(Received for publication, March 17, 1997)
From the Aryl hydrocarbon receptor nuclear translocator
(ARNT) is a component of the transcription factors, aryl hydrocarbon
receptor (AhR) and hypoxia-inducible factor 1, which transactivate
their target genes, such as CYP1A1 and erythropoietin, in
response to xenobiotic aromatic hydrocarbons and to low O2
concentration, respectively. Since ARNT was isolated as a factor
required for the nuclear translocation of AhR from the cytoplasm in
response to xenobiotics, the subcellular localization of ARNT has been of great interest. In this investigation, we analyzed the subcellular distribution of ARNT using transient expression of a fusion gene with
Aryl hydrocarbon receptor (AhR)1 (also
called TCDD receptor) is a ligand-dependent transcription
factor, which regulates the expression of various genes via binding to
its cognate binding sequence named XRE (xenobiotics responsive element)
(1-3). CYP1A1, one of the target genes of AhR, plays an
important role in the metabolism of procarcinogens, such as
benzo(a)pyrene in cigarette smoke, resulting in formation of
activated DNA binding derivatives (4). Among various classes of ligands
for AhR, the most potent is a well known environmental pollutant,
tetrachlorodibenzo-p-dioxin (TCDD). Using an animal model
system, the toxicity of TCDD has been found to cause wasting syndrome,
immunodeficiency, tumor promotion, and teratogenesis (5, 6).
The aryl hydrocarbon receptor nuclear translocator (ARNT) was
identified as a factor that rescues the aryl hydrocarbon hydroxylase activity in response to xenobiotics in Hepa-1 c4 mutant cells (7).
Since the ligand binding subunit of AhR is present in the cytoplasm of
Hepa-1 c4 mutant cells, this led to the notion that ARNT is required
for ligand-dependent nuclear translocation of AhR. Sequence
analysis showed that ARNT is a 90-kDa protein possessing the
basic-helix-loop-helix (bHLH) domain as well as the PAS domain, showing
similarity with two Drosophila proteins called Per (for
period) important for circadian rhythms and Sim (single-minded)
required for the formation of the central nervous system (7).
Subsequent cloning of the ligand-binding subunit of AhR also showed
structural similarities with ARNT in the bHLH and PAS domains (8, 9).
Various mutational analyses revealed that the formation of heteromeric
complex of ARNT and AhR mediated by bHLH and PAS domains is required
for it to have DNA binding activity (10-14). Recently, ARNT has also
been identified as a component of another transcription factor called
HIF-1 (hypoxia-inducible factor 1). HIF-1 consists of two subunits
called HIF-1 Since ARNT was first cloned as a factor required for the nuclear
translocation of AhR from the cytoplasm to the nucleus, the subcellular
localization of ARNT was believed to be cytoplasmic. In fact, most of
ARNT were recovered in the cytosolic fraction by cell fractionation.
However, recent immunohistochemical analysis has shown that ARNT is
localized predominantly in the nucleus, regardless of the presence or
absence of ligands (19, 20). It has also been reported that
overexpressed ARNT is present in both the cytoplasm and the nucleus in
the insect cell system (21). These observations led to the notion that
ARNT is localized mainly in the nucleus, but some fractions might also
be localized in the cytoplasm under certain circumstances. The
increasing importance of the physiological role of ARNT in the
regulation of gene transcription in response to various signals, such
as oxygen (22), prompted us to investigate its subcellular localization
in detail.
