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Molecular and Cellular Biology, February 1999, p. 1210-1217, Vol. 19, No. 2
Howard Hughes Medical Institute and
Department of Genetics, Duke University Medical Center, Durham,
North Carolina 27710
Received 17 August 1998/Returned for modification 14 October
1998/Accepted 27 October 1998
Protein nuclear import is generally mediated by basic nuclear
localization signals (NLSs) that serve as targets for the importin The majority of nuclear proteins are
targeted to the nucleus by basic, generally lysine-rich nuclear
localization signals (NLSs) that serve as binding sites for an NLS
receptor termed importin Human immunodeficiency virus type 1 (HIV-1) encodes two essential
regulatory proteins that are both active in the cell nucleus (reviewed
in references 8 and 11). The Tat
protein is an unusual transcriptional transactivator that dramatically
enhances the processivity of transcription directed by the viral long
terminal repeat promoter element. Tat function involves a direct
interaction between Tat and an RNA target site, termed TAR, that is
mediated by an arginine-rich RNA binding motif (ARM) that also
functions as the Tat NLS (18, 40). The Rev protein, while
equally critical for HIV-1 replication, acts posttranscriptionally to
induce the sequence-specific nuclear export of late HIV-1 mRNA species.
This RNA export activity requires a direct interaction between Rev and
a specific RNA target sequence present in these RNAs, termed the RRE.
Rev binding to the RRE is, in turn, mediated by an ARM sequence that is
somewhat similar to the ARM present in Tat (Fig. 1), although the RNA sequence
specificities of these two motifs are distinct. The Rev ARM also shares
the ability of the Tat ARM to function as an effective NLS (3, 6,
17, 27).
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Arginine-Rich Domains Present in Human
Immunodeficiency Virus Type 1 Tat and Rev Function as Direct
Importin
-Dependent Nuclear Localization Signals
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
(Imp ) NLS receptor. Imp is in turn bound by importin 
(Imp ), which targets the resultant protein complex to the nucleus. Here,
we report that the arginine-rich NLS sequences present in the human
immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to
interact with Imp and instead bind directly to Imp . Using in
vitro nuclear import assays, we demonstrate that Imp is entirely
dispensable for Tat and Rev nuclear import. In contrast, Imp proved
both sufficient and necessary, in that other -like import factors,
such as transportin, were unable to support Tat or Rev nuclear import.
Using in vitro competition assays, it was demonstrated that the target
sites on Imp for Imp , Tat, and Rev binding either are identical
or at least overlap. The interaction of Tat and Rev with Imp is
also similar to Imp binding in that it is inhibited by RanGTP but
not RanGDP, a finding that may in part explain why the interaction of
the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these
studies define a novel class of arginine-rich NLS sequences that are
direct targets for Imp and that therefore function independently of
Imp .
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
(Imp ) or karyopherin (reviewed in
references 29 and 44). Imp in
turn interacts with a second import factor, termed importin (Imp
) or karyopherin 1, that mediates docking of the resultant
ternary complex to the cytoplasmic face of the nuclear pore complex
(NPC) via a direct interaction with specific nucleoporins (5, 16,
32, 38). The subsequent translocation of this heterotrimer
through the NPC remains poorly understood but is known to require
energy and may be mediated by additional Imp -nucleoporin
interactions (39, 45). Once the heterotrimer reaches the
nuclear face of the NPC, the GTP-bound form of the Ran GTPase directly
binds to Imp , resulting in the release of Imp and the NLS
protein into the nucleoplasm (15, 25, 33). Ran, which is
found in the GDP-bound form in the cytoplasm and in the GTP-bound form
in the nucleus, is therefore a major determinant of the directionality
of nuclear import and may also provide a source of energy (21, 31,
36, 45). Once the NLS protein is released, both Imp and Imp
are separately recycled back to the cytoplasm, where they can then
participate in additional rounds of nuclear import.
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FIG. 1.
Sequence of the Tat and Rev NLS. A potential alignment
of the 9-amino-acid Tat NLS with the somewhat larger Rev NLS is shown.
