The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.     

A method for the large-scale cloning of nuclear proteins and nuclear targeting sequences on a functional basis

We describe here a selection strategy allowing the cloning of sequences that contain a functional nuclear targeting signal. Our method relies on the use of green fluorescent protein fusion proteins to identify nuclear targeting sequences. Transfected cells expressing nuclear protein fusions were isolated on the basis of their nuclear fluorescence using flow cytometry and the transfected DNAs were recovered after bacterial transformation with total DNA from pools of sorted cells. Starting from a cDNA expression library, in which only 1% of the expressed proteins were nuclear, we obtained a 70-fold enrichment in nuclear protein-encoding clones after a single round of selection. Among the 63 clones that have been partially sequenced to date, 25 (40%) corresponded to known nuclear proteins and 13 (20%) to previously uncharacterized sequences. Despite their ability to target the green fluorescent protein marker to the cell nucleus, about half of the cloned sequences did not encode canonical basic or bipartite nuclear localization signals. The method can thus be applied to the large-scale cloning of functional nuclear targeting sequences, which opens the way to a wide investigation of nuclear import mechanisms and to the identification of previously unknown nuclear proteins.     

Tus,an E. coli Protein,Contains Mammalian Nuclear Targeting and Exporting Signals

Shuttling of proteins between nucleus and cytoplasm in mammalian cells is facilitated by the presence of nuclear localization signals (NLS) and nuclear export signals (NES), respectively. However, we have found that Tus, an E. coli replication fork arresting protein, contains separate sequences that function efficiently as NLS and NES in mammalian cell lines, as judged by cellular location of GFP-fusion proteins. The NLS was localized to a short stretch of 9 amino acids in the carboxy-terminus of Tus protein. Alterations of any of these basic amino acids almost completely abolished the nuclear targeting. The NES comprises a cluster of leucine/hydrophobic residues located within 21 amino acids at the amino terminus of Tus. Finally, we have shown that purified GFP-Tus fusion protein or GFP-Tus NLS fusion protein, when added to the culture media, was internalized very efficiently into mammalian cells. Thus, Tus is perhaps the first reported bacterial protein to possess both NLS and NES, and has the capability to transduce protein into mammalian cells.     

A lily pollen ASR protein localizes to both cytoplasm and nuclei requiring a nuclear localization signal

LLA23, an abscisic acid-, stress- and ripening-induced (ASR) protein, was isolated previously from lily ( Lilium longiflorum ) pollen. Close examination of the C-terminus of this ASR protein revealed the presence of basic regions reminiscent of a nuclear localization signal (NLS). Fluorescence microscopy studies using green fluorescent protein (GFP) fusion proteins indicated that the bipartite NLS in LLA23 exhibited nuclear localization properties. Accordingly, mutations in the NLS motifs of LLA23 defined two regions, either of which was necessary for partial nuclear targeting and both of which were required for complete nuclear localization. In addition, oligonucleotide-directed mutagenesis identified lysine residues within the NLS necessary for nuclear localization. Immunogold localization confirmed that the protein was located to both the cytoplasm and nucleus of generative and vegetative cells of pollen grains; the generative nuclei showed the highest number of LLA23 labelling. The possible function of ASR proteins in both the cytoplasm and nuclei of pollen grains is discussed.     

Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport.

Large amounts of pp65 (UL83) of human cytomegalovirus are translocated to the cell nucleus during the first minutes after uptake of the tegument protein from infecting viral particles. Two stretches of basic amino acids which resembled nuclear localization signals (NLS) of both the simian virus 40 type and the bipartite type were found in the primary structure of pp65. Deletion of these sequences significantly impaired nuclear localization of the truncated proteins after transient expression. The results indicated that both elements contributed to the nuclear localization of the protein. When fused to the bacterial beta-galactosidase, only one of the two basic elements was sufficient to mediate nuclear translocation. This element consisted of two clusters of basic amino acids (boxes C and D), which were separated by a short spacer sequence. In contrast to other bipartite NLS of animal cells, both basic boxes C and D functioned independently in nuclear transport, thus resembling simian virus 40-type NLS. Yet, complete translocation of beta-galactosidase was only found in the bipartite configuration. When both boxes C and D were fused, thereby deleting the intervening sequences, the nuclear transport of beta-galactosidase was reduced to levels seen with constructs in which only one of the boxes was present. Appropriate spacing, therefore, was important but not absolutely required. This was in contrast with results for other bipartite NLS, in which spacer deletions led to complete cytoplasmic retention. The presented results demonstrate that efficient nuclear transport of pp65 is mediated by one dominant NLS and additional targeting sequences. The major NLS of pp65 is an unusual signal sequence composed of two weak NLS which function together as one strong bipartite nuclear targeting signal.     

Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity

Acinetobacter baumannii is an emerging opportunistic pathogen responsible for healthcare-associated infections. The outer membrane protein A of A. baumannii (AbOmpA) is the most abundant surface protein that has been associated with the apoptosis of epithelial cells through mitochondrial targeting. The nuclear translocation of AbOmpA and the subsequent pathology on host cells were further investigated. AbOmpA directly binds to eukaryotic cells. AbOmpA translocates to the nucleus by a novel monopartite nuclear localization signal (NLS). The introduction of rAbOmpA into the cells or a transient expression of AbOmpA–EGFP causes the nuclear localization of these proteins, while the fusion proteins of AbOmpAΔNLS–EGFP and AbOmpA with substitutions in residues lysine to alanine in the NLS sequences represent an exclusively cytoplasmic distribution. The nuclear translocation of AbOmpA induces cell death in vitro . Furthermore, the microinjection of rAbOmpA into the nucleus of Xenopus laevis embryos fails to develop normal embryogenesis, thus leading to embryonic death. We propose a novel pathogenic mechanism of A. baumannii regarding the nuclear targeting of the bacterial structural protein AbOmpA.     

Switching of cardiac troponin I between nuclear and cytoplasmic localization during muscle differentiation

The nuclear accumulation of proteins may depend on the presence of short targeting sequences, which are known as nuclear localization signals (NLSs). Here, we found that NLSs are predicted in some cytosolic proteins and examined the hypothesis that these NLSs may be functional under certain conditions. As a model, human cardiac troponin I (hcTnI) was used. After expression in cultured non-muscle or undifferentiated muscle cells, hcTnI accumulated inside nuclei. Several NLSs were predicted and confirmed by site-directed mutagenesis in hcTnI. Nuclear import occurred via the classical karyopherin-α/β nuclear import pathway. However, hcTnI expressed in cultured myoblasts redistributed from the nucleus to the cytoplasm, where it was integrated into forming myofibrils after the induction of muscle differentiation. It appears that the dynamic retention of proteins inside cytoplasmic structures can lead to switching between nuclear and cytoplasmic localization.     

Agrobacterium VirE2 Proteins Can Form a Complex with T Strands in the Plant Cytoplasm

Wild-type VirE2 and VirD2 proteins from Agrobacterium tumefaciens contain nuclear targeting sequences (NLS) that are likely involved in directing transferred T strands to the plant nucleus. An A. tumefaciens virE2 virD2ΔNLS double mutant was able to form tumors on VirE2-producing transgenic tobacco but not on wild-type tobacco. Because this mutant bacterial strain contains no known T-strand nuclear targeting signal, the data indicate that wild-type VirE2 proteins produced by the plant can interact with the T strands in the plant cytoplasm and direct them to the nucleus.     

Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins.     

Phosphorylation mediates the nuclear targeting of the maize Rab17 protein

The maize abscisic acid-responsive Rab17 protein localizes to the nucleus and cytoplasm in maize cells. In-frame fusion of Rab17 to the reporter protein β-glucuronidase (GUS) directed GUS to the nucleus and cytoplasm in transgenic Arabidopsis thaliana and in transiently transformed onion cells. Analysis of chimeric constructs identified one region between amino acid positions 66–96, which was necessary for targeting GUS to the nucleus. This region contains a serine cluster followed by a putative consensus site for protein kinase CK2 phosphorylation, and a stretch of basic amino acids resembling the simian virus 40 large T antigen-type nuclear localization signal (NLS). Mutation of two basic amino acids in the putative NLS had a weak effect on nuclear targeting in the onion cell system and did not modify the percentage of nuclear fusion protein in the Arabidopsis cells. The mutation of three amino acids in the consensus site for CK2 recognition resulted in the absence of in vitro phosphorylated forms of Rab17 and in a strong decrease of GUS enzymatic activity in isolated nuclei of transgenic Arabidopsis. These results suggest that phosphorylation of Rab17 by protein kinase CK2 is the relevant step for its nuclear location, either by facilitating binding to specific proteins or as a direct part of the nuclear targeting apparatus.     