Active transport of protein from the cytoplasm to the nucleus requires
the presence of a short amino acid moiety named nuclear localization
signal (NLS) in any part of the protein (23). NLSs of various proteins
identified so far can be classified mainly into two classes: 1) a
single cluster of basic amino acids represented by the SV40 large T
antigen NLS, and 2) a bipartite type in which two sets of adjacent
basic amino acids are separated by a stretch of approximately 10 amino
acids (24). The NLS-dependent nuclear translocation process
depends on the cytosolic fractions and can be separated mainly into two
steps: energy-independent targeting to the nuclear pore and the
energy-dependent entrance to the nucleus. Recently, four
soluble factors have been purified and implicated in nuclear protein
import (24, 25): importin- In the present study, we investigated the subcellular localization of
ARNT using a transient expression of chimeric constructs of ARNT and
Cell lines used for the study were mouse
hepatoma Hepa-1 clone Hepa1c1c7, Hepa-1 c4 mutant which lacks ARNT
expression (generously provided by Dr. O. Hankinson, UCLA), and HeLa
cells. Cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum at 37 °C with a 5%
CO2 atmosphere.
The human ARNT cDNA was prepared
by PCR amplification of reverse-transcribed products of total RNA from
HepG2 cells using specific primers and Pfu DNA polymerase
(CLONTECH), and was inserted in the pGEM-7Zf(+)
vector (Promega). The sequence of the construct was confirmed by
sequencing using fluorescein-labeled SP6 and T7 primers, AutoRead
Sequencing kits, and A.L.F. II DNA sequencer (Pharmacia Biotech Inc.).
For subsequent cloning into the Various portions of ARNT cDNA were amplified by PCR using
To construct GST-ARNT-GFP fusion genes, the GST-GFP cassette vector was
prepared as follows. After PCR amplification of the GFP cDNA (a
gift of Dr. Roger Y. Tsien, University of California, San Diego) to
generate SmaI and EcoRI sites at its 5 Electroporation was carried out using 15 µg each of
The
GST-ARNT-GFP vectors described above were introduced into the
Escherichia coli strain BL21. A single colony was picked and
cultured in LB broth/Amp until A600 reached 1.2, then isopropyl-1-thio- Microinjection experiments were performed
essentially as described previously (43). After microinjection of
samples into the cytoplasm of HeLa cells, the cells were incubated at
37 °C for 30 min before fixation with 3.7% formaldehyde.
Localization of injected GST-ARNT-GFP fusion proteins was examined by
fluorescent microscopy.
Recombinant PTAC58 and PTAC97 were expressed in BL21 as
GST fusion protein as described previously (28). The fusion proteins were purified using glutathione-Sepharose affinity chromatography. Finally, recombinant proteins of PTAC58 and PTAC97 were obtained by
cleavage with thrombin to release the GST portion.
Preparation of total cytosol of
Ehrlich ascites tumor cells was conducted as described previously (40).
Digitonin-permeabilized MDBK cells were prepared based on the method of
Adam et al. (44) as described previously (45). The testing
solution (10 µl) consisted of GST-ARNT-(39-61)-GFP and transport
buffer (20 mM HEPES (pH 7.3), 110 mM potassium
acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 0.5 mM EGTA, 2 mM dithiothreitol, and
1 µg/ml each of aprotinin, leupeptin, and pepstatin). Transport assay
was performed in the presence or absence of cytosol with 1 mM ATP, 5 mM creatine phosphate, and 20 units/ml creatine phosphokinase at 37 °C for 30 min. For the
nuclear-binding assay, recombinant PTAC58 and PTAC97 proteins were
added and incubated at 4 °C for 30 min. After incubation, the cells
were fixed and the location of GST-ARNT-GFP fusion proteins was
examined.