The Tat M1 mutant encodes glutamic acid residues in place of lysine 51 and arginine 55 while the Rev N40D mutation substitutes aspartic acid
for asparagine 40, as indicated. The Rev M6 mutant has been previously
described (27, 28) and bears an aspartic acid and a leucine
residue in place of arginines 41 to 44.
Because the NLS sequences present in Tat and Rev are arginine rich, and
therefore basic, it might be assumed that they would mediate nuclear
import via the same import pathway utilized by the prototypic basic
NLSs found in simian virus 40 (SV40) large T antigen and nucleoplasmin
(9, 23). However, several reports have shed doubt on this
hypothesis. Thus, Efthymiadis et al. (10) have reported that
the Tat NLS is able to mediate nuclear import in vitro in the absence
of both Imp and Imp , that nuclear import of a Tat NLS substrate
is not inhibited by an excess of an SV40 T antigen NLS peptide, and,
finally, that the Tat NLS fails to bind to either Imp or Imp .
These data were interpreted to suggest that the Tat NLS functioned via
an entirely novel nuclear import pathway. In the case of HIV-1 Rev,
Fankhauser et al. (12) have proposed that the nucleolar
protein B23 binds to the Rev basic domain and mediates its nuclear
import. In contrast, Henderson and Percipalle (20) have
reported that the Rev NLS can bind directly to Imp and that this
interaction is inhibited by added Imp , thus suggesting that Imp and Rev compete for overlapping binding sites on Imp . While these
authors also reported a Rev-Imp interaction, this was not dependent
on a functional Rev NLS and was therefore viewed as nonspecific.
However, because this latter article did not report any nuclear import
assays, it remained unclear whether Imp was indeed sufficient or
even necessary for Rev nuclear import.
While the reports described above might suggest that the Tat and Rev
NLSs are functionally distinct, the sequence similarity between the Tat
and Rev NLS-ARM sequences (Fig. 1) seemed more consistent with the
hypothesis that these NLSs utilize the same nuclear import pathway.
Here, we demonstrate that both the Tat and the Rev NLS directly
interact with Imp but not Imp in vitro and further demonstrate
that Imp is both necessary and sufficient for the nuclear import of
both Tat and Rev into isolated nuclei. These studies therefore
demonstrate the existence of a novel class of basic NLS sequences that
function independently of Imp .
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Bacterial expression plasmids.
Plasmids for the expression
in Escherichia coli of the recombinant glutathione
S-transferase (GST) fusion proteins GST-SV40 large T antigen
NLS (T NLS), GST-Imp , GST-Imp , GST-IBB (Imp binding domain
of Imp ), GST-M9, GST-Ran, GST-RanQ69L, GST-p10/NTF2, GST-Rev, and
GST-RevM6 have been described previously (13, 28, 42).
Plasmid pMBP-Trn, encoding the human transportin protein fused to
maltose binding protein (MBP), has also been described (13,
42). Plasmid pGST-RevN40D was made by site-directed mutagenesis, using pGST-Rev as a template, by the Quick change method (Stratagene) changing the codon at amino acid position 40 from AAT (asparagine) to
GAC (aspartic acid). Plasmid pGST-R5QR4 was constructed by annealing
two complementary oligonucleotides encoding the amino acid sequence
NH2-RRRRRQRRRR-COOH, bearing 5'-BamHI
and 3'-XhoI overhanging ends, and ligating into
BamHI- and XhoI-digested pGex5X-1. Plasmid
pGST-TatNLS, encoding the Tat amino acid sequence
49-RKKRRQRRRAHQ-60, was constructed similarly to pGST-R5QR4.
Plasmid pGST-TatM1 is similar to pGST-TatNLS, except that
oligonucleotides encoding the amino acid sequence
49-RKeRRQeRRAHQ-60 were used.
Recombinant protein expression and purification.
GST-Rev,
GST-RevM6, and GST-RevN40D proteins were purified from E. coli BL21 cells containing the relevant plasmids. Bacteria were
grown overnight to saturation and then diluted 1:10 in fresh media for
growth at 30°C for 3 h prior to induction with 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG) for 5 h.
