Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells.     

Structural Basis of High‐Affinity Nuclear Localization Signal Interactions with Importin‐α

Classical nuclear localization signals (cNLSs), comprising one (monopartite cNLSs) or two clusters of basic residues connected by a 10–12 residue linker (bipartite cNLSs), are recognized by the nuclear import factor importin‐α. The cNLSs bind along a concave groove on importin‐α; however, specificity determinants of cNLSs remain poorly understood. We present a structural and interaction analysis study of importin‐α binding to both designed and naturally occurring high‐affinity cNLS‐like sequences; the peptide inhibitors Bimax1 and Bimax2, and cNLS peptides of cap‐binding protein 80. Our data suggest that cNLSs and cNLS‐like sequences can achieve high affinity through maximizing interactions at the importin‐α minor site, and by taking advantage of multiple linker region interactions. Our study defines an extended set of binding cavities on the importin‐α surface, and also expands on recent observations that longer linker sequences are allowed, and that long‐range electrostatic complementarity can contribute to cNLS‐binding affinity. Altogether, our study explains the molecular and structural basis of the results of a number of recent studies, including systematic mutagenesis and peptide library approaches, and provides an improved level of understanding on the specificity determinants of a cNLS. Our results have implications for identifying cNLSs in novel proteins.     

KPNB1, XPO7 and IPO8 Mediate the Translocation ofNF‐κB/p65 into the Nucleus

NF‐κB/p65 is retained in the cytoplasm until it is activated in response to stress. Nuclear import of p65 is regulated by importin α in a nuclear localization signal (NLS)‐dependent manner. However, the role of importin β family members in the nuclear translocation of p65 is largely unclear. In this study, using high‐content siRNA screening, we identified three of 17 importin β family members that are involved in the nuclear import of p65. Our data showed that knockdown of KPNB1, XPO7 and IPO8 reduced the amount of nuclear p65 following tumor necrosis factor‐α (TNF‐α) stimulation, resulting in lower NF‐κB activity. KPNB1 was the major importin β receptor for p65 import, and this import was dependent on the NLS of p65. However, NLS‐mutated p65 still entered the nucleus and bound to XPO7 and IPO8. Interestingly, among the six members of the importin α family, KPNA2 was most important for p65 import. Taken together, our results show that the import of p65 mainly relies on the canonical KPNA2/KPNB1 pathway; however, p65 is also imported by an alternative pathway that is independent of its NLS. Redundant importin receptors are likely to maintain the important function of p65 according to need .      

A minimal nuclear localization signal (NLS) in human phospholipid scramblase 4 that binds only the minor NLS-binding site of importin alpha1

Importin α1 can bind classical nuclear localization signals (NLSs) in two NLS-binding sites, known as "major" and "minor." The major site is located between ARM repeats 2-4, whereas the minor site spans ARM 7-8. In this study, we have characterized the cellular localization of human phospholipid scramblase 4 (hPLSCR4), a member of the phospholipid scramblase protein family. We identified a minimal NLS in hPLSCR4 ((273)GSIIRKWN(280)) that contains only two basic amino acids. This NLS is both necessary for nuclear localization of hPLSCR4 in transfected HeLa cells and sufficient for nuclear import of a non-diffusible cargo in permeabilized cells. Mutation of only one of the two basic residues, Arg(277), correlates with loss of nuclear localization, suggesting this amino acid plays a key role in nuclear transport. Crystallographic analysis of mammalian importin α1 in complex with the hPLSCR4-NLS reveals this minimal NLS binds specifically and exclusively to the minor binding site of importin α. These data provide the first structural and functional evidence of a novel NLS-binding mode in importin α1 that uses only the minor groove as the exclusive site for nuclear import of nonclassical cargos.     

Induced fit, folding, and recognition of the NF-kappaB-nuclear localization signals by IkappaBalpha and IkappaBbeta

Protein structure prediction codes based on the associative memory Hamiltonian were used to probe the binding modes between the nuclear localization signal (NLS) polypeptide of NF-kappaB and the inhibitors IkappaBalpha and IkappaBbeta. Experimentally, it is known that the NLS polypeptide is unstructured in the NF-kappaB complex with DNA but it forms an extended helical structure with the NLS (residues 301-304) between the two helices in the NF-kappaB/IkappaBalpha complex. The simulations included the NF-kappaB(p65) and (p50) NLS polypeptides and various mutants alone and in the presence of IkappaBalpha and IkappaBbeta. The simulations predict that the NLS polypeptide by itself binds tightly to IkappaBalpha and IkappaBbeta. In the NF-kappaB (p50/p65) heterodimer, the p50 NLS is predicted to remain free to bind to importin alpha. In the interaction with IkappaBalpha, both p65 NLSs are predicted to be bound. In IkappaBbeta, the NLS polypeptide binds to two binding sites, as seen in the crystal structure, with one site heavily favored for stable binding.     