To
determine the subcellular localization of human ARNT, we constructed
the fusion genes
To identify the region of ARNT required for nuclear
localization, various portions of cDNA for ARNT were synthesized
using PCR and ligated to the modified
To
confirm the capacity for nuclear localization, we next examined the
fate of recombinant proteins microinjected into the cytoplasm of HeLa
cells. The cDNA of GFP(S65T), possessing amino acid substitution
from Ser65 to Thr to give a stronger fluorescence intensity
(46), was inserted into the region downstream of GST gene to give a
fusion gene of GST-GFP. The fusion protein was obtained by expression in BL21 in the presence of
isopropyl-1-thio- To identify which amino
acid residue(s) are important for the accessory activity to give a full
NLS, mutational analysis in the region of 46-61 was performed. Among
these, we developed great interest in the Ser residue at 57, since
various transcription factors possess a Ser residue in their NLS, which
is often phosphorylated in the activated form (47). Substitution of the
Ser57 to Ala (S57A; Fig. 4A) or
Thr (S57T; Fig. 4B) was performed, and the mutated fragments
were analyzed by microinjection of GST-ARNT-GFP fusion proteins. As
shown in Figs. 3 and 4, nuclear localization of these mutants and the
wild type proteins were indistinguishable, indicating that
Ser57 does not participate in the NLS activity.
We next asked possible roles of basic amino acids Lys58 and
Arg61 by replacing them to Ala (K58A and R61A,
respectively). Notably, K58A mutant lost efficient nuclear
translocation activity in microinjection assay (Fig. 4C).
R61A mutant also drastically reduced the NLS activity; the fluorescence
intensity was almost equal between the cytoplasm and nucleus (Fig.
4D). The double mutant K58A/R61A completely lost its
translocation activity in the microinjection assay (Fig.
4E). These findings suggest that these two basic amino acid
residues are necessary for efficient nuclear translocation activity.
We are also interested in amino acid residues 48-54 of ARNT, since
this region is apparently rich in acidic amino acids (5 out of 7). To
evaluate the possible requirement of the negative charge for efficient
NLS activity, we have also introduced amino acid substitutions of
Asp48 for Asn, Asp52 for Asn, and
Glu54 for Gln (Fig. 5A). No
apparent effect of these mutations was observed, suggesting that the
negative charge of the cluster of acidic amino acids in the center of
ARNT-(39-61) is not important for efficient NLS activity. We also
changed five out of five negative residues to neutral residues and
found the same observation (data not shown). However, deletion of this
region abolished the NLS activity (Fig. 5B), indicating that
these amino acids were also required as spacers to give enough length
between the two separated clusters of basic amino acids.
We next investigated whether the NLS
of ARNT is recognized by the components of the nuclear pore targeting
complex, PTAC58/PTAC97. To evaluate the involvement of these factors in
the nuclear targeting process, we analyzed various recombinant fusion
proteins of GST-ARNT-GFP using an in vitro nuclear transport
assay (Fig. 6). We used GST-NLSc-GFP fusion protein as a
control substrate and observed clear nuclear accumulation or targeting
to the nuclear rim incubated with cytosol in the presence of ATP (Fig.
6B) or with purified PTAC58/PTAC97 (Fig. 6C),
respectively. When incubated with cell extracts of Ehrlich tumor in the
presence of ATP at 37 °C, GST-ARNT-(39-61)-GFP localized to the
nucleus, confirming that the prepared recombinant protein can be a good
substrate for this assay as well (Fig. 6E). In addition,
incubation of GST-ARNT-(39-61)-GFP with PTAC58/PTAC97 enabled
targeting of the recombinant protein to the nuclear rim (Fig.
6F), suggesting that the inserted fragment ARNT-(39-61) is
recognized by these factors as was the classical SV40-like NLS.
Since the two basic amino acids Lys58 and Arg61
are needed for efficient nuclear translocation as judged by
microinjection analysis (Fig. 4), the effect of double mutation of
these two amino acids to Ala on the nuclear transport activity in
vitro was investigated. When incubated with cell extract in the
presence of ATP, mutated GST-ARNT-GFP did not accumulate in the nucleus
at all (Fig. 6G). Furthermore, ARNT with double mutations
K58A/R61A drastically reduced the nuclear rim targeting activity (Fig.
6H).