The proteins were then purified on glutathione-Sepharose 4B beads
(Pharmacia Biotech, Inc.), dialyzed into storage buffer A (100 mM NaCl,
10 mM HEPES [pH 7.4], 1 mM dithiothreitol [DTT], 10% glycerol),
and frozen in aliquots at 
80°C. For RNA mobility shift assays, the GST-Rev protein was further purified over a Q-Sepharose Fast Flow column (Pharmacia Biotech, Inc.), eluted in 500 mM NaCl, dialyzed into
storage buffer B (50 mM NaCl, 10 mM KCl, 10 mM HEPES [pH 7.4], 10%
glycerol, 2 mM DTT), and frozen in single-use aliquots at 80°C. The
GST-IBB, GST-T NLS, GST-R5QR4, GST-M9, GST-TatNLS, and GST-TatM1 fusion
proteins were purified by the standard batch method recommended by the
glutathione-Sepharose bead supplier (Pharmacia Biotech, Inc.), dialyzed
into storage buffer A, and frozen at 80°C.
, Imp (Rch1), Ran, RanQ69L, and p10/NTF2
were expressed, purified, cleaved, and stored as described previously
(42). For protein binding experiments to the Tat NLS and Rev
protein, the recombinant Imp protein was further purified by
affinity chromatography on a column of GST-IBB protein covalently
linked to Affi-Gel active ester agarose (Affi-10; Bio-Rad Laboratories)
at a 5-mg/ml concentration.
For peptide competition experiments, the GST-T NLS, GST-IBB,
GST-TatNLS, and GST-Rev proteins were expressed in bacteria and purified by the standard batch procedure onto glutathione-Sepharose 4B
beads (Pharmacia Biotech, Inc.). The glutathione-Sepharose resin-bound
fusion proteins were quantitated by the Bradford assay reagent (Bio-Rad
Laboratories) and cleaved by the appropriate protease (factor Xa or
thrombin) at 25°C for 12 h. For final quantitation, 80%
efficiency of cleavage by proteases was assumed. The supernatants were
treated with 1,5 Dansyl-Glu-Gly-Arg chloromethyl ketone HCl (Calbiochem) to inactivate the protease and were then aliquoted and
frozen at 80°C. The integrity of all recombinant proteins used in
this study was confirmed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE).
Protein affinity chromatography.
Purified proteins (GST-Tat
or GST-Rev) were coupled at 4- to 8-mg/ml concentrations onto Affi-Gel
10 active ester agarose beads (Bio-Rad Laboratories) in coupling buffer
(500 mM NaCl, 10 mM HEPES [pH 7.4], 10% glycerol). Columns of
10-µl bed volume were constructed in 100-µl borosilicate pipettes
and equilibrated with 50 column volumes of AC buffer (50 mM NaCl, 10 mM
HEPES [pH 7.4], 0.1 mM DTT, 10% glycerol) injected with a 100-µl
Hamilton syringe. Approximately 2 µg of Imp or Imp was loaded
onto the columns in 25 µl of AC buffer, and another 25 µl of AC
buffer was then added and collected to constitute the flow-through
fraction. The columns were then washed with 3 column volumes of AC
buffer, and bound proteins were eluted with 50 µl of 800 mM
MgCl2. The entire flow-through and eluate (bound) fractions
were analyzed by SDS-12% PAGE (Ready gels; Bio-Rad Laboratories) and
visualized by Coomassie blue R-250 (Life Technologies, Inc.) staining.
RanGTP release experiments.
For the RanGTP and -GDP release
experiment, 5 µg of Imp was incubated with ~20 µg of
GST-TatNLS protein in a total volume of 50 µl of AC buffer. Next,
~50 µl of equilibrated glutathione-Sepharose 4B beads (Pharmacia
Biotech, Inc.) was added, and the reactions were spun at 250 × g for 1 min. A total of 100 µl of Ran buffer (20 mM
NaHPO4, 50 mM NaCl, 1 mM Mg acetate) was then added to the
beads, which were then incubated for 30 min at 25°C in the presence
of buffer alone or in buffer with RanGTP or RanGDP. The Q69L mutant of
Ran, which is unable to hydrolyze bound nucleotides (2), was
used for these release experiments. RanQ69L (5 µg) and 2 mM GTP or
GDP were premixed in Ran binding buffer prior to addition to the Ran
binding buffer-bead mixture. The reactions were then spun at 250 × g for 1 min, the supernatant was discarded, and 50 µl
of 1% SDS sample loading buffer was added. The reactions were then
spun at 500 × g for 1 min, and the supernatant was
analyzed by SDS-12% PAGE and visualized by Coomassie blue R-250 staining.