Rapid mitochondrial dysfunction mediates TNF‐alpha‐induced neurotoxicity

Tumor necrosis factor alpha (TNF‐α) is known to exacerbate ischemic brain injury; however, the mechanism is unknown. Previous studies have evaluated the effects of TNF‐α on neurons with long exposures to high doses of TNF‐α, which is not pathophysiologically relevant. We characterized the rapid effects of TNF‐α on basal respiration, ATP production, and maximal respiration using pathophysiologically relevant, post‐stroke concentrations of TNF‐α. We observed a reduction in mitochondrial function as early as 1.5 h after exposure to low doses of TNF‐α, followed by a decrease in cell viability in HT‐22 cells and primary neurons. Subsequently, we used the HT‐22 cell line to determine the mechanism by which TNF‐α causes a rapid and profound reduction in mitochondrial function. Pre‐treating with TNF‐R1 antibody, but not TNF‐R2 antibody, ameliorated the neurotoxic effects of TNF‐α, indicating that TNF‐α exerts its neurotoxic effects through TNF‐R1. We observed an increase in caspase 8 activity and a decrease in mitochondrial membrane potential after exposure to TNF‐α which resulted in a release of cytochrome c from the mitochondria into the cytosol. These novel findings indicate for the first time that an acute exposure to pathophysiologically relevant concentrations of TNF‐α has neurotoxic effects mediated by a rapid impairment of mitochondrial function.

     

Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding

Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to 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.     

Insulin‐like growth factor‐1 abrogates microglial oxidative stress and TNF‐α responses to spreading depression

Spreading depression (SD), the most likely cause of migraine aura and perhaps migraine, occurs with increased oxidative stress (OS). SD increases reactive oxygen species (ROS), and ROS, in turn, can signal to increase neuronal excitability, which includes increased SD susceptibility. SD also elevates tumor necrosis factor‐α (TNF‐α), which increases neuronal excitability. Accordingly, we probed for the cellular origin of OS from SD and its relationship to TNF‐α, which might promote SD, using rat hippocampal slice cultures. We observed significantly increased OS from SD in astrocytes and microglia but not in neurons or oligodendrocytes. Since insulin‐like growth factor‐1 (IGF‐1) mitigates OS from SD, we determined the cell types responsible for this effect. We found that IGF‐1 significantly decreased microglial but not astrocytic OS from SD. We also show that IGF‐1 abrogated the SD‐induced TNF‐α increase. Furthermore, TNF‐α application increased microglial but not astrocytic OS, an effect abrogated by IGF‐1. Next, we showed that SD increased SD susceptibility, and does so via TNF‐α. This work suggests that microglia promote SD via increased and interrelated ROS and TNF‐α signaling. Thus, IGF‐1 mitigation of microglial ROS and TNF‐α responses may be targets for novel therapeutics development to prevent SD, and perhaps migraine.

     

Interdependent Nucleocytoplasmic Trafficking and Interactions of Dis3 with Rrp6, the Core Exosome and Importin‐α3

Subcellular compartmentalization of exoribonucleases (RNAses) is an important control mechanism in the temporal and spatial regulation of RNA processing and decay. Despite much progress towards understanding RNAse substrates and functions, we know little of how RNAses are transported and assembled into functional, subcellularly restricted complexes. To gain insight into this issue, we are studying the exosome‐binding protein Dis3, a processive 3′ to 5′ exoribonuclease. Here, we examine the interactions and subcellular localization of the Drosophila melanogaster Dis3 (dDis3) protein. N‐terminal domain mutants of dDis3 abolish associations with the ‘core’ exosome, yet only reduce binding to the ‘nuclear’ exosome‐associated factor dRrp6. We show that nuclear localization of dDis3 requires a C‐terminal classic nuclear localization signal (NLS). Consistent with this, dDis3 specifically co‐precipitates the NLS‐binding protein importin‐α3. Surprisingly, dDis3 constructs that lack or mutate the C‐terminal NLS retain importin‐α3 binding, suggesting that the interaction is indirect. Finally, we find that endogenous dDis3 and dRrp6 exhibit coordinated nuclear enrichment or exclusion, suggesting that dDis3, Rrp6 and importin‐α3 interact in a complex independent of the core. We propose that the movement and deposition of this complex is important for the subcellular compartmentalization and regulation of the exosome core.     

Dissection of a nuclear localization signal

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.     

Impaired clustered protocadherin‐α leads to aggregated retinogeniculate terminals and impaired visual acuity in mice

Clustered protocadherins (cPcdhs) comprising cPcdh‐α, ‐β, and ‐γ, encode a large family of cadherin‐like cell‐adhesion molecules specific to neurons. Impairment of cPcdh‐α results in abnormal neuronal projection patterns in specific brain areas. To elucidate the role of cPcdh‐α in retinogeniculate projections, we investigated the morphological patterns of retinogeniculate terminals in the lateral geniculate (LG) nucleus of mice with impaired cPcdh‐α. We found huge aggregated retinogeniculate terminals in the dorsal LG nucleus, whereas no such aggregated terminals derived from the retina were observed in the olivary pretectal nucleus and the ventral LG nucleus. These aggregated terminals appeared between P10 and P14, just before eye opening and at the beginning of the refinement stage of the retinogeniculate projections. Reduced visual acuity was observed in adult mice with impaired cPcdh‐α, whereas the orientation selectivity and direction selectivity of neurons in the primary visual cortex were apparently normal. These findings suggest that cPcdh‐α is required for adequate spacing of retinogeniculate projections, which may be essential for normal development of visual acuity.

