Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Majadahonda 28220, Madrid, Spain1
Author for correspondence: Agustín Portela. Fax +34 91 5097919. e-mail aportela{at}isciii.es
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Abstract |
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Introduction |
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Protein import and export are signal-mediated events that occur through the nuclear pore complex. There are different nuclear import and export pathways, each involving the interaction of the target protein with specific cell receptors. In this report, a protein sequence that acts as a signal for protein import will be referred to as a nuclear-import signal (NIS), whichever cell receptor is used for import into the nucleus. The term nuclear-localization signal (NLS) will only be used to designate the NIS that has been shown to bind to the NLS receptor (importin and importin
; also known as karyopherin
and karyopherin
), whereas the term nuclear-export signal (NES) will designate protein sequences that direct nuclear export, independently of the export pathway used. In addition to these transport signals, it has been shown that some proteins contain retention signals, either nuclear (NRS) or cytoplasmic (CRS), that also contribute to the determination of subcellular localization (all these aspects have been reviewed by Jans & Hübner, 1996
; Nigg, 1997
; Izaurralde & Adam, 1998
; Görlich, 1998
; Mattaj & Englmeier, 1998
).
A full understanding of the mechanisms that regulate the nucleo-cytoplasmic transport of RNPs would require the identification of the signals that regulate the intracellular localization of isolated NP. However, despite several reports on this issue, the identification of these signals is far from complete. Davey et al. (1985) concluded, from experiments with Xenopus oocytes, that the region required for NP nuclear accumulation was located between residues 327 and 345. However, this conclusion was questioned by results showing that proteins containing substitutions or deletions within this region still accumulated in the nuclei of mammalian cells (Neumann et al., 1997
; Wang et al., 1997
; Digard et al., 1999
; Mena et al., 1999
). Moreover, it was also found that the NP contains an NLS within the 20 N-terminal residues of NP (ONeill & Palese, 1995
; Neumann et al., 1997
; Wang et al., 1997
). Unexpectedly, an NP derivative lacking the two karyophilic signals mentioned above localized mostly to the cell nucleus, indicating the presence of additional NISs in the NP (Neumann et al., 1997
; Wang et al., 1997
). Furthermore, it has been reported recently that the NP contains an actin-binding domain, and it has been suggested that this domain could act functionally as a CRS to cause the cytoplasmic retention of RNPs later in infection (Digard et al., 1999
).
It is well documented that nuclear transport can be controlled by phosphorylation (Hunter & Karin, 1992 ; Jans & Hübner, 1996
; Whittaker & Helenius, 1998
). The NP is a phosphorylated protein (Privalsky & Penhoet, 1977
; Kistner et al., 1989
) and we have demonstrated that the major phosphorylation site of the A/Victoria/3/75 NP is serine-3 (Arrese & Portela, 1996
). The fact that this residue is conserved in practically all NPs sequenced to date (see Arrese & Portela, 1996
, and references therein) and the observations that the N-terminal 13 amino acids of the NP include the binding site for members of the importin
family (ONeill et al., 1995
; Wang et al., 1997
) and that inhibitors and activators of protein kinases can alter the subcellular localization of recombinant NP (Neumann et al., 1997
) suggest that phosphorylation/dephosphorylation of serine-3 may control some of the NP nuclear-traffic events. This hypothesis, however, remains to be demonstrated.
In this report we have carried out a systematic analysis to identify the regions involved in NP subcellular localization. Thus, we have analysed the intracellular localization of a number of NP derivatives, including deleted NP polypeptides, fusion proteins and proteins containing amino acid substitutions at the NP major phosphorylation site.
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Methods |
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Monoclonal antibody M/58/p44/E, which recognizes the A/Victoria/3/75 NP protein (López et al., 1986 ), and rabbit antisera raised against maltose-binding protein-fusion proteins containing the N-terminal 77 amino acids or the C-terminal 120 amino acids of the NP have been described previously (Arrese & Portela, 1996
). For immunofluorescence analysis, the rabbit antisera were enriched in anti-NP antibodies by following the procedure described by Gu et al. (1994)
. Briefly, the antisera were passed through a column containing His-tagged NP and the bound antibodies were eluted by successive washes with 4 M MgCl2 and with 50 mM diethylamine15 mM NaCl. The eluted antibodies were then dialysed against PBS. Anti-actin monoclonal antibody and anti-green fluorescent protein (GFP) polyclonal serum were purchased from Amersham and Clontech, respectively.
