From the Nuclear Signaling Laboratory, Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, A.C.T. 2601, Australia
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ABSTRACT |
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The different classes of conventional
nuclear localization sequences (NLSs) resemble one another in that
NLS-dependent nuclear protein import is
energy-dependent and mediated by the cytosolic NLS-binding
importin/karyopherin subunits and monomeric GTP-binding protein
Ran/TC4. Based on analysis of the nuclear import kinetics mediated by
the NLS of the human immunodeficiency virus accessory protein Tat using
in vivo and in vitro nuclear transport assays and confocal laser scanning microscopy, we report a novel nuclear import pathway. We demonstrate that the Tat-NLS, not recognized by
importin 58/97 subunits as shown using an enzyme-linked immunosorbent assay-based binding assay, is sufficient to target the 476-kDa heterologous -galactosidase protein to the nucleus in
ATP-dependent but cytosolic factor-independent fashion.
Excess SV40 large tumor antigen (T-ag) NLS-containing peptide had no
significant effect on the nuclear import kinetics implying that the
Tat-NLS was able to confer nuclear accumulation through a pathway
distinct from conventional NLS-dependent pathways.
Nucleoplasmic accumulation of the Tat-NLS-
-galactosidase fusion
protein, in contrast to that of a T-ag-NLS-containing fusion protein,
also occurred in the absence of an intact nuclear envelope, implying
that the Tat-NLS conferred binding to nuclear components. This is in
stark contrast to known NLSs such as those of T-ag which confer nuclear
entry rather than retention. Significantly, the ability to accumulate in the nucleus in the absence of an intact nuclear envelope was blocked
in the absence of ATP, as well as by nonhydrolyzable ATP and GTP
analogs, demonstrating that ATP is required to effect release from a
complex with insoluble cytoplasmic components. Taken together, the
results demonstrate that, dependent on ATP for release from cytoplasmic
retention, the Tat-NLS is able to confer nuclear entry and binding to
nuclear components. These unique properties indicate that Tat
accumulates in the nucleus through a novel import pathway.
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INTRODUCTION |
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To enter the eukaryotic cell nucleus, proteins larger than 45 kDa
require targeting signals called nuclear localization sequences (NLSs)1 defined as the
sequences sufficient and necessary for nuclear localization of their
respective proteins (1, 2). NLSs appear to fall into several classes,
including those homologous to the NLS of the simian virus SV40 large
tumor-antigen (T-ag) consisting of a single stretch of basic residues
(1-3), those termed bipartite NLSs comprising two clusters of basic
amino acids separated by a spacer of 10-12 amino acids (1, 4) and
those resembling the NLS of the yeast homeodomain protein Mat2
(NKIPIKDLLNPQ13 (5)). All of these types of NLS are similar
in terms of the transport process and the cytosolic factors mediating
it (see Refs. 1, 2, and 6), whereby NLS-containing proteins are initially bound by a heterodimer consisting of proteins of about 60 and
95 kDa, variously named importin
/
(7), importin 58/97 (8), and
karyopherin
/
(9). The smaller importin/karyopherin subunit binds
the NLS specifically, whereas the larger subunit both enhances the
affinity of the complex for the NLS (6, 9-11) and mediates the docking
of the cargo-carrier complex to the nuclear pore complex (NPC). The
second, energy-dependent step involves transfer of the
cargo-carrier complex to the nucleoplasmic side and requires GTPase
activity on the part of the monomeric GTP-binding protein/GTPase
Ran/TC4 and other factors such as NTF2 (see Refs. 1, 2, and
12-14).
While the conventional NLSs mentioned above appear to be recognized by
importin/karyopherin and transported to the nucleus as outlined above,
recent studies have revealed two novel nuclear protein import pathways
which are mediated by quite distinct targeting signals and do not
appear to involve the importin 58/97 complex (15-17). Nuclear import
of the nuclear-cytoplasmic shuttling hnRNP protein A1 is mediated by an
importin-97-homolog transportin (karyopherin 2), which recognizes
the A1 "M9" NLS but does not interact with the more conventional
NLSs referred to above (15, 16). In contrast, nuclear import of the
shuttling hnRNP K protein through the NPC conferred by the "KNS"
NLS-sequence does not appear to require a soluble cytosolic receptor or
Ran (17). Conventional NLSs, as well as the M9 and KNS NLSs, do not
mediate nuclear accumulation by conferring binding to nuclear
components, but function exclusively as nuclear entry signals
(1, 6, 15).
