Showing papers on "Importin published in 2003"
TL;DR: A model whereby lesion-induced upregulation of axonal importin beta may enable retrograde transport of signals that modulate the regeneration of injured neurons is suggested.
470 citations
TL;DR: Exportin 6 (Exp6) is identified as a novel family member from higher eukaryotes and it is shown that it mediates nuclear export of profilin·actin complexes and functions not only as the nucleotide exchange factor for actin, but also as a cofactor of actin export.
Abstract: Active macromolecular transport between the nucleus and cytoplasm proceeds through nuclear pore complexes and is mostly mediated by transport receptors of the importin β-superfamily. Here we identify exportin 6 (Exp6) as a novel family member from higher eukaryotes and show that it mediates nuclear export of profilin·actin complexes. Exp6 appears to contact primarily actin, but the interaction is greatly enhanced by the presence of profilin. Profilin thus functions not only as the nucleotide exchange factor for actin, but can also be regarded as a cofactor of actin export and hence as a suppressor of actin polymerization in the nucleus. Even though human and Drosophila Exp6 share only ∼20% identical amino acid residues, their function in profilin·actin export is conserved. A knock-down of Drosophila Exp6 by RNA interference abolishes nuclear exclusion of actin and results in the appearance of nuclear actin paracrystals. In contrast to a previous report, we found no indications of a major and direct role for CRM1 in actin export from mammalian or insect nuclei.
313 citations
TL;DR: The different fate of mature and immature capsids after passing the nuclear pore indicates that the outcome of a nuclear import event may be regulated within the nuclear basket.
Abstract: While studying the import of the hepatitis B virus genome into the nucleus of permeabilized tissue culture cells, we found that viral capsids were imported in intact form through the nuclear pore into the nuclear basket. Import depended on phosphorylation of the capsid protein and was mediated by the cellular transport receptors importin α and β. Virus-derived capsids that contained the mature viral genome were able to release the viral DNA and capsid protein into the nucleoplasm. The uncoating reaction was independent of Ran, a GTP-binding enzyme responsible for dissociating other imported cargoes from the inner face of the nuclear pore. Immature capsids that did not contain the mature viral genome reached the basket but did not release capsid proteins nor immature genomes into the nucleoplasm. The different fate of mature and immature capsids after passing the nuclear pore indicates that the outcome of a nuclear import event may be regulated within the nuclear basket.
257 citations
TL;DR: It is shown that unidirectional nuclear transport can be faithfully simulated without the assumption of a vectorial NPC passage; transport receptors only need to reversibly cross NPCs and switch their affinity for cargo in response to the RanGTP gradient.
Abstract: Here, we analyse the RanGTPase system and its coupling to receptor-mediated nuclear transport. Our simulations predict nuclear RanGTP levels in HeLa cells to be very sensitive towards the cellular energy charge and to exceed the cytoplasmic concentration ≈1000-fold. The steepness of the RanGTP gradient appears limited by both the cytoplasmic RanGAP concentration and the imperfect retention of nuclear RanGTP by nuclear pore complexes (NPCs), but not by the nucleotide exchange activity of RCC1. Neither RanBP1 nor the NPC localization of RanGAP has a significant direct impact on the RanGTP gradient. NTF2-mediated import of Ran appears to be the bottleneck for maximal capacity of Ran-driven nuclear transport. We show that unidirectional nuclear transport can be faithfully simulated without the assumption of a vectorial NPC passage; transport receptors only need to reversibly cross NPCs and switch their affinity for cargo in response to the RanGTP gradient. A significant RanGTP gradient after nuclear envelope (NE) breakdown can apparently exist only in large cytoplasm. This indicates that RanGTP gradients can provide positional information for mitotic spindle and NE assembly in early embryonic cells, but hardly any in small somatic cells.
224 citations
TL;DR: The sterol regulatory element–binding protein 2 (SREBP-2), a nuclear transcription factor that is essential for cholesterol metabolism, enters the nucleus through a direct interaction of its helix-loop-helix leucine zipper domain with importin-β, which changes its conformation to reveal a pseudo-twofold symmetry on its surface structure so that it can accommodate a symmetric dimer molecule.