The sequence [EKRKI(E/R)(K/L/R/S/T)] is a nuclear localization signal for importin 7 binding (NLS7)

BackgroundNuclear translocation of large proteins is mediated through specific protein carriers, collectively named karyopherins (importins, exportins and adaptor proteins). Cargo proteins are recognized by importins through specific motifs, known as nuclear localization signals (NLS). However, only the NLS recognized by importin α and transportin (M9 NLS) have been identified so farMethodsAn unsupervised in silico approach was used, followed by experimental validation.ResultsWe identified the sequence EKRKI(E/R)(K/L/R/S/T) as an NLS signal for importin 7 recognition. This sequence was validated in the breast cancer cell line T47D, which expresses importin 7. Finally, we verified that importin 7-mediated nuclear protein transport is affected by cargo protein phosphorylation.ConclusionsThe NLS sequence for importin 7 was identified and we propose this approach as an identification method of novel specific NLS sequences for β-karyopherin family members.General significanceElucidating the complex relationships of the nuclear transporters and their cargo proteins may help in laying the foundation for the development of novel therapeutics, targeting specific importins, with an immediate translational impact.     

A plant in vitro system for the nuclear import of proteins

This paper reports the development of an in vitro system that allows the direct assay of protein import into plant nuclei. In this assay the import of fluorescently labelled karyophilic protein substrates into nuclei isolated from evacuolated tobacco BY-2 suspension cells is monitored. It is demonstrated that import of the fluorescently labelled peptide conjugates is rapid, saturable and nuclear localization signal (NLS)-dependent. Exclusion of high molecular weight (70 kDa) dextran and substrates carrying mutated NLS sequences further underline the specificity of this system. Nuclear translocation of karyophilic import substrates in tobacco, similar to mammalian systems, is inhibited by the non-hydrolysable GTP analogue GTP-γ-S. In contrast, protein uptake is not blocked by wheat germ agglutinin, N-ethyl-maleinimide and iodoacetic acid. Furthermore, it is shown that nuclear import of proteins is only partially inhibited by low temperature (0–4°C). The in vitro nuclear import assay does not depend on exogenously added ATP or cytosolic factors. However, a block of nuclear import with GTP-γ-S could be overcome by the addition of cytosolic extract, suggesting the dependence on cytosolic factors or proteins. These data indicate that the characteristics of nuclear protein import in plant and mammalian cells are similar, but may be, at least in some respects, also different from each other.     

One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA-binding domain

The nuclear localization sequences (NLSs) of the Ac transposase (TPase) protein have been characterized by indirect immunofluorescence detection of TPase deletion derivatives and TPase/β-glucuronidase (GUS) fusion proteins in transiently transfected Petunia cells. The TPase contains three NLSs near its amino-terminal end, NLS(44–62), NLS(159–178) and NLS(174–206), each of which is sufficient to redirect GUS to the nucleus. Deletion of the N-terminal 102 TPase residues including NLS(44–62) results in strongly reduced nuclear import of the truncated TPase. NLS(44–62) and NLS(159–178) are bipartite NLSs, whereas the structure of NLS(174–206) does not allow a classification into one of the three major NLS categories. NLS(174–206) overlaps with the basic DNA-binding domain of TPase. A substitution of two amino acids in this segment (HiS₁₉₁→Arg and Arg₁₉₃→His) results in a total loss of DNA-binding activity, but retains reduced NLS activity. Accordingly, the two functions can be separated. In addition, we show that a NLS-deficient 71 kDa TPase derivative is co-imported into the nucleus in the presence of wildtype TPase.     

Agrobacterium VirE2 gets the VIP1 treatment in plant nuclear import.

Agrobacterium tumefaciens transforms plant cells by targeting a large single-stranded DNA molecule (T-strand) to the plant nucleus. The host cell contribution to nuclear import and transformation is the focus of several current articles. Recently, plant proteins have been identified that promote nuclear import of the T-strand. In particular, VIP1 might couple transformation to the importin-dependent nuclear import pathway and deliver the T-strand to chromatin, thereby promoting integration into the host genome.     