We have been interested for some time in the nuclear import of viral proteins (6, 11), and in particular the human immunodeficiency virus type 1 (HIV-1) Tat protein, which is a potent activator of viral gene expression and replication (see Ref. 18). Tat accumulates predominantly in the nucleus and nucleolus (19-21) through possession of an amino-terminal stretch of basic amino acid residues purported to be the NLS (GRKKRRQRRRAP59, single-letter amino acid code; basic residues highlighted in bold type) (22, 23), which is highly conserved among HIV-I isolates. The Tat-NLS resembles similar highly basic amino-terminal sequences of the HIV-1 Rev (RQARRNRRRRWRERQRQ51 (24)) and the HTLV-1 (human T-cell leukemia virus) Rex (MPKTRRRPRRSQRKRPPTP119) proteins, both of which have been shown to constitute functional NLSs (24-27).
To gain insight into Tat targeting function as a possible paradigm of
this class of viral targeting signal, this study examines the nuclear
import kinetics of a -galactosidase fusion protein carrying Tat
amino acids 48-59 in vivo and in vitro at the
single cell level, comparing results to those for fusion proteins
carrying a classical NLS, that of T-ag (3). We find that the Tat-NLS, in contrast to the classical T-ag-NLS, confers nuclear accumulation through an import pathway which appears to require ATP but not cytosolic factors such as importin. Experiments using cells in which
the nuclear envelope was permeabilized with CHAPS indicate that Tat
fusion proteins can bind to both insoluble cytoplasmic and nuclear
factors and that ATP is required to effect release from cytoplasmic
retention and relocation to the nucleus. In contrast, the T-ag fusion
protein binds neither cytosolic nor nuclear factors under the same
conditions. We conclude that the Tat-NLS is able to target
-galactosidase to the nucleus through a novel import pathway.
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MATERIALS AND METHODS |
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Chemicals and Reagents--
The detergent CHAPS was from
Boehringer Mannheim and AMP-PNP from Calbiochem. The bacterial strains
for karyopherin (Kap60) and
(Kap95) fusion protein expression
(9) were provided by Michael Rexach. Other reagents were from the
sources previously described (6, 11, 28-31).
Cell Culture-- Cells of the HTC rat hepatoma tissue culture (a derivative of Morris hepatoma 7288C) line were cultured as described previously (28, 29).
-Galactosidase Fusion Proteins--
The plasmid expressing
the Tat-NLS-
-galactosidase fusion protein Tat-NLS-
-Gal was
derived by oligonucleotide insertion into the SmaI
restriction endonuclease site of the plasmid vector pPR2 (29). The
resultant fusion protein comprises Tat amino acids 48-59
(GRKKRRQRRRAP59) fused amino-terminal to the
Escherichia coli
-galactosidase enzyme sequence (amino
acids 9-1023). The T-ag
-galactosidase fusion protein
(T-ag-CcN-
-Gal) used in the comparative studies contains T-ag amino
acids 111-135, including the CcN motif (comprising protein kinase
CK2 and cdk phosphorylation sites and the
NLS) fused amino-terminal to
-galactosidase amino acids
9-1023 (28, 29). 1 mM isopropyl-
-thiogalactoside was
used to induce expression of fusion proteins in E. coli.
They were purified by affinity chromatography and labeled using the
sulfhydryl labeling reagent 5-iodoacetamidofluorescein (Molecular
Probes) as described (29).
Nuclear Import Kinetics-- Nuclear import kinetics at the single cell level were measured using either microinjected (in vivo) or mechanically perforated (in vitro) HTC cells in conjunction with confocal laser scanning microscopy (CLSM) (6, 11, 28-31). In the case of microinjection, HTC cells were fused with polyethylene glycol about 1 h prior to microinjection to produce polykaryons (6, 11, 28, 29, 31). Reticulocyte lysate (Promega) was used as the source of cytosol for the in vitro assay (6, 28, 30, 31). Image analysis of CLSM files using the NIH Image public domain software and curve-fitting were performed as described (6, 11, 30, 31).