We have shown that human ARNT is localized to the nucleus when
analyzed by transient expression of the chimeric constructs of ARNT
cDNA and bacterial Although it has been pointed out previously that the basic amino acid
sequence (Arg-Ala-Ile-Lys-Arg-Arg-Pro; amino acids 39-45) of human
ARNT, which is similar to the NLS of SV40 large T antigen, might be a
candidate for an NLS (20), our analysis presented here clearly showed
that the basic amino acids were necessary, but not sufficient alone for
the full activity needed for nuclear localization of ARNT. Most
importantly, we have shown the absolute requirement of the additional
amino acids spanning from 46 to 61 (Gly-Leu-AspPhe-Asp-Asp-Asp-Gly-Glu-Gly-Asn-Ser-Lys-Phe-Leu-Arg) for efficient nuclear localization. Both deletion and point mutation analyses of the C-terminal part of this region revealed that two basic
amino acids, Lys at 58 and Arg at 61 were important for NLS activity.
We also analyzed the possible involvement of the acidic amino acids
present in the central region (positions 48-54) of the NLS of ARNT.
Apparently, deletion of this region diminished its NLS activity; thus,
these amino acids were also found out to be necessary for the nuclear
localization activity. On the other hand, when their negative charges
were altered to neutral, the mutant did not show any changes in the
nuclear localization activity. These results strongly suggest that the
central region (48-54) of the minimal NLS of ARNT is required as a
spacer to give enough length between the two separated portions of the
clusters of basic amino acids as was present in the nucleoplasmin NLS. Although the role of negative charges in the central part of the ARNT
NLS is still unknown, they may enhance the exposure of NLS to the
surface of the whole protein. In this sense, the NLS of the human ARNT
resembles the consensus of bipartite NLS (48), but a clear difference
from the consensus did exist in the C-terminal portions, where two
basic amino acids required for the full NLS activity were separated by
a two amino acid insertion. Here, we propose that the ARNT NLS (39-61)
should be categorized to be a novel variant of the bipartite type. To
date, two protein sequences closely related to the human ARNT have been
reported; one is the mouse homologue of ARNT (49, 50), while the other
is a novel member of this family named ARNT2, which is expressed in
limited organs in both adult and developing embryo (51). As is shown in
Fig. 7, all the amino acids required for the NLS
activity of ARNT were conserved among these three proteins, confirming
the importance of these amino acids.
The novel variant bipartite type of ARNT NLS was also recognized by the
two components of nuclear targeting complex, PTAC58 and PTAC97.
Concomitant with the deficiency of nuclear localization activity of the
K57A/R60A mutant of ARNT NLS, the mutant did not target the nuclear rim
in the presence of PTAC58 and PTAC97, confirming the requirement of
these basic amino acids for the first step of nuclear localization: the
recognition of NLS by its receptor to bring it to the nuclear pore.
These observations also suggested that PTAC58 (importin- In summary, we have identified the NLS of human ARNT/HIF-1 We thank Dr. O. Hankinson for providing cell
line Hepa-1 c4 mutants and Dr. R. Y. Tsien for the cDNA of GFP.
Department of Biochemistry,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
-galactosidase and microinjection of recombinant proteins containing
various fragments of ARNT in the linker region of glutathione S-transferase/green fluorescent protein. We found a clear
nuclear localization of ARNT in the absence of exogenous ligands to
AhR, and identified the nuclear localization signal (NLS) of amino acid
residues 39-61. The characterized NLS consists of 23 amino acids, and
can be classified as a novel variant of the bipartite type on the basis
of having two separate regions responsible for efficient nuclear
translocation activity, but considerable deviation of the sequence from
the consensus of the classical bipartite type NLSs. Like the well
characterized NLS of the SV40 T-antigen, this variant bipartite type of
ARNT NLS was also mediated by the two components of nuclear pore
targeting complex, PTAC58 and PTAC97, to target to the nuclear rim in
an in vitro nuclear transport assay.
and HIF-1
(15); the former is a new member of the
bHLH/PAS protein family, while the latter was found to be identical to
ARNT (16). Physiologically, HIF-1 responds to a low O2
concentration and transactivates many genes, including erythropoietin
(17) and vascular endothelial growth factor (18).