Abstract: The sterol regulatory element-binding protein 2 (SREBP-2), a nuclear transcription factor that is essential for cholesterol metabolism, enters the nucleus through a direct interaction of its helix-loop-helix leucine zipper domain with importin-beta. We show the crystal structure of importin-beta complexed with the active form of SREBP-2. Importin-beta uses characteristic long helices like a pair of chopsticks to interact with an SREBP-2 dimer. Importin-beta changes its conformation to reveal a pseudo-twofold symmetry on its surface structure so that it can accommodate a symmetric dimer molecule. Importin-beta may use a similar strategy to recognize other dimeric cargoes.
212 citations
TL;DR: This work has studied nuclear import of purified HIV‐1 RTCs in primary macrophages and found that importin 7, an import receptor for ribosomal proteins and histone H1, is involved in the process.
Abstract: Human immunodeficiency virus type 1 (HIV-1), like other lentiviruses, can infect non-dividing cells. This property depends on the active nuclear import of its intracellular reverse transcription complex (RTC). We have studied nuclear import of purified HIV-1 RTCs in primary macrophages and found that importin 7, an import receptor for ribosomal proteins and histone H1, is involved in the process. Nuclear import of RTCs requires, in addition, energy and the components of the Ran system. Depletion of importin 7 from cultured cells by small interfering RNA inhibits HIV-1 infection. These results provide a new insight into the molecular mechanism for HIV-1 nuclear import and reveal potential targets for therapeutic intervention.
190 citations
TL;DR: It is shown by site-directed mutagenesis that the very C-terminal arm repeats 8 and 9 of importin α5 form a unique binding site for STAT1 homodimers and STAT1-STAT2 heterodIMers, which means that interferon-activated, dimerized signal transducers and activators of transcription (STAT1 and STAT2) directly bind to importin β via a dimeric nonclassical NLS.
185 citations
TL;DR: The data suggest that the critical early function in spindle formation regulated by importin α is TPX2‐mediated microtubule nucleation.
Abstract: The importin alpha-regulated microtubule-associated protein TPX2 is known to be critical for meiotic and mitotic spindle formation in vertebrates, but its detailed mechanism of action and regulation is not understood. Here, the site of interaction on TPX2 for importin alpha is mapped. A TPX2 mutant that cannot bind importin alpha is constitutively active in the induction of microtubule-containing aster-like structures in Xenopus egg extract, demonstrating that no other importin alpha or RanGTPase target is required to mediate microtubule assembly in this system. Further, recombinant TPX2 is shown to induce the formation and bundling of microtubules in dilute solutions of pure tubulin. In this purified system, importin alpha prevents TPX2-induced microtubule formation, but not TPX2-tubulin interaction or microtubule bundling. This demonstrates that TPX2 has more than one mode of interaction with tubulin and that only one of these types of interaction is abolished by importin alpha. The data suggest that the critical early function in spindle formation regulated by importin alpha is TPX2-mediated microtubule nucleation.
174 citations
TL;DR: The various strategies used to provide plasmid DNA with nuclear localization sequences are reviewed, and the possibility of developing efficient gene delivery systems based on these strategies is discussed.
171 citations
TL;DR: It is concluded that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination, and cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate inThe nucleus.
Abstract: The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding/bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein in vitro and in vivo, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMPβ. The SRY-R133W mutant shows reduced IMPβ binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMPβ binding, whereas SRY-R75N and SRY-R76P show normal IMPβ binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.
152 citations
TL;DR: Recent experimental approaches and results are discussed that have begun to provide molecular insight into the mechanisms by which transport complexes traverse the nuclear pore complex, and significant gaps in understanding that remain are pointed out.
Abstract: Nucleocytoplasmic transport is mediated by shuttling receptors that recognize specific signals on protein or RNA cargoes and translocate the cargoes through the nuclear pore complex. Transport receptors appear to move through the nuclear pore complex by facilitated diffusion, involving repeated cycles of binding to and dissociation from nucleoporins with phenylalanine-glycine motifs. We discuss recent experimental approaches and results that have begun to provide molecular insight into the mechanisms by which transport complexes traverse the nuclear pore complex, and point out the significant gaps in understanding that remain.
TL;DR: Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly.
Abstract: Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin β negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin β is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin β down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin β to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin β 45–462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin β-regulation of NPC assembly. Excess full-length importin β and β 45–462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin β NPC block, which maps downstream of GTPγS and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 μM RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin β. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly.
TL;DR: This work reports the identification of a second nucleoporin binding region in the NH2-terminal half of importin β, which is important for its nuclear import function.