In vitro nuclear uptake assays.
In vitro nuclear uptake
assays were performed using digitonin-permeabilized HeLa cells, as
previously described (1, 42). Recombinant purified GST-Rev,
GST-Tat, and GST-T NLS fusion moieties were fluorescein labeled (FLOUS;
Boehringer Mannheim) and used at 2 µM final concentrations. The
import factor proteins Imp , Imp , MBP-transportin, Ran, and
p10/NTF2 were used at 2 µM final concentrations. For competition
assays, fluorescein-labeled proteins were used at a 500 nM final
concentration, and rabbit reticulocyte lysate (Promega) was used as
cytosol. Competing peptides were used at an ~60-fold molar excess
(~30 µM). Competition assays were performed in a total volume of 70 µl for 20 min at 18°C. Images were digitally captured with a Leica
DMRB fluorescence microscope and converted to 8-bit gray scale with
Adobe Photoshop 4.0 software.
Rev-RRE mobility shift assays.
The GST-Rev mobility shift
assay on the HIV-1 RRE was performed essentially as previously
described (28), with the exception that 0.5% Triton X-100
was added to the binding buffer and a different nonspecific RNA
competitor (4 µg of 16S rRNA; Boehringer Mannheim) was used. Thirty
nanograms of GST-Rev, 500 ng of Imp or transportin, and 1 µg of
RanQ69L were used per reaction. RanQ69L protein was preincubated with 1 mM GTP or GDP in the presence of 0.1 mM MgCl2 prior to
addition. All proteins were added simultaneously to the 32P-CTP-labeled RRE probe and incubated for 20 min on ice
before loading into a 6% Tris-glycine-5% glycerol (acrylamide/bis
ratio, 80:1) amino-gel. The gel was run at 180 V at 25°C and was
visualized by autoradiography after drying.
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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To examine whether either Imp or Imp is required for the
nuclear import of HIV-1 Tat or Rev, we performed in vitro nuclear uptake assays using suspended, digitonin-permeabilized HeLa cells, as
previously described (42). The permeabilization procedure used, which involves digitonin treatment followed by the isolation of
intact nuclei on a sucrose cushion (42), results in a
preparation of nuclei with only limited residual cytoplasm. In
comparison to cells that have been digitonin permeabilized on
coverslips, this procedure has the advantage that cytoplasmic import
factors are more effectively depleted but has the disadvantage that
lack of nuclear import gives a lack of any detectable signal, rather than a cytoplasmic halo (1, 35, 42). The import substrates used were FLUOS-labeled proteins consisting of GST fused to full-length wild-type or mutant (M6 or N40D) Rev, to wild-type or M1 mutant forms
of the Tat NLS (Tat amino acids 49 to 60), or to the SV40 large T
antigen NLS (T NLS). The T NLS is known to be dependent on both Imp and Imp for nuclear import (38).
Nuclear import reactions were performed in a buffer containing
recombinant human Ran and p10/NTF2, as well as a source of energy, as
previously described (42). As shown in Fig.