Plasmids containing NP gene derivatives downstream of the T7 RNA polymerase promoter of vector pGEM-3.
Plasmid pGEM-NP, which contains a cDNA encoding the NP gene of the influenza A/Victoria/3/75 virus cloned downstream of the T7 RNA polymerase promoter of plasmid pGEM-3 (Promega), has been described previously (de la Luna et al., 1989 ; Mena et al., 1994
). pGEM-NP derivatives encoding deleted NPs have also been described previously (Albo et al., 1995
). Plasmid
13 was prepared by oligonucleotide-directed mutagenesis as described by Mena et al. (1999)
. Plasmids NP-S3D and NP-S3E were prepared by site-directed mutagenesis, with plasmid pGEM-NP as the template, as described previously for plasmid NP-S3A (Arrese & Portela, 1996
). These latter plasmids encode full-length NP proteins that contain Asp (NPS3D), Glu (NPS3E) or Ala (NPS3A) instead of Ser at position 3 of the NP gene.
Plasmids expressing GFPNP fusion proteins.
Fusion proteins containing GFP were obtained by subcloning specific NP gene fragments into the plasmid vectors pEGFP-C1 or pEGFP-N1 (Clontech), which contain a jellyfish GFP gene optimized for maximum fluorescence downstream of a CMV promoter. The NP gene-specific fragments were obtained by PCR by using plasmid pGEM-NP as the template and two oligonucleotides that contained NP gene sequences. In addition to these sequences, one of the oligonucleotides contained an Asp718 restriction site and the other oligonucleotide included, after the NP gene sequence, a stop codon and a restriction site for the enzyme XbaI. The PCR fragments obtained were digested with these restriction enzymes and cloned between the same restriction sites present in the vector pEGFP-C1 (Clontech) to yield fusion proteins that contained GFP at the N terminus of the chimeric polypeptide. The plasmids that encode fusion proteins GFPGST1 and GFPGST2 contained GFP followed by the N-terminal 80 or 160 residues of the bacterial glutathione S-transferase (GST) protein, respectively, and they were prepared by the same approach used to prepare the chimeric GFPNP polypeptides.
To obtain plasmid S3wtGFP, an NP gene-specific fragment corresponding to the first 80 amino acids was obtained by PCR with two oligonucleotides that included restriction sites for the enzymes EcoRI and Asp718. The PCR product was then digested with these two restriction enzymes and cloned between the same two restriction sites present in the vector pEGFP-N1, so that a GFP-fusion polypeptide was obtained that contained the NP protein sequence at the N terminus. Plasmids containing the same NP fragment but with an Ala (S3AGFP), Asp (S3DGFP) or Glu (S3EGFP) residue at position 3 were prepared by following similar procedures.
In all the plasmids that encoded chimeric proteins, the integrity of the NP gene sequence was confirmed by sequencing. The sequences of the oligonucleotides used for mutagenesis will be provided on request.
Subcellular localization of NP derivatives expressed from pGEM-derived plasmids.
Cultures of Vero and/or COS-1 cells growing on glass coverslips were infected with vTF7-3 and transfected individually with the indicated pGEM-NP-derived plasmid and processed for indirect immunofluorescence as described previously (Mena et al., 1999 ).
Detection of GFP-fusion proteins by direct fluorescence.
COS-1 cells growing on coverslips were transfected individually with plasmids encoding GFP-fusion proteins by using liposomes. At 24 h post-transfection, the cells were fixed with 4 % paraformaldehyde in PBS for 10 min at room temperature, incubated in the presence of Hoechst 33258 dye (0·5 µg/µl) for 30 min and visualized with a fluorescence microscope.
Nuclear and cytoplasmic fractionation.
The procedure described by Avalos et al. (1997) was used. Briefly, transfected COS-1 cells were lysed in a solution containing 1% Nonidet P-40, 1% Triton X-100 and 0·5% sodium deoxycholate. The cell lysate was then passed 20 times through a 25-gauge hypodermic needle and centrifuged at 1000 g for 5 min. The supernatant (cytoplasmic) and the pellet (nuclear) fractions were resuspended in SDS sample buffer and analysed by SDSPAGE and Western blotting as described previously (Arrese & Portela, 1996
).
