Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

A nucleus-localization-deficient mutant serves as a dominant-negative inhibitor of gut-enriched Krüppel-like factor function

Cancer cells differ from normal cells in many aspects, including loss of differentiation and uninhibited cell proliferation. Recent studies have suggested that gut-enriched Krüppel-like factor (GKLF) played an important role in the regulation of cell growth in the colon. Studies from this laboratory have shown that GKLF protein predominantly expressed in the cytoplasm but not the nucleus of colon cancer cells, suggesting that impaired nuclear translocation of GKLF might contribute to cancer formation. In this report, a region containing putative nuclear localization signal (NLS) of GKLF (PKRGRR; amino acids 385-390) was investigated. Mutation of KR to WT had no effect on the inhibitory properties of GKLF on cyclin D1 promoter activity and [(3)H]thymidine uptake in HT-29 cells, whereas mutation of RR to GL abolished GKLF function completely. Additional mutation analyses demonstrated that Arg(390) is the most critical moiety within this region that mediated GKLF function and its nucleus localization. Cotransfection of Arg(390) mutant (RR/RS) completely inhibited wild-type GKLF function, and GFP-RR/RS GKLF fusion proteins failed to translocate to the nucleus. The results from this study demonstrate that Arg(390) confers the NLS of GKLF and that the nucleus-localization-deficient mutant serves as dominant-negative inhibitor of GKLF function.     

Nuclear and nucleolar localization of 18-kDa fibroblast growth factor-2 is controlled by C-terminal signals

Members of high (22-, 22.5-, 24-, and 34-kDa) and low (18-kDa) molecular mass forms of fibroblast growth factor-2 (FGF-2) regulate cell proliferation, differentiation, and migration. FGF-2s have been previously shown to accumulate in the nucleus and nucleolus. Although high molecular weight forms of FGF-2 contain at least one nuclear localization signal (NLS) in their N-terminal extension, the 18-kDa FGF-2 does not contain a standard NLS. To determine signals controlling the nuclear and subnuclear localization of the 18-kDa FGF-2, its full-length cDNA was fused to that of green fluorescent protein (GFP). The fusion protein was primarily localized to the nucleus of COS-7 and HeLa cells and accumulated in the nucleolus. The subcellular distribution was confirmed using wild type FGF-2 and FGF-2 tagged with a FLAG epitope. A 17-amino acid sequence containing two groups of basic amino acid residues separated by eight amino acid residues directed GFP and a GFP dimer into the nucleus. We systematically mutated the basic amino acid residues in this nonclassical NLS and determined the effect on nuclear and nucleolar accumulation of 18-kDa FGF-2. Lys(119) and Arg(129) are the key amino acid residues in both nuclear and nucleolar localization, whereas Lys(128) regulates only nucleolar localization of 18-kDa FGF-2. Together, these results demonstrate that the 18-kDa FGF-2 harbors a C-terminal nonclassical bipartite NLS, a portion of which also regulates its nucleolar localization.     

Characterization of nuclear localizing sequences derived from yeast ribosomal protein L29.

Two particular seven-amino-acid segments from yeast ribosomal protein L29 caused a non-nuclear reporter protein to associate almost exclusively with the yeast nucleus. The two L29-derived nuclear localizing sequences were identical in five of the seven residues, many of which were basic amino acids. Generally, localization of the reporter protein was most impaired by replacement of the basic residues. A particular Arg residue was unique; substitution by any amino acid including Lys diminished nuclear localization of the reporter protein. In L29 the corresponding Arg 25----Lys substitution within the nuclear localizing sequence distal to the N-terminus was without effect, as evidence by normal rates of ribosome assembly and cell growth. However, the analogous Arg 8----Lys substitution within the localizing sequence proximal to the N-terminus led to greatly reduced rates of ribosome assembly and cell growth. Finally, when both localizing sequences contained the Arg----Lys substitution a still greater decrease in ribosome assembly and cell growth was observed. These results were as expected if the two short peptide sequences functioned in nuclear localization and/or assembly of yeast ribosomal protein L29.     

The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19- amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.     

