A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth

The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the (32)EGDL(35) residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the (32)EGDL(35) was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of (32)EGDL(35) responsible for nuclear localization are important for the inhibitory activity of NV.     

Chromatin binding of RCC1 during mitosis is important for its nuclear localization in interphase

RCC1, a guanine nucleotide exchange factor of the small GTPase Ran, plays various roles throughout the cell cycle. However, the functions of RCC1 in biological processes in vivo are still unclear. In particular, although RCC1 has multifunctional domains, the biological significance of each domain is unclear. To examine each domain of RCC1, we established an RCC1 conditional knockout chicken DT40 cell line and introduced various RCC1 mutants into the knockout cells. We found that nuclear reformation did not occur properly in RCC1-deficient cells and examined whether specific RCC1 mutants could rescue this phenotype. Surprisingly, we found that neither the nuclear localization signal nor the chromatin-binding domain of RCC1 is essential for its function. However, codisruption of these domains resulted in defective nuclear reformation, which was rescued by artificial nuclear localization of RCC1. Our data indicate that chromatin association of RCC1 during mitosis is crucial for its proper nuclear localization in the next interphase. Moreover, proper nuclear localization of RCC1 in interphase is essential for its function through its nucleotide exchange activity.     

A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN

Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrate.     

Rad52 multimerization is important for its nuclear localization in Saccharomyces cerevisiae

Rad52 is essential for all homologous recombination and DNA double strand break repair events in Saccharomyces cerevisiae. This protein is multifunctional and contains several domains that allow it to interact with DNA as well as with different repair proteins. However, it has been unclear how Rad52 enters the nucleus. In the present study, we have used a combination of mutagenesis and sequence analysis to show that Rad52 from S. cerevisiae contains a single functional pat7 type NLS essential for its nuclear localization. The region containing the NLS seems only to be involved in nuclear transport as it plays no role in repair of MMS-induced DNA damage. The NLS in Rad52 is weak, as monomeric protein species that harbor this NLS are mainly located in the cytosol. In contrast, multimeric protein complexes wherein each subunit contains a single NLS(Rad52) sort efficiently to the nucleus. Based on the results we propose a model where the additive effect of multiple NLS(Rad52) sequences in a Rad52 ring-structure ensures efficient nuclear localization of Rad52.     

NXT1 is necessary for the terminal step of Crm1-mediated nuclear export

Soluble factors are required to mediate nuclear export of protein and RNA through the nuclear pore complex (NPC). These soluble factors include receptors that bind directly to the transport substrate and regulators that determine the assembly state of receptor-substrate complexes. We recently reported the identification of NXT1, an NTF2-related export factor that stimulates nuclear protein export in permeabilized cells and undergoes nucleocytoplasmic shuttling in vivo (Black, B.E., L. Lévesque, J.M. Holaska, T.C. Wood, and B.M. Paschal. 1999. Mol. Cell. Biol. 19:8616-8624). Here, we describe the molecular characterization of NXT1 in the context of the Crm1-dependent export pathway. We find that NXT1 binds directly to Crm1, and that the interaction is sensitive to the presence of Ran-GTP. Moreover, mutations in NXT1 that reduce binding to Crm1 inhibit the activity of NXT1 in nuclear export assays. We show that recombinant Crm1 and Ran are sufficient to reconstitute nuclear translocation of a Rev reporter protein from the nucleolus to an antibody accessible site on the cytoplasmic side of the NPC. Further progress on the export pathway, including the terminal step of Crm1 and Rev reporter protein release, requires NXT1. We propose that NXT1 engages with the export complex in the nucleoplasm, and that it facilitates delivery of the export complex to a site on the cytoplasmic side of NPC where the receptor and substrate are released into the cytoplasm.     

Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development

Mfn2 is a mitochondrial fusion protein with bioenergetic functions implicated in the pathophysiology of neuronal and metabolic disorders. Understanding the bioenergetic mechanism of Mfn2 may aid in designing therapeutic approaches for these disorders. Here we show using endoplasmic reticulum (ER) or mitochondria‐targeted Mfn2 that Mfn2 stimulation of the mitochondrial metabolism requires its localization in the ER, which is independent of its fusion function. ER‐located Mfn2 interacts with mitochondrial Mfn1/2 to tether the ER and mitochondria together, allowing Ca²⁺ transfer from the ER to mitochondria to enhance mitochondrial bioenergetics. The physiological relevance of these findings is shown during neurite outgrowth, when there is an increase in Mfn2‐dependent ER‐mitochondria contact that is necessary for correct neuronal arbor growth. Reduced neuritic growth in Mfn2 KO neurons is recovered by the expression of ER‐targeted Mfn2 or an artificial ER‐mitochondria tether, indicating that manipulation of ER‐mitochondria contacts could be used to treat pathologic conditions involving Mfn2.     

Reversible cAMP-dependent change in nuclear localization of microtubule-associated protein-1 analogues

Intranuclear immunofluorescent staining by monoclonal and polyclonal antibodies against microtubule-associated protein-1 (MAP-1) on SV-3Y1 cells disappeared when the cells were treated with 1 mM db-cAMP and 1 mM theophylline for 20-30 min at 37 degrees C. The nuclear dots of immunofluorescence disappeared and reappeared repeatedly on successive incubation of the cells with and without these drugs. The same phenomenon was induced by treatment of the cells with 6 mM theophylline or 6 mM papaverine which inhibits the cAMP-hydrolysing enzyme. The following results seem to support the hypothesis that cAMP-induced transfer of antigenic molecules from the nucleus to the cytoplasm is mediated by microtubules: Partial staining of the nucleus during the transitional period. Bright staining of the cytoplasm on treated cells in contrast to nuclear staining on control cells. Disappearance of the nuclear staining not only by the monoclonal antibody but also by the polyclonal antibody. Complete prevention of disappearance of nuclear dots induced by these drugs by pretreatment of the cells with colchicine (1 microgram/ml) or colcemid (1 microgram/ml).     

Ligand binding induces a conformational change in ifnar1 that is propagated to its membrane-proximal domain

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.     

PBX1 intracellular localization is independent of MEIS1 in epithelial cells of the developing female genital tract

While studies have highlighted the role of HOXA9-13 and PBX1 homeobox genes during the development of the female genital tract, the molecular mechanisms triggered by these genes are incompletely elucidated. In several developmental pathways, PBX1 binds to MEINOX family members in the cytoplasm to be imported into the nucleus where they associate with HOX proteins to form a higher complex that modulates gene expression. This concept has been challenged by a recent report showing that in some cell cultures, PBX1 nuclear localization might be regulated independently of MEINOX proteins (Kilstrup-Nielsen et al., 2003). Our work gives the first illustration of this alternative mechanism in an organogenesis process. Indeed, we show that PBX1 is mostly cytoplasmic in epithelial endometrial cells of the developing female genital tract despite the nuclear localization of MEIS1. We thus provide evidence for a control of PBX1 intracellular distribution which is independent of MEINOX proteins, but is cell cycle correlated.     

Identification of a bipartite nuclear localization sequence necessary for nuclear import of 5-lipoxygenase.

5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. This enzyme can reside either in the cytoplasm or the nucleus; its subcellular distribution is influenced by extracellular factors, and its nuclear import correlates with changes in leukotriene synthetic capacity. To identify sequences responsible for the nuclear import of 5-lipoxygenase, we transfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors encoding various 5-lipoxygenase constructs fused to green fluorescent protein. Overexpression of wild type 5-lipoxygenase with or without fusion to green fluorescent protein resulted in a predominantly intranuclear pattern of fluorescence, similar to the distribution of native 5-lipoxygenase in primary alveolar macrophages. Within the 5-lipoxygenase protein is a sequence (Arg(638)-Lys(655)) that closely resembles a bipartite nuclear localization signal. Studies using deletion mutants indicated that this region was necessary for nuclear import of 5-lipoxygenase. Analysis of mutants containing specific amino acid substitutions within this sequence confirmed that it was this sequence that was necessary for nuclear import of 5-lipoxygenase and that a specific arginine residue was critical for this function. As nuclear import of 5-lipoxygenase may regulate leukotriene production, natural or induced mutations in this bipartite nuclear localization sequence may also be important in affecting leukotriene synthesis.     

