植物细胞质介导GFP-NLS蛋白入核转运的HeLa细胞系统的建立

细胞核作为细胞中重要的遗传物质存储、复制和转录的结构,牵涉着大量信息和物质的传输活动,尤其是蛋白质的入核转运一直以来都是研究的热点问题之一。本文利用病毒SV40抗原蛋白中的核定位信号（nuclear localization signal,NLS）标记GFP蛋白,通过拟南芥细胞质的介导,利用HeLa细胞核建立起了研究蛋白质入核转运的半细胞体系。结果显示,植物细胞质结合NLS片段能改变GFP在HeLa细胞核内外的分布,实现对目标蛋白入核过程的介导,使GFP-NLS最后定位于细胞核内。这也意味着通过HeLa细胞建立起的半细胞体系能为蛋白入核转运研究提供一个有效的研究体系。     

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.     

Characterization of an unusual importin alpha binding motif in the borna disease virus p10 protein that directs nuclear import.

Nuclear import of many cellular and viral proteins is mediated by short nuclear localization signals (NLS) that are recognized by intracellular receptor proteins belonging to the importin/karyopherin alpha and beta families. The primary structure of NLS is not well defined, but most contain at least three basic amino acids and harbor the relative consensus sequence K(K/R)X(K/R). We have studied the nuclear import of the Borna disease virus p10 protein that lacks a canonical oligobasic NLS. It is shown that the p10 protein exhibits all characteristics of an actively transported molecule in digitonin-permeabilized cells. Import activity was found to reside in the 20 N-terminal p10 amino acids that are devoid of an NLS consensus motif. Unexpectedly, p10-dependent import was blocked by a peptide inhibitor of importin alpha-dependent nuclear translocation, and the transport activity of the p10 N-terminal domain was shown to correlate with the ability to bind to importin alpha. These findings suggest that nuclear import of the Borna disease virus p10 protein occurs through a nonconventional karyophilic signal and highlight that the cellular importin alpha NLS receptor proteins can recognize nuclear targeting signals that substantially deviate from the consensus sequence.     

Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins

Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV-40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import.     

Development of a self-assembling nuclear targeting vector system based on the tetracycline repressor protein

The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR.tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4- and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2-8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetRNLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.     

Inhibition of Nuclear Protein Import by a Monoclonal Antibody against a Novel Class of Nuclear Pore Proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab E2-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS--albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small non-nuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex.     

A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.     

Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase II

Fission Yeast DNA topoisomerase II (165 kD) consists of an enzymatically active 125-kD core, approximately 10-kD NH2-terminal and 30-kD COOH-terminal domains. The question addressed in the present study is what is the role of the topo II termini. Although deletion of either the NH2 or the COOH terminus is viable, deletion of both termini is lethal; the termini share an essential role for viability. We show here that topo II phosphorylation sites are localized in the terminal domains, but dephosphorylated topo II is still active. The topo II terminal sequences are required for nuclear localization; topo II double terminal deletion mutants are deficient for nuclear targeting, whereas wild-type and single deletion mutant topo IIs are transported into the nucleus with different efficiencies. Functional subdomains in the NH2 terminus are further dissected; we identified a 15 amino acid nuclear localization sequence (NLS) which is essential for viability and nuclear localization when the COOH terminus is deleted. This NLS could be substituted with SV-40 large T-antigen NLS. Two other functional subdomains were found; a non-essential acidic stretch which is phosphorylated and apparently enhances the nuclear localization and an essential hydrophilic stretch of unknown function. Motifs similar to these three NH2-terminal subdomains are also found in the COOH terminus. Our results support the possibility that phosphorylation of topo II does not play an essential role in fission yeast.     

Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein

We previously reported that the nuclear localization signal (NLS) peptides stimulate the in vitro phosphorylation of several proteins, including a 34 kDa protein. In this study, we show that this specific 34 kDa protein is a novel murine leucine-rich acidic nuclear protein (LANP)-like large protein (mLANP-L). mLANP-L was found to have a basic type NLS. The co-injection of Q69LRan-GTP or SV40 T-antigen NLS peptides prevented the nuclear import of mLANP-L. mLANP-L NLS bound preferentially to Rch1 and NPI-1, but not to the Qip1 subfamily of importin alpha. These findings suggest that mLANP-L is transported into the nucleus by Rch1 and/or NPI-1.     

Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide the first evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.     

Envelope membrane proteins that interact with chloroplastic precursor proteins.

The post-translational transport of cytoplasmically synthesized precursor proteins into chloroplasts requires proteins in the envelope membranes. To identify some of these proteins, label transfer cross-linking was performed using precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase (prSSU) that was blocked at an early stage of the transport process. Two envelope proteins were identified: an 86-kD protein and a 75-kD protein, both present in the outer membrane. Labeling of both proteins required prSSU and could not be accomplished with SSU lacking a transit peptide. Labeling of the 75-kD protein occurred only when low levels of ATP were present, whereas labeling of the 86-kD protein occurred in the absence of exogenous ATP. Although both labeled proteins were identified as proteins of the outer envelope membrane, the labeled form of the 75-kD protein could only be detected in fractions containing mixed envelope membranes. Based on these observations, we propose that prSSU first binds in an ATP-independent fashion to the 86-kD protein. The energy-requiring step is association with the 75-kD protein and assembly of a translocation contact site between the inner and outer membrane of the chloroplastic envelope.     