In in vitro experiments where the ATP dependence of transport was tested, apyrase pretreatment was used to hydrolyze endogenous ATP in both cytosol (10 min at room temperature with 800 units/ml) and perforated cells (15 min at 37 °C with 0.2 unit/ml) (6, 30, 32), and transport assays were performed in the absence of the ATP regenerating system (28, 30) which was otherwise used. Where the dependence on the GTP-binding protein Ran was tested, cytosolic extract was treated with 860 µM GTPELISA-based Binding Assay--
An ELISA-based binding assay (6,
11, 31) was used to examine the binding affinity between importin
subunits (mouse importin 58 and 97 glutathione S-transferase
(GST)- fusion proteins, expressed as described (8, 11)) and Tat or T-ag
fusion proteins. This involved coating 96-well microtiter plates with
-galactosidase fusion proteins, hybridization with increasing
concentrations of importin subunits, and detection of bound
importin-GST using goat anti-GST primary, and alkaline
phosphatase-coupled rabbit anti-goat secondary antibodies, and the
substrate p-nitrophenyl phosphate (6, 11). Absorbance
measurements were performed over 90 min using a plate reader (Molecular
Devices), and values were corrected by subtracting absorbance both at 0 min and in wells incubated without importin (6, 11, 31). To quantitate importin binding specifically to the NLSs, quantitation was performed in identical fashion for
-galactosidase itself, and the values were
subtracted from those for the respective fusion proteins (6, 11, 31).
Fusion proteins were also subjected to a parallel
-galactosidase
ELISA (see Refs. 6, 11, and 31) to correct for any differences in
coating efficiencies and enable a true estimate of bound importin (6,
11). Measurements for the NLS binding affinity of the karyopherin
subunits were performed in identical fashion to those for importin
58/97 using GST-fusion proteins expressed in E. coli
(9).
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RESULTS AND DISCUSSION |
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The NLS of HIV-1 Tat Is Capable of Targeting a Heterologous Protein
to the Nucleus--
To examine the ability of the HIV-1 Tat basic
region to target a large (476-kDa) heterologous protein
(-galactosidase from E. coli) to the nucleus, a plasmid
was derived expressing fusion protein Tat-NLS-
-Gal containing Tat
amino acids 48-59 (see "Materials and Methods") fused
amino-terminal to
-galactosidase amino acids 9-1023. Its nuclear
import kinetics were measured in vivo and in
vitro using microinjected cells of the HTC line (6, 11, 28, 29,
31) and mechanically perforated HTC cells (6, 28, 30, 31),
respectively, and compared with those for a fusion protein
(T-ag-CcN-
-Gal) carrying the T-ag NLS and
-galactosidase itself.
The Tat-NLS targeted
-galactosidase to the nucleus in both assay
systems (Figs. 1 and
2), Tat-NLS-
-Gal accumulating maximally to levels about 2-fold those in the cytoplasm (Figs. 1B and 2B; Table
I). The extent of maximal accumulation of
Tat-NLS-
-Gal was markedly lower than that of T-ag-CcN-
-Gal which
attained levels over 5-fold those in the cytoplasm (Table I). The
transport rate of Tat-NLS-
-Gal in vivo was markedly
higher (rate constant (k) of 0.3) than that of
T-ag-CcN-
-Gal (k = 0.125) (see Table I). As observed
previously (28, 29),
-galactosidase was completely excluded from the
nucleus both in vivo and in vitro (Fn/cmax < 0.65, Figs. 1B and
2B; Table I). Although Tat nucleolar localization has been
reported using transfection systems (19-21), we did not observe
anything other than nucleoplasmic accumulation (Figs. 1 and 2 and see
below). That the lack of nucleolar accumulation is unlikely to be an
artifact of the systems used is indicated by our previous studies
examining nucleolar import of other proteins in vitro
(30),2 and we conclude that
Tat amino acids 48-59 do not confer nucleolar localization.