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Results and Discussion |
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We decided to use GFP, a 27 kDa protein not known to contain any sequence involved in nuclear targetting, as the reporter protein. Due to its small size, GFP can diffuse passively through the nuclear pore complex and, in fact, the protein distributes uniformly throughout the cytoplasm and nucleus (pattern N+C) when expressed in mammalian cells (Ogawa et al., 1995 ; Pines, 1995
; Carey et al., 1996
). However, the intracellular distribution of GFP changes when it is fused to a protein containing a functional NLS or NES (Pines, 1995
; Carey et al., 1996
), and we observed that a fusion protein (GFPNP0) containing the full-length NP attached to the C terminus of GFP was localized exclusively to the cell nucleus (Fig. 2
).
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Firstly, five fusion proteins (GFPNP1 to GFPNP5) were prepared that spanned the whole length of NP and that contained the NP fragments at the end of the GFP gene (Fig. 2a). It was then demonstrated that the fusion proteins were of the predicted size and that they were efficiently expressed in transfected COS-1 cells (Fig. 2b
). As summarized in Fig. 2(a)
, four cellular staining patterns were observed: the three patterns described above (N, N>>C and N+C) and a fourth pattern that corresponded to cells displaying exclusively cytoplasmic fluorescence (pattern C). From these results, it was considered that the NP contained at least two signals involved in nuclear accumulation, one located within the N-terminal 160 amino acids and the other one in the region (residues 80320) spanned by proteins GFPNP2 and GFPNP4.
To delineate further the signals involved in NP subcellular localization, nine more recombinant GFP-fusion proteins (GFPNP6 to GFPNP14) were prepared (Fig. 2a). Eight of these proteins contained 80 amino acid-long NP inserts and one included the C-terminal 99 residues of NP. All recombinant proteins accumulated efficiently in transfected cells (Fig. 2b
) and their subcellular distributions are summarized and illustrated in Fig. 2(a
, c
).
From the results obtained with all the fusion proteins, it was concluded that the NP contains at least three karyophilic signals. One of them was mapped to the first 80 amino acids of the protein, since GFPNP6 but not GFPNP7 displayed the same nuclear (N) staining pattern as GFPNP1. This signal probably corresponds to the NLS identified within the N-terminal 20 residues of NP (Wang et al., 1997 ; Neumann et al., 1997
). The second of the signals involved in the nuclear phenotype of NP was that located between residues 80 and 320, which is the NP region covered by proteins GFPNP2 and GFPNP4. It has been impossible to narrow the location of this signal down since none of the five recombinant polypeptides that included short fragments of this NP region localized preferentially to the nucleus. It is, however, suggested that the karyophilic signal within this region would be located within the 80 amino acids (residues 160240) shared by proteins GFPNP2 and GFPNP4, since Weber et al. (1998)
showed the presence of an NIS in the NP region extending from residues 198 to 216. A third signal involved in NP nuclear localization was located between residues 320 and 400, since most of the cells (70%) expressing protein GFPNP10 displayed a clear N C pattern. This NP region includes the signal identified in Xenopus oocytes (Davey et al., 1985
) and this report shows, for the first time, that this region includes a karyophilic signal that is also active in mammalian cells.
Does the NP contain an NES and/or a CRS?
Strikingly, it was observed that three fusion proteins (GFPNP5, GFPNP8 and GFPNP12) were excluded from the nuclei of transfected cells (Fig. 2a, c
), a result that strongly suggests the presence of an NES and/or CRS in the NP protein.
Considering the NP sequences included in these cytosolic fusion proteins, it is concluded that the NP contains at least two signals that drive NP cytoplasmic accumulation. One signal resides between residues 240 and 400 and the other one is tentatively assigned to the region extending from amino acid 160 to 200, which corresponds to the NP fragment shared by cytosolic proteins GFPNP8 and GFPNP12. Further experiments are required to determine whether these regions include an NES and/or a CRS. It is worth noting that the NP contains an actin-binding domain that may act functionally as a CRS (Digard et al., 1999 ), since NP proteins with mutations that lower the affinity for actin display increased nuclear accumulation. Since the studies carried out by Digard et al. (1999)
indicate that this domain is around residue 340 of the NP, it is tempting to speculate that the most C-terminal of the two NP signals that drive cytoplasmic accumulation may correspond to the domain involved in binding actin (Digard et al., 1999
).