Abstract: Proteins containing a classical NLS are transported into the nucleus by the import receptor importin β, which binds to cargoes via the adaptor importin α. The import complex is translocated through the nuclear pore complex by interactions of importin β with a series of nucleoporins. Previous studies have defined a nucleoporin binding region in the NH2-terminal half of importin β. Here we report the identification of a second nucleoporin binding region in its COOH-terminal half. Although the affinity of the COOH-terminal region for nucleoporins is dramatically weaker than that of the NH2-terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin β to a similar extent (∼50%). An importin β mutant with a combination of mutations in the NH2- and COOH-terminal regions is completely inactive for nuclear import. Thus, importin β possesses two nucleoporin binding sites, both of which are important for its nuclear import function.
TL;DR: It is shown that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin α and β (α/β) and restored by addition of Ran-GTP, which is identical to that used in classical NLS-driven nuclear transport.
Abstract: The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin α and/or importin β to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin α and β (α/β) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin α/β-mediated regulation of XCTK2 microtubule binding. Our data show that importin α/β directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.
TL;DR: It is proposed that Gle2 may act to deliver TAP to Nup98 and that this may represent the first in a series of interactions between an export complex and a nucleoporin.
TL;DR: This report unraveled nuclear import mechanisms of Smad3 and Smad4 that are dependent on their interaction with FG-repeat-containing nucleoporins such as CAN/Nup214, without the involvement of importin molecules that are responsible for most of the known nuclear import events.
TL;DR: By regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly, RanGTP is hypothesized to maintain the spindleAssembly factors in the active state by binding to importin β, part of thenuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle Assembly factors.
Abstract: Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin β, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 we identified nuclear targeting information, microtubule-binding and Aurora A binding activities. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevented Aurora A activation and recruitment to microtubules. In addition we identified nuclear targeting information in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly.
TL;DR: It is shown that nuclear import of the major capsid protein, hexon, is mediated by protein VI, a structural protein located underneath the 12 vertices of the adenoviral capsid and that it links hexon to the nuclear import machinery via an importin α/β‐dependent mechanism.
Abstract: Replication and assembly of adenovirus occurs in the nucleus of infected cells, requiring the nuclear import of all viral structural proteins. In this report we show that nuclear import of the major capsid protein, hexon, is mediated by protein VI, a structural protein located underneath the 12 vertices of the adenoviral capsid. Our data indicate that protein VI shuttles between the nucleus and the cytoplasm and that it links hexon to the nuclear import machinery via an importin α/β-dependent mechanism. Key nuclear import and export signals of protein VI are located in a short C-terminal segment, which is proteolytically removed during virus maturation. The removal of these C-terminal transport signals appears to trigger a functional transition in protein VI, from a role in supporting hexon nuclear import to a structural role in virus assembly.
TL;DR: Results from cytoskeletal disruption and the addition of an alternate nuclear localization sequence (NLS) to mAhR-YFP suggest that XAP2 does not physically tether the complex in the cytoplasm, and XAP1 represses the transactivation potential of the AhR, in contrast to previously published reports.
TL;DR: The results strongly suggest that the molecular mechanism of the nuclear import of SXR was different from that of another xenosensor, the constitutively active receptor, whose translocation into the nucleus is mediated by a leucine-rich xenochemical response signal in its ligand binding domain.
Abstract: The steroid and xenobiotic receptor (SXR) is an orphan nuclear receptor that plays a key role in the regulation of xenobiotic response by controlling the expression of drug metabolizing and clearance enzymes. We observed that pregnane X receptor (PXR), the mouse ortholog of SXR, was retained in the cytoplasm of hepatic cells of untreated mice, whereas PXR was translocated to the nucleus after administration of a ligand, pregnenolone 16α-carbonitrile. To understand the molecular mechanisms underlying the xenochemical-dependent nuclear translocation of SXR, we identified the signal sequence of SXR that regulates its nuclear translocation; using an in vitro expression system, we allocated the nuclear localization signal (NLS) to amino acid residues 66 to 92 within the DNA binding domain of SXR. The NLS of SXR is characterized as the bipartite type, and is recognized by the three molecular species of importin α: Rch1 (PTAC58), NPI1, and Qip1, in the presence of PTAC97 of importin β to target the nuclear pore. The nuclear translocation of SXR was observed as an essential regulatory event for transcription of its target genes such asCYP3A4. These results strongly suggest that the molecular mechanism of the nuclear import of SXR was different from that of another xenosensor, the constitutively active receptor, whose translocation into the nucleus is mediated by a leucine-rich xenochemical response signal in its ligand binding domain.