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Independence of Nuclear Uptake Conferred by the Tat-NLS on
Cytosolic Factors--
Conventional NLS-mediated nuclear protein
import in vitro is dependent on energy (6, 28, 30, 32) and
the addition of exogenous cytosol (6-10, 28, 30, 32). The latter
supplies the importin 58/97 NLS-binding/NPC-docking dimer (7-9) as
well as the GTPase Ran (12, 13) and interacting proteins (see Refs. 1,
2, and 14), which are essential for nuclear accumulation. Analogously,
M9-mediated nuclear import of hnRNP A1 requires the cytosolic
NLS-binding transportin protein and Ran (15, 16, 34). Nuclear import of
Tat-NLS--Gal was found to be dependent on ATP but not on exogenous
cytosol, in contrast to that of T-ag-CcN-
-Gal which required both
ATP and cytosol (Fig. 2; Table I). Interestingly, accumulation of
Tat-NLS-
-Gal in the presence of ATP but without cytosol was 50%
increased compared with that in the presence of cytosol, implying that
the latter inhibited transport. The nonhydrolyzable GTP analog GTP
S
was able to inhibit nuclear accumulation of both Tat-NLS-
-Gal and
T-ag-CcN-
-Gal in the presence of the ATP regenerating system; the
nonhydrolyzable ATP analog AMP-PNP similarly inhibited nuclear
transport (Table I). Nuclear accumulation of Tat fusion proteins thus
appears to be an active process. GTP/GDP could not substitute for ATP
to permit nuclear accumulation (see Table I), which, together with its
cytosolic independence, implies that a role for Ran in Tat-NLS-mediated
nuclear import is unlikely (see Refs. 13, 35, and 36). That other
GTP-binding proteins appear to play a role in nuclear protein import
(see Refs. 37 and 38) may constitute the basis of the inhibition of
Tat-NLS-mediated nuclear import by GTP
S.
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Nuclear Accumulation of Tat-NLS--Gal in the Absence of an Intact
Nuclear Envelope--
Detergents such as CHAPS can be used to
perforate the nuclear envelope to enable molecules to diffuse freely
between cytoplasm and nucleoplasm; nuclear accumulation under these
conditions occurs through binding to nuclear components (6). As
observed previously (6), T-ag-CcN-
-Gal did not accumulate in the
nucleus under these conditions (Fig. 4,
top right panel; Table I), instead showing equilibration
between nuclear and cytoplasmic compartments in the absence of a
barrier to diffusion. In contrast, Tat-NLS-
-Gal accumulated quite
well in the absence of cytosol, but only in the presence of ATP (Fig.
4, left panels; Table I). Surprisingly, despite the absence
of a barrier to diffusion, Tat-NLS-
-Gal exhibited quite marked
nuclear exclusion due to cytoplasmic association in the absence of ATP
(Fig. 4, bottom right panel; Table I). GTP/GDP could not
substitute for ATP in terms of facilitating nuclear accumulation (Table
I), while both GTP and ATP analogs inhibited accumulation in the
presence of the ATP regenerating system. The results indicate that, in
contrast to the conventional T-ag-NLS, which, although not preventing
nuclear entry in the absence of an intact nuclear envelope, does not
confer nuclear accumulation (see also Ref. 6), the Tat-NLS is able to
confer nuclear accumulation in the absence of an intact nuclear
envelope. In the absence of ATP hydrolysis, Tat-NLS-
-Gal appears to
exhibit high affinity for an insoluble cytoplasmic factor, while in its presence, it can accumulate in the nucleus through binding to nucleoplasmic components. Consistent with these conclusions,
interaction of the complete Tat molecule with either cytoplasmic or
nuclear components, varying according to the phase of HIV-1 infection, has been reported (38).
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Lack of Recognition of the Tat-NLS by the Conventional NLS-binding
Importin 58/97 Dimer--
To test directly whether importin subunits
could recognize the Tat-NLS, we used a previously established, specific
ELISA-based binding assay (6, 11, see "Materials and Methods").
Tat-NLS--Gal and T-ag-CcN-
-Gal fusion proteins were coated onto
microtiter plates, incubated with increasing amounts of importin
58-GST, importin 97-GST, or importin 58/97-GST complex, and binding was then quantitated using antibodies specific to GST and an alkaline phosphatase-labeled secondary antibody as previously described (11).