2, the GST-Rev, GST-TatNLS, and GST-T NLS
substrates all failed to effectively enter nuclei when incubated in
buffer alone, i.e., in the absence of both Imp and Imp (panels
A, G, and J). However, in the presence of recombinant Imp , both the
GST-Rev and the GST-TatNLS substrates displayed readily observable
nuclear import (panels B and K), although the GST-T NLS substrate was
still not detectably imported (panel H). Further addition of
recombinant Imp induced GST-T NLS import as expected (panel I) but
did not appreciably inhibit or enhance nuclear import of either the
GST-Rev or the GST-TatNLS substrate (panels C and L). The nuclear
import of GST-TatNLS and GST-Rev in the absence of added Imp was
specific in that mutations known to inactivate Rev NLS function (Rev M6
and Rev N40D) or Tat NLS function (TatM1) in vivo (17, 18,
27) also blocked Imp -dependent import in vitro (panels E, F,
and M). This import was also specific for Imp in that the related
nuclear import factor transportin (4, 13, 37) was not able
to mediate the nuclear import of these substrates (panel D). We
therefore conclude that Imp is able to mediate the in vitro nuclear
import of substrates containing either the Rev NLS or the Tat NLS in the absence of functionally detectable levels of Imp .
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20 µm.
The Rev and Tat NLS both directly bind Imp .
The
observation that Imp alone can mediate both Rev NLS and Tat NLS
function in vitro (Fig. 2) implies a direct interaction between these
NLSs and Imp . Initially, we examined whether the Rev NLS can bind
to either Imp or Imp in vitro, using an affinity chromatography
assay. A direct and specific interaction between Rev and Imp was
indeed observed (Fig. 3A, lanes 7 and 8, and Fig. 3B, lanes 1 and 2), which was inhibited by both the M6 NLS mutation (Fig. 3A, lanes 5 and 6) and the less severe N40D NLS mutation
(Fig. 3B, lanes 3 and 4). No interaction between Imp and Rev (Fig.
3A, lanes 3 and 4) under conditions where the SV40 T NLS bound Imp effectively (Fig. 3C, lanes 1 and 2) was detected. Finally, a
recombinant protein consisting of GST fused to a highly arginine-rich
sequence (R5QR4) failed to bind to either Imp
or Imp in vitro (Fig. 3C, lanes 5 to 8) and also failed to
import in vitro in the presence of Imp plus or minus Imp (data
not shown). Therefore, this simple basic sequence is not sufficient for
Imp binding even though its charge is comparable to that of the Rev
NLS (Fig. 1). We conclude that the interaction between Rev and Imp is both direct and specific.
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and
Imp . As may be observed, wild-type Tat, but not the Tat M1 mutant,
proved able to directly bind to Imp (Fig. 4, lanes 1 to 4) but not to Imp (lanes 5 and 6). Therefore, the Tat NLS shares the ability of the Rev NLS to directly interact with Imp in vitro.
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The Imp -Tat NLS interaction is inhibited by RanGTP.
As
described in the introduction, Imp and related import factors, such
as transportin, bind import substrates in the cytoplasm where RanGTP
levels are low and then release these substrates, including Imp ,
into the nucleoplasm when they encounter nuclear RanGTP (21, 29,
33, 44). If the interaction between the Tat NLS and Imp indeed leads to the productive nuclear import of Tat, then this
interaction should also be released by RanGTP but not by RanGDP. As
shown in Fig. 4B, RanGTP indeed proved to be an effective inhibitor of
the Imp -Tat NLS interaction while added RanGDP had no detectable
effect. Similar data showing inhibition of Rev NLS binding to Imp
were previously reported (20) and have been confirmed in
this laboratory (data not shown).
Tat, Rev, and Imp bind to overlapping sites on Imp .
The interaction between Imp and Imp is mediated by a short
sequence, termed the Imp binding or IBB domain, located near the
Imp amino terminus (14, 46). Importantly, the IBB domain can function as an effective Imp -dependent but Imp -independent NLS both in vitro and in vivo. Because the interaction between Imp and the Rev NLS or the Tat NLS is inhibited by RanGTP, a property that
is also shared by the Imp -IBB interaction (21), it
appeared possible that these three NLS signals might bind to the same,
or at least overlapping, regions on Imp . To examine this question,
we asked whether nuclear import of GST-Rev, GST-Tat NLS, or GST-IBB
would be specifically inhibited by an ~60-fold molar excess of a Rev
NLS, Tat NLS, or IBB peptide. Relevant controls included the GST-T NLS
substrate, import of which is Imp dependent and should therefore be
inhibited by the IBB peptide (27), and an SV40 T NLS
peptide, which should only inhibit GST-T NLS import. A final control is
the GST-M9 substrate, which is imported into the nucleus by the
distinct transportin import factor (4, 13, 37) and should
therefore be unaffected by all four peptides. These in vitro nuclear
uptake experiments were performed using isolated HeLa cell nuclei with
reticulocyte lysate as a source of native import factors.