     

Interaction of the Vp3 nuclear localization signal with the importin alpha 2/beta heterodimer directs nuclear entry of infecting simian virus 40

For nuclear entry of large nucleoprotein complexes, it is thought that one key nuclear localization signal (NLS) of a protein component becomes exposed to mediate importin recognition. We show that the nuclear entry of simian virus 40 involves a dynamic interplay between two distinct interiorly situated capsid NLSs, the Vp1 NLS and the Vp3 NLS, and the selective exposure and importin recognition of the Vp3 NLS. The Vp3 NLS-null mutants assembled normally into virion-like particles (VLP) in mutant DNA-transfected cells. When used to infect a new host, the null VLP entered the cell normally but was impaired in viral DNA nuclear entry due to a lack of recognition by the importin alpha 2/beta heterodimer, leading to reduced viability. Both Vp3 and Vp1 NLSs directed importin interaction in vitro, but the Vp1 NLS, which overlaps the Vp1 DNA binding domain, did not bind importins in the presence of DNA. The results suggest that certain canonical NLSs within a nucleoprotein complex, such as the Vp1 NLS, can be masked from functioning by binding to the nucleic acid component and that the availability of an NLS that is not masked and can become exposed for importin binding, such as the Vp3 NLS, is a general feature of the nuclear entry of the nucleoprotein complexes, including those of other animal viruses.     

Dissecting the roles of Cse1 and Nup2 in classical NLS‐cargo release in vivo

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.     

Efficiency of importin alpha/beta-mediated nuclear localization sequence recognition and nuclear import. Differential role of NTF2.

Little quantitative, kinetic information is available with respect to the process of nuclear import of conventional nuclear localization sequence (NLS)-containing proteins, which initially involves recognition and docking at the nuclear pore by importin alpha/beta. This study compares the binding and nuclear import properties of mouse (m) and yeast (y) importin (IMP) subunits with respect to the NLSs from the SV40 large tumor antigen (T-ag), and the Xenopus laevis phosphoprotein N1N2. m- and y-IMPalpha recognized both NLSs, with y-IMPalpha exhibiting higher affinity. m-IMPbeta greatly enhanced the binding of m-IMPalpha to the T-ag and N1N2 NLSs, but y-IMPbeta did not significantly affect the affinity of y-IMPalpha for the T-ag NLS. In contrast, y-IMPbeta enhanced y-IMPalpha binding to the NLS of N1N2, but to a lesser extent than the enhancement of m-IMPalpha binding by m-IMPbeta. NLS-dependent nuclear import was reconstituted in vitro using the different importin subunits together with the transport factors Ran and NTF2. Whereas T-ag NLS-mediated nuclear import did not exhibit an absolute requirement for NTF2, N1N2 NLS-mediated transport strictly required NTF2. High levels of NTF2 inhibited nuclear accumulation conferred by both NLSs. We conclude that different NLSs possess distinct nuclear import properties due to differences in recognition by importin and requirements for NTF2.     

Nuclear localization signal and protein context both mediate importin alpha specificity of nuclear import substrates

The "classical" nuclear protein import pathway depends on importin alpha and importin beta. Importin alpha binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin beta-dependent import pathway. In humans, only one importin beta is known to interact with importin alpha, while six alpha importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular alpha importins. Whether the NLS is sufficient to mediate importin alpha specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin alpha3, and the less specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and protein context contribute to the specificity of importin alpha binding and transport.     

Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localization signals by karyopherin alpha

BACKGROUND: Karyopherin alpha (importin alpha) is an adaptor molecule that recognizes proteins containing nuclear localization signals (NLSs). The prototypical NLS that is able to bind to karyopherin alpha is that of the SV40 T antigen, and consists of a short positively charged sequence motif. Distinct classes of NLSs (monopartite and bipartite) have been identified that are only partly conserved with respect to one another but are nevertheless recognized by the same receptor. RESULTS: We report the crystal structures of two peptide complexes of yeast karyopherin alpha (Kapalpha): one with a human c-myc NLS peptide, determined at 2.1 A resolution, and one with a Xenopus nucleoplasmin NLS peptide, determined at 2.4 A resolution. Analysis of these structures reveals the determinants of specificity for the binding of a relatively hydrophobic monopartite NLS and of a bipartite NLS peptide. The peptides bind Kapalpha in its extended surface groove, which presents a modular array of tandem binding pockets for amino acid residues. CONCLUSIONS: Monopartite and bipartite NLSs bind to a different number of amino acid binding pockets and make different interactions within them. The relatively hydrophobic monopartite c-myc NLS binds extensively at a few binding pockets in a similar manner to that of the SV40 T antigen NLS. In contrast, the bipartite nucleoplasmin NLS engages the whole array of pockets with individually more limited but overall more abundant interactions, which include the NLS two basic clusters and the backbone of its non-conserved linker region. Versatility in the specific recognition of NLSs relies on the modular.