Comparable with previous measurements (6, 11, 31) the apparent
dissociation constant (KD) of T-ag-CcN-
-Gal for
importin 58 and 58/97 was 45 and 9.6 nM, respectively. In contrast, Tat-NLS-
-Gal exhibited no detectable binding of either 58 or 58/97 above that of
-galactosidase alone. No binding by importin
97-GST to either T-ag-CcN-
-Gal or Tat-NLS-
-Gal could be detected.
Similar results were obtained for the karyopherin subunits (9). The
lack of binding of Tat-NLS-
-Gal by importin/karyopherin subunits was
thus consistent with our in vitro transport results indicating that nuclear accumulation of the Tat fusion protein does not
require cytosolic factors.
Mechanism of Tat-NLS-conferred Nuclear Accumulation--
The
observation that Tat-NLS--Gal may have high affinity for an
insoluble cytoplasmic factor in the absence of ATP inspired us to test
whether cytoplasmic retention could be overcome by increasing the
relative concentration of Tat-NLS-
-Gal. Assays were accordingly
performed using up to 18-fold higher concentrations of Tat-NLS-
-Gal
(where the amount of labeled protein was kept constant, and final
Tat-NLS-
-Gal concentration was adjusted through the addition of
unlabeled protein) than the standard concentration (4 × 10
7 M) used in the in vitro assay.
Measurements in the presence of ATP showed a small (~25%) increase
in maximal accumulation (Fn/cmax of 3.8) at
1.6 × 10
6 compared with at 4 × 10
7 M (Fn/cmax of
2.9), but a significant reduction at higher concentrations (Fn/cmax of 1.6 at 7.2 × 10
6
M). This implied that rather than a cytoplasmic retention
factor, some other transport component is limiting, e.g. the
number of sites for Tat-NLS binding within the nucleus may be
titratable (see also below).
Novel Nuclear Import Pathway Conferred by the Tat-NLS-- The results above indicate that the Tat-NLS is capable of targeting a large heterologous protein to the nucleus through a pathway which is dependent on ATP hydrolysis but independent of the transport components mediating conventional NLS-dependent nuclear accumulation including importin and probably Ran. In contrast to conventional NLSs such as that of T-ag (see Ref. 6), the Tat-NLS appears to mediate binding to cytoplasmic components (see Ref. 38) in the absence of ATP, as well as conferring passage through the nuclear envelope, and the ability to bind to nuclear components (see Ref. 38) in the presence of ATP. It seems reasonable to propose that the Tat-NLS is recognized by a carrier/receptor protein which mediates nuclear entry and binding to nuclear components, as well as binding to insoluble cytoplasmic structures in the absence of ATP.
At the sequence level, the Tat-NLS is more closely related, especially in terms of the preponderance of positive charge, to the more conventional importin/karyopherin ![]() |
ACKNOWLEDGEMENTS |
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We thank Michael Rexach for providing the bacterial strains for karyopherin subunit expression, Imre Pavo and Gabor Toth for peptides P101 and P101T, and Patricia Jans for skilled technical assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: c/
Nuclear Signaling
Laboratory, Division for Biochemistry and Molecular Biology, John
Curtin School of Medical Research, Australian National University, P.O.
Box 334, Canberra City, A.C.T. 2601, Australia. Tel.: 00616-2494188; Fax: 00616-2490415; E-mail: daj224{at}leonard.anu.edu.au.
1
The abbreviations used are: NLS, nuclear
localization sequence; T-ag, simian virus SV40 large tumor-antigen;
NPC, nuclear pore complex; Tat, human immunodeficiency virus type 1 Tat
protein; KD, apparent dissociation constant; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; GST,
glutathione S-transferase; CLSM, confocal laser scanning microscopy; HTC, hepatoma tissue culture; ELISA, enzyme-linked immunosorbent assay; AMP-PNP, adenylyl imidodiphosphate; GTPS, guanosine 5
-3-O-(thio)triphosphate; hnRNP, heterogeneous
ribonucleoprotein.
2 A. Efthymiadis, L. J. Briggs, and D. A. Jans, unpublished results.
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REFERENCES |
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