(IBB) with Imp is mediated by identical, or at least
overlapping, Imp sequences and that these three protein-protein
interactions are therefore mutually incompatible. While the observed
inhibition of Tat and Rev nuclear import by added IBB peptide (Fig. 5)
might appear to contradict the earlier finding that added full-length Imp has no evident effect on Tat and Rev import (Fig. 2), it should
be recalled that the IBB peptide was added at an ~60-fold molar
excess over Imp , while the full-length Imp was added at an
equimolar level.
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Imp can inhibit the Rev-RRE interaction.
While Tat remains
in the cell nucleus after import, Rev continuously shuttles between the
nucleus and cytoplasm in the process of mediating HIV-1 late mRNA
export from the nucleus (8, 11, 30). It has, however,
remained unclear why the interaction of Rev with the RRE RNA, which is
mediated by an ARM sequence extensively overlapping with the Rev NLS
(3, 28), is efficient in the cell nucleus yet is released in
the cell cytoplasm. One possible explanation for this
compartmentalization is that Imp might inhibit the Rev-RRE
interaction by competing with the RRE for Rev binding, a process that
should only occur in the cytoplasm where RanGTP levels are low
(21, 25).
results in the inhibition of
this interaction, Rev-RRE binding can be rescued by the further
addition of RanGTP, but not of RanGDP (Fig. 6). No inhibition of the
Rev-RRE interaction was observed upon addition of an equivalent level
of the Imp -like import factor transportin. We therefore conclude
that Imp indeed has the potential to specifically inhibit the
Rev-RRE interaction in the cytoplasm, but not the nuclei, of HIV-1
infected cells.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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As described in more detail in the introduction, nuclear import of
lysine-rich NLS sequences, such as the SV40 T NLS, requires an indirect
interaction between the NLS and the Imp nuclear import factor that
is mediated by the Imp NLS receptor (29, 44). The role
of Imp in the process of nuclear import therefore appears to be
solely that of targeting the NLS to Imp . Several lines of evidence
have suggested that NLS sequences that directly interact with Imp ,
and that therefore would be Imp independent, might exist. First,
the short IBB motif present in Imp , which is necessary and
sufficient for Imp binding, has itself been shown to function as an
Imp -dependent, Imp -independent NLS when attached to a substrate
protein (14, 46). Second, the NLS found in the yeast
nucleocytoplasmic shuttle protein Nab2 has been shown to mediate
protein nuclear import via a direct interaction with Imp when
expressed in mammalian cells (42). Third, it is now apparent
that Imp is unusual, and possibly unique, in relying on the Imp adaptor protein to recognize target NLS sequences. In contrast, other
Imp -related nuclear import factors, such as transportin, are now
known to bind to their cognate NLS sequences directly (4, 13,
37).
In this article, we present evidence demonstrating that both the Tat
NLS and the Rev NLS in fact function as direct Imp -dependent NLSs.
Specifically, we have shown that the in vitro nuclear import of
substrates bearing the Tat and Rev NLS is dependent on Imp but
independent of Imp under conditions where SV40 T NLS import requires both added proteins (Fig. 2). We have further demonstrated a
highly specific, direct interaction between Imp , but not Imp ,
and both the Tat NLS and the Rev NLS under conditions where the SV40 T
NLS binds Imp but not Imp (Fig. 3 and 4A). The interaction
between Imp and the Tat NLS was found to be potently inhibited by
RanGTP, but not RanGDP, as predicted for a functionally relevant Imp
interaction (Fig. 4B) (21). Finally, we have demonstrated that the Tat NLS, the Rev NLS and the Imp IBB motif can all act as competitive inhibitors of all forms of Imp - but not
transportin-dependent nuclear import, thus strongly suggesting that
these proteins bind to an identical or overlapping surface on Imp (Fig. 5). Collectively, these data identify a novel class of Imp
-independent basic NLS sequences that are functionally distinct from
Imp -dependent NLSs, such as the monopartite SV40 T NLS and the
bipartite nucleoplasmin NLS (9, 23).