Interestingly, according to the location of the NP signals involved in the subcellular localization of the protein, each of the signals that causes cytoplasmic accumulation is in close proximity to a karyophilic signal. It is thus tempting to speculate that these two types of signals may overlap, so that the same protein domain could confer bidirectional transport of the corresponding protein across the nuclear envelope, as has been reported for the ~40 amino acid domains M9, KNS and HNS present in hnRNP A1, hnRNP K and HuR, respectively (Siomi & Dreyfuss, 1995 ; Michael et al., 1995
, 1997
; Fan & Steitz, 1998
).
The close proximity of the cytoplasmic accumulation signals and the NISs probably explains in part the apparently contradictory results obtained with some of the GFP-fusion proteins. For example, the nuclear protein GFPNP2 and the cytoplasmic protein GFPNP8 would contain, according to the data mentioned above, both a karyophilic and a cytoplasmic accumulation domain. To explain the different subcellular localization of these two proteins, it is proposed that the karyophilic signal is properly folded and exposed in protein GFPNP2 so that it dominates over the cytoplasmic accumulation signal (which may also be partially masked) whereas, because of conformational effects, the opposite would be true for protein GFPNP8.
Amino acid substitutions at residue 3 of the NP alter the functionality of the N-terminal NLS
It was decided to investigate the possible role of phosphorylation of serine 3 in modulating nuclear translocation of NP. Thus, we prepared plasmids encoding full-length NP containing substitutions at position 3 that were supposed to mimic the unphosphorylated (mutation S3A) and the negatively charged, phosphorylated (mutations S3D and S3E) forms of the protein. The wild-type NP and the three serine-altered mutant proteins accumulated to similar levels in transfected cells (Fig. 3a) and displayed indistinguishable fluorescence patterns, which included cells showing exclusively nuclear staining and cells showing the N C pattern (Fig. 3a
). The three mutant proteins were also tested for their functionality in a system in which expression of a synthetic influenza virus-like CAT RNA is achieved in COS-1 cells that express the NP and the three subunits of the viral polymerase from cloned cDNAs (Mena et al., 1994
). As previously demonstrated for the S3A mutant (Arrese & Portela, 1996
), it was observed that the two proteins containing acidic substitutions were also functional in the system and yielded CAT activities ranging from 50 to 90% of that obtained with the wild-type NP (data not shown). These results indicated that neither the subcellular distribution of the full-length protein nor NP activity, as regarding its role during replication/transcription of the viral genome, are modified drastically by the mutations introduced at position 3.
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Considering that the charged acidic amino acids may mimic the phosphorylated serine at position 3, the results suggest that phosphorylation of this residue impairs the functionality of the N-terminal NLS of NP. The situation may be similar to that found for the simian virus 40 (SV40) T antigen (and other proteins), in which phosphorylation diminishes the rate of protein nuclear import, probably as a consequence of reducing the affinity of the NIS for its cellular receptor (Jans & Hübner, 1996 ; Rihs et al., 1991
). The diminished activity of the NP N-terminal NLS may be relevant during certain stages of the virus life-cycle. For example, late in infection when the RNPs have been exported to the cytoplasm, a diminished functionality of the N-terminal NLS would contribute to the prevention of re-entry of the RNPs into the nucleus.
Summary
The results presented here, together with the previous studies mentioned above, emphasize the complexity of the signals that regulate nucleo-cytoplasmic transport of NP. The picture emerging is that NP contains three signals involved in nuclear accumulation and two signals that lead to cytoplasmic accumulation. Considering that masking/unmasking of these signals during the infectious cycle follows a strict temporal order, we postulate that phosphorylation and interactions of the NP protein with other factors (RNA, actin and other viral proteins) play a critical role in regulating the ordered exposure of the NP subcellular signals.
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Acknowledgments |
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References |
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Received 30 June 1999;
accepted 21 September 1999.