Interaction of yeast importin alpha with the NLS of prothymosin alpha is insufficient to trigger nuclear uptake of cargos

A proliferation-related human protein prothymosin alpha displays exclusively nuclear localization when produced in human and Saccharomyces cerevisiae cells, whereas its isolated bipartite NLS confers nuclear targeting of the GFP reporter in human but not in yeast cells. To test whether this observation is indicative of the existence of specific requirements for nuclear targeting of proteins in yeast, a set of prothymosin alpha deletion mutants was constructed. Subcellular localization of these mutants fused to GFP was determined in yeast and compared with their ability to bind yeast importin alpha (Srp1p) in vitro. The NLS of prothymosin alpha turned out to be both necessary and sufficient to provide protein recognition by importin alpha. However, the NLS-importin alpha interaction did not ensure nuclear targeting of prothymosin alpha derivatives. This defect could be complemented by adding distinct prothymosin alpha sequences to the NLS-containing import substrate, possibly by providing binding site(s) for additional components of the yeast nuclear import machinery.     

Molecular Control of Nuclear and Subnuclear Targeting of the Plant CDK Inhibitor ICK1 and ICK1-Mediated Nuclear Transport of CDKA

ICK1 is the first member of a family of plant cyclin-dependent kinase (CDK) inhibitors. It has been shown that ICK1 is localized in the nuclei of transgenic Arabidopsis plants. Since cellular localization is important for the functions of cell cycle regulators, a comprehensive analysis was undertaken to identify specific sequences regulating the cellular localization of ICK1. Deletion and site-specific mutants fused to the green fluorescent protein (GFP) were used in transgenic Arabidopsis plants and transfected tobacco cells. Surprisingly, three separate sequences in the N-terminal, central and C-terminal regions of ICK1 could independently confer nuclear localization of the GFP fusion proteins. The central nuclear localization signal NLS^ICK1 could transport the much larger GUS (β-glucuronidase)-GFP fusion protein into nuclei, while the other two sequences were unable to. These results suggest that NLS^ICK1 is a strong NLS that actively transports the fusion protein into nuclei, while the other two sequences are either a weaker NLS or confer the nuclear localization of GFP indirectly. It was further observed that the N-terminal sequence specifies a punctate pattern of subnuclear localization, while the C-terminal sequence suppresses it. Furthermore, co-expression of ICK1 and Arabidopsis CDKA, tagged with different GFP variants, showed that ICK1 could mediate the transport of CDKA into nuclei while a mutant ICK1^1–162 that does not interact with CDKA lost this ability. These results illustrate how the nuclear localization of ICK1 is regulated and also suggest a possible role of ICK1 in regulating the cellular distribution of CDKA.     

Novel nuclear targeting coiled-coil protein of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> showing Ca<Superscript>2+</Superscript>-independent,Mg<Superscript>2+</Superscript>-dependent DNase I activity

HP0059, an uncharacterized gene of Helicobacter pylori, encodes a 284-aa-long protein containing a nuclear localization sequence (NLS) and multiple leucine-rich heptad repeats. Effects of HP0059 proteins in human stomach cells were assessed by incubation of recombinant HP0059 proteins with the AGS human gastric carcinoma cell line. Wild-type HP0059 proteins showed cytotoxicity in AGS cells in a concentration-dependent manner, whereas NLS mutant protein showed no effect, suggesting that the cytotoxicity is attributed to host nuclear localization. AGS cells transfected with pEGFP-HP0059 plasmid showed strong GFP signal merged to the chromosomal DNA region. The chromosome was fragmented into multiple distinct dots merged with the GFP signal after 12 h of incubation. The chromosome fragmentation was further explored by incubation of AGS chromosomal DNA with recombinant HP0059 proteins, which leaded to complete degradation of the chromosomal DNA. HP0059 protein also degraded circular plasmid DNA without consensus, being an indication of DNase I activity. The DNase was activated by MgCl₂, but not by CaCl₂. The activity was completely blocked by EDTA. The optimal pH and temperature for DNase activity were 7.0–8.0 and 55°C, respectively. These results indicate that HP0059 possesses a novel DNase I activity along with a role in the genomic instability of human gastric cells, which may result in the transformation of gastric cells.     

A nuclear localization domain in the hnRNP A1 protein

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta- galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.