The data presented in this manuscript partly contradict the report of
Efthymiadis et al. (10), which noted that Tat NLS function
is both Imp and Imp independent. This earlier result may
reflect the use of the SV40 T NLS as a control for in vitro factor
dependence. If the permeabilized cells used by these workers retained
Imp activity but lacked functional Imp , then it would appear
that the Tat NLS differed from the SV40 T NLS in being factor
independent. However, our data both explain and reproduce (Fig. 5) this
group's finding that Tat NLS function is not inhibited by an excess of
an SV40 T NLS peptide. In addition, our data confirm the observation by
Henderson and Percipalle (20) that the Rev NLS can directly
bind Imp and now show, for the first time, that Rev NLS function is
indeed independent of Imp . Finally, we have not observed any
evidence in support of the hypothesis of Fankhauser et al.
(12) that Rev NLS function is mediated by the B23 protein.
While it remains formally possible that B23 could affect Rev nuclear
import, it is clearly not essential (Fig. 2).
At least six NLS sequences able to directly interact with Imp have
now been reported, namely, the Tat and Rev NLSs (Fig. 1), the Imp IBB, the Rex NLS, and the NLSs found in the T-cell protein tyrosine
phosphatase and in the yeast protein Nab2 (14, 35, 41, 42,
46). Inspection of these six sequences reveals that they are all
arginine rich. Thus, the ~40-amino-acid IBB motif conserved in all
six human Imp homologs contains nine conserved arginine residues,
four of which are sequentially arranged (14, 46), in a
manner reminiscent of the Tat and Rev NLSs (Fig. 1), while the
~18-amino-acid Rex NLS contains seven arginine residues
(35). Finally, the Nab2 NLS contains eight arginine residues, located over a 31-amino-acid core sequence, and mutation of
two of these has been shown to result in a loss of Nab2 NLS function
(42). In contrast, basic NLS sequences of the Imp
-dependent type are lysine rich and generally contain at least two,
and more often three or more, lysine residues (7, 9, 23,
29). While the number of examples of Imp -dependent, Imp
-independent NLSs remains too small for confident prediction, it
nevertheless seems possible that basic NLSs may be subdivided into at
least two types, a more common, lysine-rich, Imp -dependent class
and a less common, arginine-rich, Imp -independent class. The
recently reported (7) structure of a complex of Imp with
the SV40 T NLS provides a molecular explanation for the requirement for lysine residues in Imp -dependent NLSs, as these have been found to
make critical hydrophobic contacts with Imp that could not be
formed by arginine residues.
Immediately prior to submission of this article, Jäkel and
Görlich (22) reported that several ribosomal proteins,
including particularly rpL23a, contained NLSs that could function as
Imp -dependent, Imp -independent NLSs. Interestingly, the rpL23a NLS was mapped to a 32-amino-acid sequence that contained eight arginine residues. Nevertheless, the rpL23a NLS appears to be functionally distinct from the NLSs present in the Tat and Rev proteins. Specifically, the rpL23a NLS was reported to bind to a region
on Imp that is distinct from the Imp IBB binding site. In
contrast, we observed that the IBB effectively competed the in vitro
nuclear import of both the Tat NLS and the Rev NLS, and vice versa
(Fig. 5), thus strongly suggesting that the Tat and Rev NLS bind to a
site on Imp that overlaps the IBB site. Secondly, the rpL23a NLS
was reported to function as a target not only for Imp but also for
transportin and two other Imp -like factors, termed RanBP5 and
RanBP7. In contrast, we did not observe any nuclear import of a Rev NLS
substrate into isolated nuclei in the presence of recombinant
transportin (Fig. 2), even though we have previously shown that
transportin is fully able to mediate nuclear import of substrates
bearing the cognate M9 NLS under these assay conditions (13,
42). In addition, the finding that an IBB peptide can entirely
block nuclear import of both Rev and Tat NLS substrates in vitro in the
presence of an added cytoplasmic extract that can fully support M9 NLS
nuclear import (Fig. 5) demonstrates that Imp is necessary, as well as sufficient (Fig. 2), for Tat and Rev NLS function. The data of
Jäkel and Görlich (22) would therefore seem to
imply the existence of a third class of basic NLSs that differ from the Tat and Rev NLS, as well as from the IBB and Rex NLS (14, 35, 46), in terms of both their binding site on Imp and their ability to also use other nuclear import factors, such as transportin.