A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export

XAB1/Gpn1 is a GTPase that associates with RNA polymerase II (RNAPII) in a GTP-dependent manner. Although XAB1/Gpn1 is essential for nuclear accumulation of RNAPII, the underlying mechanism is not known. A XAB1/Gpn1-EYFP fluorescent protein, like endogenous XAB1/Gpn1, localized to the cytoplasm but it rapidly accumulated in the cell nucleus in the presence of leptomycin B, a chemical inhibitor of the nuclear transport receptor Crm1. Crm1 recognizes short peptides in substrate proteins called nuclear export sequences (NES). Here, we employed site-directed mutagenesis and fluorescence microscopy to assess the functionality of all six putative NESs in XAB1/Gpn1. Mutating five of the six putative NESs did not alter the cytoplasmic localization of XAB1/Gpn1-EYFP. However, a V302A/L304A double mutant XAB1/Gpn1-EYFP protein was clearly accumulated in the cell nucleus, indicating the disruption of a functional NES. This functional XAB1/Gpn1 NES displays all features present in most common and potent NESs, including, in addition to Φ1-Φ4, a critical fifth hydrophobic amino acid Φ0. Therefore, in human Gpn1 this NES spans amino acids 292-LERLRKDMGSVAL-304. XAB1/Gpn1 NES is remarkably conserved during evolution. XAB1/Gpn1 NES was sufficient for nuclear export activity, as it caused a complete exclusion of EYFP from the cell nucleus. Molecular modeling of XAB1/Gpn1 provided a mechanistic reason for NES selection, as functionality correlated with accessibility, and it also suggested a mechanism for NES inhibition by intramolecular masking. In conclusion, we have identified a highly active, evolutionarily conserved NES in XAB1/Gpn1 that is critical for nucleo-cytoplasmic shuttling and steady-state cytoplasmic localization of XAB1/Gpn1.     

Molecular characterization of yeast regulatory gene CAT3 necessary for glucose derepression and nuclear localization of its product

The yeast regulatory gene CAT3 has an essential function for the depression of several glucose-repressible enzymes. Therefore, cat3 mutants are unable to grow on maltose or on non-fermentable carbon sources. Unlike the point mutants isolated previously, cat3 null allele strains also failed to utilize raffinose or galactose as sole carbon sources. Sequencing of an 1.6-kb HindIII-BglII fragment complementing cat3 mutations revealed an open reading frame of 322 codons, size of which is in good agreement with the 1.3-kb size of mRNA. No significant similarities with previously sequenced genes could be detected. CAT3-lacZ fusions confirmed the proposed reading frame. A CAT3-lacZ fusion encoding 307 amino acids of CAT3 was able to complement the growth defects of cat3 point mutants and null allele strains. Assay of beta-galactosidase activity under different growth conditions indicated a constitutive expression of the CAT3 gene product. Cellular fractionation studies showed the nuclear localization of the CAT3 protein.     

A conformational change in heparan sulfate 3-O-sulfotransferase-1 is induced by binding to heparan sulfate

The 3-O-sulfation of glucosamine by heparan sulfate 3-O-sulfotransferase-1 (3-OST-1) is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). In this paper, we present evidence of a conformational change that occurs in 3-OST-1 upon binding to heparan sulfate. The intrinsic fluorescence of 3-OST-1 was increased in the presence of HS, suggesting a conformational change. This apparent conformational change was further investigated using differential chemical modification of 3-OST-1 to measure the solvent accessibility of the lysine residues. 3-OST-1 was treated with acetic anhydride in either the presence or absence of HS using both acetic anhydride and hexadeuterioacetic anhydride under nondenaturing and denaturing conditions, respectively. The relative reactivity of the lysine residues to acetylation and [2H] acetylation in the presence or absence of HS was analyzed by measuring the ratio of acetylated and deuterioacetylated peptides using matrix-assisted laser desorption ionization mass spectrometry. The solvent accessibilities of the lysine residues were altered differentially depending on their location. In particular, we observed a group of lysine residues in the C-terminus of 3-OST-1 that become more solvent accessible when 3-OST-1 binds to HS. This observation indicates that a conformational change could be occurring during substrate binding. A truncated mutant of 3-OST-1 that lacked this C-terminal region was expressed and found to exhibit a 200-fold reduction in sulfotransferase activity. The results from this study will contribute to our understanding of the interactions between 3-OSTs and HS.