Nuclear targeting is required for hepatoma-derived growth factor-stimulated mitogenesis in vascular smooth muscle cells

We recently identified hepatoma-derived growth factor (HDGF) as a nuclear targeted vascular smooth muscle cell (VSM) mitogen that is expressed in developing vascular lesions. In the present study, VSM in culture express endogenous HDGF only in the nucleus and target a green fluorescent protein (GFP)-HDGF fusion to the nucleus. To define the features of the HDGF molecule that are essential for nuclear localization and mitogenic function, deletion and site-directed mutagenesis were performed. Deletion analysis identified the carboxyl-terminal half of HDGF to be responsible for nuclear targeting in VSM. Overexpression of tagged HDGF proteins with point mutations in the putative bipartite nuclear localization sequence in the carboxyl terminus demonstrated that single Lys --> Asn mutations randomized HDGF expression to both the nucleus and cytoplasm similar to the empty vector. Importantly, the Lys --> Asn mutation of all three lysines blocked nuclear entry. Point mutation of a p34(cdc2) kinase consensus motif within the nuclear localization sequence had no effect on nuclear targeting. Moreover, nuclear entry was essential for the HDGF mitogenic effect, as transfection with the triple Lys --> Asn mutant HA-HDGF significantly attenuated bromodeoxyuridine uptake when compared with transfection with wild type HA-HDGF. We conclude that HDGF contains a true bipartite nuclear localization sequence with all three lysines necessary for nuclear targeting. Nuclear targeting of HDGF is required for HDGF stimulation of DNA replication in VSM.     

Bipartite Nuclear Localization Signals in the C Terminus of Human Topoisomerase IIα

DNA topoisomerase IIα is the intracellular target for several important chemotherapeutic agents, and drug-resistant human tumor cell lines have been described in which deletions in the C-proximal region of this enzyme are associated with its cytoplasmic localization. We have identified multiple potential bipartite nuclear localization signal (NLS) sequences in this region using a modified definition of the motif, and in the present study, we have expressed five of these as fusion proteins with β-galactosidase. Only one sequence (spanning amino acids 1454 to 1497) was sufficient to cause strong nuclear localization. Subsequent mutation analyses indicated that this NLS sequence was bipartite and that both domains contain more than two basic amino acids. Substitution of the lysine residue at position 1492 in the second basic domain with glutamine resulted in a fusion protein that localized inefficiently to the nucleus, indicating that all three basic residues in this domain are necessary. Our results confirm that a broader definition is required to detect all potential bipartite NLS motifs in a polypeptide sequence, although functional tests are still essential for identification of those sequences actually capable of directing nuclear localization.     

pH-sensitive interactions between IgG and a mutated IgG-binding protein based upon two B domains of protein A from Staphylococcus aureus.

A fusion protein, consisting of the N-terminal 81 amino acids from an inactive bovine DNase I (Q38,E39-E38,Q39) and two sequential synthetic IgG-binding domains based upon domain B of Protein A from Staphylococcus aureus has been shown to bind to porcine IgG with a similar affinity and pH profile to Protein A. The same residue in each B domain (Tyr111 and Tyr169) has been mutated by cassette mutagenesis to Ser, Glu, His, Lys or Arg and the effect of the mutation on binding interactions with porcine IgG investigated. The evidence presented suggests that the interactions at the B domain are highly sensitive to the presence of a charged residue.     

A Nuclear Targeting Determinant for SATB1, a Genome Organizer in the T Cell Lineage

SATB1 is a nuclear protein which acts as a cell-type specific genome organizer and gene regulator essential for T cell differentiation and activation. Several functional domains of SATB1 have been identified. However, the region required for nuclear localization remains unknown. To delineate this region, we employed sequence analysis to identify phylogenetically diverse members of the SATB1 protein family, and used hidden Markov model (HMM)-based analysis to define conserved regions and motifs in this family. One of the regions conserved in SATB1- and SATB2-like proteins in mammals, fish, frog and bird, is located near the N-terminus of family members. We found that the N-terminus of human SATB1 was essential for the nuclear localization of the protein. Furthermore, fusing residues 20-40 to a cytoplasmic green fluorescence protein (GFP) fused to pyruvate kinase (PK) was sufficient to quantitatively translocate the pyruvate kinase into the nucleus. The nuclear targeting sequence of human SATB1 (residues 20-40) is novel and does not contain clusters of basic residues, typically found in ‘classical’ nuclear localization signals (NLSs). We investigated the importance of four well-conserved residues (Lys29, Arg32, Glu34, and Asn36) in this nuclear targeting sequences. Remarkably, full-length SATB1 harboring a single point mutation at either Lys29 or Arg32, but not Glu34 or Asn36, did not enter the nucleus. Our results indicate that SATB1 N-terminal residues 20-40 represent a novel determinant of nuclear targeting.     