An unresolved question is why Tat and Rev would evolve arginine-rich,
Imp -independent NLSs rather than the more common Imp -dependent
NLS. Two possibilities suggest themselves. Firstly, both Tat and Rev
are short proteins (86 and 116 amino acids, respectively) that are
expressed by a virus containing a small genome (8, 11). Both
proteins are also RNA binding proteins that contain ARMs. It is
therefore possible that these proteins have simply evolved to also use
the preexisting ARMs as NLS sequences rather than acquire an
additional, lysine-rich NLS that would require an increase in both
protein size and complexity. On the other hand, it is also known that
the various forms of Imp (at least five distinct variants are
encoded in the human genome) are expressed at widely divergent levels
in different tissues and also display very different affinities for
distinct NLS sequences (26, 34, 43). Potential difficulties
in achieving efficient nuclear import in all of the various tissues
infected by HIV-1 in vivo could therefore be avoided by targeting Imp
directly, rather than relying on one or more forms of Imp as an intermediary.
While the reasons why Tat and Rev contain an unusual Imp
-independent NLS are presently unclear, this finding does have
potential implications for the cytoplasmic release of the RRE
containing RNAs that are exported from the nucleus by the Rev protein.
Specifically, as shown in Fig. 6, Imp can effectively inhibit the
Rev-RRE interaction, but only in the absence of RanGTP. Because RanGTP is found at high levels in the cell nucleus but only at low levels in
the cytoplasm (21, 25, 29), this finding provides a
potential mechanism to explain why the Rev-RRE interaction is efficient in the nucleus, where RanGTP would prevent the Rev-Imp interaction, but is released in the cytoplasm, where little RanGTP is found. This
may therefore represent another example of the known critical role of
Ran in determining the directionality of nucleocytoplasmic transport pathways.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Box 3025, Room 426, CARL Building, Research Drive, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-3369. Fax: (919) 681-8979. E-mail: Culle002{at}mc.duke.edu.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Nagoshi, E., Yoneda, Y.
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Bunn, C. F., Neidig, J. A., Freidinger, K. E., Stankiewicz, T. A., Weaver, B. S., McGrew, J., Allison, L. A.
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Kurisaki, A., Kose, S., Yoneda, Y., Heldin, C.-H., Moustakas, A.
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Vongsamphanh, R., Fortier, P.-K., Ramotar, D.
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Gujral, A., Burton, D. W., Terkeltaub, R., Deftos, L. J.
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Yoshida, K., Blobel, G.
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Sherman, M. P., de Noronha, C. M. C., Heusch, M. I., Greene, S., Greene, W. C.
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Lippai, M., Tirián, L., Boros, I., Mihály, J., Erdélyi, M., Belecz, I., Máthé, E., Pósfai, J., Nagy, A., Udvardy, A., Paraskeva, E., Görlich, D., Szabad, J.
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Fahrenkrog, B., Hübner, W., Mandinova, A., Panté, N., Keller, W., Aebi, U.
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Cullen, B. R.
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Lai, M.-C., Lin, R.-I., Huang, S.-Y., Tsai, C.-W., Tarn, W.-Y.
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Titov, A. A., Blobel, G.
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Hubner, S., Smith, H. M. S., Hu, W., Chan, C. K., Rihs, H.-P., Paschal, B. M., Raikhel, N. V., Jans, D. A.
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Takizawa, C. G., Weis, K., Morgan, D. O.
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Kehlenbach, R. H., Assheuer, R., Kehlenbach, A., Becker, J., Gerace, L.
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