A long synthetic peptide containing a nuclear localization signal and its flanking sequences of SV40 T-antigen directs the transport of IgM into the nucleus efficiently

Synthetic short peptides containing only the nuclear localization signal (NLS) direct the transport of nonnuclear proteins into the nucleus. As a conjugate of the synthetic peptide with immunoglobulin M (IgM) did not enter the nucleus, there was believed to be a size limit for nuclear transport of NLS-conjugated proteins. However, we found that IgM conjugated with purified nucleoplasmin, a nuclear protein of Xenopus oocytes, rapidly accumulated in the nucleus. For direct comparison with the short peptide, we prepared a long peptide containing the NLS and its flanking sequences of SV40 large T-antigen and its mutated long peptide, in which possible phosphorylation sites located at the amino terminal of the NLS were changed to alanine. Kinetic experiments showed that wild-type long peptide-IgM conjugates were almost entirely taken up into the nucleus within 30 min after their injection, whereas almost 60 min was required for the mutated long peptide-IgM conjugates to enter the nucleus of all the cells examined, and there was no apparent accumulation of short peptide-IgM conjugates in the nucleus within 60 min. These results indicate that even when the kinetics of transport are affected by amino acid substitutions, the long peptide directs the transport of large molecules such as IgM into the nucleus.     

The C-terminal C1 cassette of the N -methyl-D-aspartate receptor 1 subunit contains a bi-partite nuclear localization sequence

The N -methyl-D-aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N-terminal extracellular domain, the transmembrane regions, and the C-terminal intracellular domain. The N-terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation. The intracellular C-terminal domain is involved in regulating receptor activity and subcellular localization. Our recent experiments indicated that the intracellular C-terminal domain, when expressed independently, localizes almost exclusively in the nucleus. An examination of the amino acid sequence reveals the presence of a putative nuclear localization sequence (NLS) in the C1 cassette of the NR1 intracellular C-terminus. Using an expression vector designed to test whether a putative NLS sequence is a valid, functional NLS, we have demonstrated that a bi-partite NLS does in fact exist within the NR1-1 C-terminus. Computer algorithms identified a putative helix-loop-helix motif that spanned the C0C1 cassettes of the C-terminus. These data suggest that the NR1 subunit may represent another member of a family of transmembrane proteins that undergo intramembrane proteolysis, releasing a cytosolic peptide that is actively translocated to the nucleus leading to alterations in gene regulation.     

The VirD2 protein of Agrobacterlum tumefaciens carries nuclear localization signals important for transfer of T-DNA to plants

Agrobacterium tumefaciens is able to transfer a piece of DNA, the T-DNA, to the nucleus of the plant cell. The VirD2 protein is required for the production of the T-DNA, it is tightly linked to the T-DNA and it is thought to direct it to the plant genome. Two nuclear localization signals (NLS), one in the N-terminal part and one in the C-terminal part of the VirD2 protein, have been shown to be able to target marker proteins to the plant nucleus. Here we analyze nuclear entry of the T-DNA complex using a new and very sensitive assay for T-DNA transfer. We show that optimal T-DNA transfer requires the VirD2 NLS located in the C-terminal part of the protein, whereas mutations in the N-terminal NLS coding sequence seem to have no effect on T-DNA transfer.     

The VirD2 protein of Agrobacterlum tumefaciens carries nuclear localization signals important for transfer of T-DNA to plants

Agrobacterium tumefaciens is able to transfer a piece of DNA, the T-DNA, to the nucleus of the plant cell. The VirD2 protein is required for the production of the T-DNA, it is tightly linked to the T-DNA and it is thought to direct it to the plant genome. Two nuclear localization signals (NLS), one in the N-terminal part and one in the C-terminal part of the VirD2 protein, have been shown to be able to target marker proteins to the plant nucleus. Here we analyze nuclear entry of the T-DNA complex using a new and very sensitive assay for T-DNA transfer. We show that optimal T-DNA transfer requires the VirD2 NLS located in the C-terminal part of the protein, whereas mutations in the N-terminal NLS coding sequence seem to have no effect on T-DNA transfer.     

Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization

Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability.     

Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27.

Previous work has shown that the herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 localizes to the cell nucleus and that certain mutant ICP27 polypeptides localize preferentially in nucleoli. To map the signals in ICP27 which mediate its nuclear localization, we identified the portions of ICP27 which can direct a cytoplasmic protein, pyruvate kinase (PK), to nuclei. Our results demonstrate that ICP27 contains multiple nuclear localization signals (NLSs) that function with differing efficiencies. First, ICP27 possesses a strong NLS, mapping to residues 110 to 137, which bears similarity to the bipartite NLSs found in Xenopus laevis nucleoplasmin and other proteins. Second, ICP27 possesses one or more weak NLSs which map to a carboxyl-terminal portion of the protein between residues 140 and 512. Our PK-targeting experiments also demonstrate that ICP27 contains a relatively short sequence, mapping to residues 110 to 152, that can function as a nucleolar localization signal (NuLS). This signal includes ICP27's strong NLS as well as 15 contiguous residues which consist entirely of arginine and glycine. This latter sequence is very similar to an RGG box, a putative RNA-binding motif found in a number of cellular proteins which are involved in nuclear RNA processing. To confirm the results of the PK-targeting experiments, we mutated the ICP27 gene by deleting sequences encoding either the strong NLS or the RGG box. Deletion of the strong NLS (residues 109 to 138) resulted in an ICP27 molecule that was only partially defective for nuclear localization, while deletion of the RGG box (residues 139 to 153) resulted in a molecule that was nuclear localized but excluded from nucleoli. Recombinant HSV-1s bearing either of these deletions were unable to replicate efficiently in Vero cells, suggesting that ICP27's strong NLS and RGG box carry out important in vivo functions.