The carboxyl terminus of RNA helicase A contains a bidirectional nuclear transport domain

Human RNA helicase A was recently identified to be a shuttle protein which interacts with the constitutive transport element (CTE) of type D retroviruses. Here we show that a domain of 110 amino acids at the carboxyl terminus of helicase A is both necessary and sufficient for nuclear localization as well as rapid nuclear export of glutathione S-transferase fusion proteins. The import and export activities of this domain overlap but are separable by point mutations. This bidirectional nuclear transport domain (NTD) has no obvious sequence homology to previously identified nuclear import or export signals. However, the Ran-dependent nuclear import of NTD was efficiently competed by excess amounts of the nuclear localization signal (NLS) peptide from simian virus 40 large T antigen, suggesting that import is mediated by the classical NLS pathway. The nuclear export pathway accessed by NTD is insensitive to leptomycin B and thus is distinct from the leucine-rich nuclear export signal pathway mediated by CRM1.     

Identification of an unconventional nuclear localization signal in human ribosomal protein S2

Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-beta-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-beta-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric beta-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importinbeta binding site fused to VP22 blocks nuclear import of rpS2-beta-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importinalpha/beta and transportin.     

The role of X/Y linker region and N-terminal EF-hand domain in nuclear translocation and Ca2+ oscillation-inducing activities of phospholipase Czeta, a mammalian egg-activating factor

Sperm-specific phospholipase C-zeta (PLCzeta) causes intracellular Ca(2+) oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCzeta, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCzeta mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374-381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg(376), Lys(377), Arg(378), Lys(379), or Lys(381) with glutamate, whereas Ca(2+) oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys(299) and/or Lys(301) in the C terminus of X domain, or Trp(13), Phe(14), or Val(18) in the N terminus of EF1. Ca(2+) oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374-383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296-309 or 1-19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2-4 or C2 domain. Thus, PLCzeta is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCdelta1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.     

Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains.

The spike glycoprotein G of vesicular stomatitis virus (VSV) induces membrane fusion at low pH. We used linker insertion mutagenesis to characterize the domain(s) of G glycoprotein involved in low-pH-induced membrane fusion. Two or three amino acids were inserted in frame into various positions in the extracellular domain of G, and 14 mutants were isolated. All of the mutants expressed fully glycosylated proteins in COS cells. However, only seven mutant G glycoproteins were transported to the cell surface. Two of these mutants, D1 and A6, showed wild-type fusogenic properties. The mutant A2 had a temperature-sensitive defect in the transport of the mutant G glycoprotein to the cell surface. The other four mutants, H2, H5, H10, and A4, although present in cell surface, failed to induce cell fusion when cells expressing these mutant glycoproteins were exposed to acidic pH. These four mutant G proteins could form trimers, indicating that the defect in fusion was not due to defective oligomerization. One of these mutations, H2, is within a region of conserved, uncharged amino acids that has been proposed as a possible fusogenic sequence. The mutation in H5 was about 70 amino acids downstream of the mutation in H2, while mutations in H10 and A4 were about 300 amino acids downstream of the mutation in H2. Conserved sequences were also noted in the H10 and A4 segment. The results suggest that in the case of VSV G glycoprotein, the fusogenic activity may involve several spatially separated regions in the extracellular domain of the protein.     

Nuclear localization signals, but not putative leucine zipper motifs, are essential for nuclear transport of hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only known protein of hepatitis delta virus and was previously shown to localize in the nucleoplasm of infected liver cells. In this study, nuclear localization signals of HDAg were defined by expressing various domains of the antigen in both hepatic and nonhepatic cells as beta-galactosidase fusion proteins. A cytochemical staining assay demonstrated that a domain from amino acid residues 35 to 88 of HDAg was able to facilitate transport to the nucleus of the originally cytoplasm-localized protein beta-galactosidase. Two nuclear localization signals, NLS1 and NLS2, which are similar to those of simian virus 40 T antigen and polyomavirus T antigen, respectively, were identified. Either NLS1 or NLS2 alone was sufficient for the nuclear transport of HDAg. However, a fusion protein (N65Z) containing beta-galactosidase and the N-terminal 65 amino acids of HDAg, containing NLS1, was localized exclusively in the cytoplasm and perinuclear region. A possible hydrophobic subdomain between amino acid residues 50 and 65 may block the function of NLS1. Nevertheless, N65Z could enter the nuclei of transfected cells when it was coexpressed with full-length HDAg. Entry into the nucleus may be mediated by the coiled-coil structure rather than the putative leucine zipper motif located between amino acid residues 35 and 65. The existence of two independent nuclear localization signals may ensure the proper functioning of HDAg in the multiplication of delta virus in the nucleus. In addition, two putative casein kinase II sites (SRSE-5 and SREE-126) that may be important in controlling the rate of nuclear transport were found in HDAg.