(26-30), importin-
(31-35), the
GTPase Ran/TC4 (36, 37), and pp15 (38). Importin has also been called
karyopherin or nuclear pore-targeting complex (PTAC). The first step in
the selection of protein targeting the nuclear pore is thought to be
recognition of the NLS by the 58-kDa cellular protein importin-
,
which associates with the 97-kDa cellular factor importin
(39, 40).
This NLS recognition complex docks to the nuclear pore complex via
importin-
(41) and subsequently is translocated through the pore by
an energy-dependent, Ran-dependent mechanism
(37). Although the association of importin-
(PTAC58) and
importin-
(PTAC97) with SV40 T NLS has been investigated extensively, not much is known about other NLSs of nucleoproteins, including transcription factors (42).
-galactosidase (
-Gal), clarifying nuclear localization of the
ARNT protein. Subsequent analysis of various portions of ARNT using
-Gal fusions as well as fusion protein with GST-GFP gave the minimum
NLS consisting of amino acids 39-61 of ARNT. The identified region
differed from the classical type of NLS reported so far, which prompted
us to analyze interaction with PTAC58 and PTAC97.
Cell Cultures
-Gal expression vector, the
NcoI site at the initiation codon of ARNT was modified to
the BglII site using pBglII linker (Takara), and another BglII site was created in front of the stop codon by
PCR-mediated mutation. To construct the expression vectors of
-Gal
fusion proteins with various portions of ARNT, an artificial
BglII site was created in front of the stop codon of
-galactosidase gene of pSV
-Gal vector (Promega) by PCR-mediated
mutation. The BglII-BglII fragment of the ARNT
cDNA was ligated to the BglII site of the modified
-Gal control vector to generate
-Gal/ARNT-(1-789) vector.
-Gal/ARNT-(1-789) vector as a template and Pfu DNA
polymerase with specific sets of primers to generate artificial
BglII sites at both ends. Sequences of the primers used for
the preparation of fragments of ARNT were as follows: F1 (GTC TGG TGT
CAA AAA CAG ATC TGC ATG) and R2 (TTC AGA TCT
ACC TAG TTG TGG CCT CTG GAT) for ARNT-(1-485); F1 and R1 (TTC
AGA TCT TTT CAG TTC CTG ATC AGT GAG) for ARNT-(1-165); F1
and R7 (TTC AGA TCT TCT CTC TTT ATC CGC AGA GCT) for
ARNT-(1-88); F1 and R22 (TTC AGA TCT CCT CAA AAA TTT ACT
GTT CCC) for ARNT-(1-61); F2 (TAT AAG ATC TGC CAT TTG ATC
TTG GAG GCA) and R2 for ARNT-(166-485); F3 (TAT AAG ATC
TGC CCC ACA GCT AAT TTA CCC) and R3 (CCT GCC CGG TTA TTA TTA
AGA TCT TTC) for ARNT-(486-789); F6 (TAT AAG ATC
TGC CTT GCC AGG GAA AAT CAC) and R1 for ARNT-(89-165); F25 (TAT
AAG ATC TGC AGG GCT ATT AAG CGG CGA) and HR01 (TTC
AGA TCT CCT CCC TTC TCC ATC ATC ATC A) for ARNT-(39-55);
F25 and R22 for ARNT-(39-61); F25 and R28 (TTC AGA TCT CCT
CAA AAA TTT AGT GTT CCC) for ARNT-(39-61)/S57T; F25 and R29 (TTC
AGA TCT CCT CAA AAA TTT AGC GTT CCC) for
ARNT-(39-61)/S57A; F25 and R35 (TTC AGA TCT CGC CAA AAA
TTT ACT GTT CCC) for ARNT-(39-61)/R61A; F25 and R36 (TTC AGA
TCT CCT CAA AAA TGC ACT GTT CCC) for ARNT-(39-61)/K58A; F25 and
