ran

Cited by 70 publications

References 58 publications

Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

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

Nuclear Import of Adenovirus DNA in Vitro Involves the Nuclear Protein Import Pathway and hsc70

Contact Info

Product

Resources

About