TL;DR: Both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre‐mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection, indicating a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus.
Abstract: Alternative splicing of precursor mRNA is often regulated by serine/arginine‐rich proteins (SR proteins) and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by transportin‐SRs (TRN‐SRs), importin β‐like nuclear transporters. Here we identify and characterize a non‐SR protein, RNA‐binding motif protein 4 (RBM4), as a novel substrate of TRN‐SR2. TRN‐SR2 interacts specifically with RBM4 in a Ran‐sensitive manner. TRN‐SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C‐terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre‐mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus.
TL;DR: It is concluded that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhancesnuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.
Abstract: The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser(112) in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.
TL;DR: In this paper, the authors applied dynamic force spectroscopy to study the interaction of Ran with the nuclear import receptor importin beta1 (impbeta) at the single-molecule level and found that the complex alternates between two distinct conformational states of different adhesion strength.
Abstract: Several million macromolecules are exchanged each minute between the nucleus and cytoplasm by receptor-mediated transport. Most of this traffic is controlled by the small GTPase Ran, which regulates assembly and disassembly of the receptor-cargo complexes in the appropriate cellular compartment. Here we applied dynamic force spectroscopy to study the interaction of Ran with the nuclear import receptor importin beta1 (impbeta) at the single-molecule level. We found that the complex alternates between two distinct conformational states of different adhesion strength. The application of an external mechanical force shifts equilibrium toward one of these states by decreasing the height of the interstate activation energy barrier. The other state can be stabilized by a functional Ran mutant that increases this barrier. These results support a model whereby functional control of Ran-impbeta is achieved by a population shift between pre-existing alternative conformations.
TL;DR: It is demonstrated that association of procaspase-2 into higher order structures, rather than its nuclear localization, is required for caspases-2 activation and its ability to induce apoptosis.
TL;DR: The data indicate that Nup2p increases the overall rate of nuclear trafficking by coordinating nuclear import termination and importin recycling as a concerted process.
Abstract: The yeast nucleoporin Nup2p is associated primarily with the nuclear basket of nuclear pore complexes and is required for efficient importin-α:β-mediated nuclear protein import as well as efficient nuclear export of Kap60p/importin-α. Residues 1–51 of Nup2p bind tightly to Kap60p and are required for Nup2p function in vivo. We have determined the 2.6 Å resolution crystal structure of a complex between this region of Nup2p and the armadillo repeat domain of Kap60p. Nup2p binds along the inner concave groove of Kap60p, but its interaction interface is different from that employed for nuclear localization signal (NLS) recognition although there is some overlap between them. Nup2p binds Kap60p more strongly than NLSs and accelerates release of NLSs from Kap60p. Nup2p itself is released from Kap60p by Cse1p:RanGTP only in the presence of the importin-β binding (IBB) domain of Kap60p. These data indicate that Nup2p increases the overall rate of nuclear trafficking by coordinating nuclear import termination and importin recycling as a concerted process.
TL;DR: A model where the auto-inhibitory activity of importin α is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of Importin α to the cytoplasm is proposed.
TL;DR: It is found that NLS-plasmid DNA conjugates with covalent bonding by diazo coupling through PEG chain (MW 3400) stimulated complexation with the nuclear transport proteins importin alpha and importin beta, suggesting that stimulation of plasmid nuclear localization cannot be achieved merely by changing spacer length or chemically modifying plasmids-NLS peptide conjugate.
TL;DR: Insight is provided into how changes in binding to cellular NLS sequences could impact cellular function and the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes are created.
TL;DR: It is shown that the pUL84 nuclear import required importin α, importin β, and Ran, thus following the classical importin-mediated import pathway, and a complex structure appears to be essential for Importin α binding and import activity.
Abstract: The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin α protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin α, importin β, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin α proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin α binding and import activity.
TL;DR: This study uses site-directed mutagenesis coupled with in vitro binding assays and in vivo analyses to investigate the intramolecular interaction of the N-terminal I BB domain and the NLS binding pocket of Saccharomyces cerevisiae importin α, Srp1p and finds that mutations within the IBB domain that decrease the binding affinity for the auto-inhibitory sequence impact importinα function in vivo.