R37 (TTC AGA TCT CGC CAA AAA TGC ACT GTT CCC TTC TCC ATC ATC ATC) for ARNT-(39-61)/K58A/R61A; F29 (TAT AAG ATC TGC
AGG GCT ATT AAG CGG CGA CCA GGG CTG AAT TTT) and R32 (TTC AGA
TCT CCT CAA AAA TTT ACT GTT CCC TTG TCC ATT ATC ATC) for
ARNT-(39-61)/D48N/D52N/E54Q. After cleavage with BglII, the
fragments were ligated to the BglII site of the
-Gal
control vector to give in-frame fusion genes. To obtain the fragment of
ARNT-(39-45), two oligonucleotides, F7 (GAT CTC TAG GGC TAT TAA GCG
GCG ACC AA) and R8 (GAT CTT GGT CGC CGC TTA ATA GCC CTA GA), were
annealed and phosphorylated using T4 polynucleotide kinase. The
resultant fragments were ligated at the BglII site of the
-Gal control vector. To generate the fragment
ARNT-(1-88;
39-45), two fragments named A and B were synthesized by
PCR. The sequence of the primers used are as follows: F1 and AR50
(AAT CTA GAC CCT GGA CAA TGG CTC CTC C) for fragment A;
AR51 (GGT CTA GAT TTT GAT GAT GAT GGA GAA GGG) and R7 for
fragment B. These fragments were digested with XbaI and
ligated to generate ARNT-(1-88;
39-45). For the preparation of
ARNT-(39-61;
48-54), two oligonucleotides, F32 (GAT CTC TAG GGC TAT
TAA GCG GCG ACC AGG GCT GGG GAA CAG TAA ATT TTT GAG GA) and R39 (GAT
CTC CTC AAA AAT TTA CTG TTC CCC AGC CCT GGT CGC CGC TTA ATA GCC
CTA GA), were annealed and phosphorylated.
and 3
flanks, the resulting fragments were subcloned into the pGEM-7Zf(+)
vector. The vector was cleaved with SmaI and XhoI
and subcloned into pGEX-5X-2 vector (Pharmacia). For construction of
in-frame fusion proteins, the resultant vector was cleaved with
XmaI, treated with Klenow fragment (Takara) in the presence
of 0.1 mM dNTPs to blunt the ends, followed by re-ligation
and transformation to generate the GST-GFP vector. Various portions of
ARNT cDNA described above were inserted at the BamHI
site of the GST-GFP2 vector. The direction of inserts was determined by
sequencing. To construct GST-NLSc-GFP vector, the core sequence of NLS
of SV40 large T antigen generously provided by Dr. Tsuneoka (Kurume
University, Fukuoka, Japan), the coding sequence of which was 5
-AAG
CTT GCC ATG GGG TGG CCC ACT CCT CCA AAA AAG AGA AAG GTA GAA GAC CCC
GGG-3
, was ligated with GFP cDNA at the SmaI site and
subcloned into the pGEM-7Zf(+) vector. The
BamHI-EcoRI fragment of the resultant was ligated to the pGEX 2T (Pharmacia) vector to give the GST-NLSc-GFP vector.
-Gal fusion protein expression vectors and cells (3.5 × 106) in 400 µl of K-PBS buffer at 960 microfarads/450 V
with Gene Pulser (Bio-Rad). The electroporated cells were seeded onto a 10-cm plastic dish and incubated at 37 °C under an atmosphere with a
5% CO2 content for 48 h. In situ staining
of
-Gal was carried out as follows; the cells were washed twice with
PBS, followed by fixation with 0.2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.0), 1 mM
MgCl2 for 15 min; they were stained with 0.2%
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside in 10 mM sodium phosphate buffer (pH 7.0) containing 1 mM MgCl2, 150 mM NaCl, and 3.3 mM each of potassium ferrocyanide and ferricyanide at
37 °C overnight.
-D-galactopyranoside was added to
1 mM and incubated at 20 °C for 14 h with vigorous shaking. The cells were collected by centrifugation at 3,500 rpm for 15 min, washed with saline, and resuspended in a lysis buffer, 50 mM Tris-HCl buffer (pH 8.3) containing 500 mM
NaCl, 1 mM EDTA, 2 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride. These cells were lysed by
two rounds of freeze/thaw treatment, then subjected to sonication. The
resultant samples were centrifuged at 12,000 rpm at 4 °C for 30 min,
and the soluble fractions were collected and subjected to batch
purification procedures using glutathione-Sepharose 4B resin
(Pharmacia). The purified protein was dialyzed against buffer
containing 20 mM HEPES (pH 7.3), 100 mM
potassium acetate, and 2 mM dithiothreitol.
Transient Expression of Chimeric Constructs of -Gal and
Full-length ARNT in HeLa, Hepa-1, and Hepa-1 c4 Cells
-Gal and ARNT under the control of the SV40
enhancer/promoter. Since the molecular mass of the
-Gal is large
enough (120 kDa) to prevent passage through the nuclear pore by
diffusion, bacterial
-Gal has been widely used as a reporter gene
for the determination of subcellular localization of expressed protein.
The expression vector of
-Gal/ARNT-(1-789) was transfected to three
cell lines, including HeLa, Hepa-1, and ARNT-deficient Hepa-1 c4 mutant
cells, by means of electroporation. Representative profiles of
expressed fusion proteins visualized by in situ staining of
-Gal with 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside are shown in Fig. 1.
As expected, no staining of the nucleus was observed for the expressed
-Gal alone (Fig. 1A), while the fusion of
-Gal with
the NLS of SV40 large T antigen (
-Gal/SV40 NLS) gave strong nuclear
localization in transfected cells (Fig. 1B). When the
chimeric gene of
-Gal/ARNT-(1-789) was expressed, the fused product
was clearly localized in the nucleus of all three cell lines tested
(Fig. 1C), which agreed well with the results obtained by
immunohistochemical analysis (19, 20).
Fig. 1.
Subcellular localization of
-Gal/ARNT-(1-789) fusion protein in HeLa, Hepa-1, and Hepa-1 c4
mutant cells. An expression vector of
-Gal/ARNT-(1-789) fusion
gene was delivered into the indicated cells by means of
electroporation. After a 48-h incubation at 37 °C, the cells were
fixed and stained with 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside solution. The subcellular
localization of the fusion proteins were examined by microscopy.
A,
-Gal control vector; B,
-Gal/SV40 NLS, a
fusion of
-Gal with the NLS of SV40 large T antigen; C,
-Gal/ARNT-(1-789). The hatched boxes in the PAS region
represent PAS A and PAS B direct repeat. The dotted and
solid boxes represent bHLH and clusters of basic amino
acids, respectively.
[View Larger Version of this Image (26K GIF file)]
-Gal vector as described
above. The chimeric constructs were introduced into HeLa cells, and
their localization was examined (Fig. 2). Deletion of
the transactivation domain located in the C-terminal portion (Fig.
2B) and/or PAS domain (Fig. 2C) did not affect
the nuclear localization of
-Gal/ARNT fusion proteins. In contrast,
fusion proteins of either
-Gal/ARNT-(166-485) (Fig. 2D)
or
-Gal/ARNT-(486-789) (Fig. 2E) showed cytoplasmic localization, confirming the absence of NLS in these regions. On the
other hand, the fusion protein containing the bHLH region of the
ARNT-(1-165) showed strong nuclear staining, suggesting the presence
of NLS in this region (Fig. 2C). Since the NLS identified so
far contained a cluster(s) of basic amino acids, two candidates of NLS
of ARNT can be estimated, one of which is located between 39 and 45 (Arg-Ala-Ile-Lys-Arg-Arg-Pro), while the other is between 99 and 104 (Arg-Arg-Arg-Arg-Asn-Lys) in the bHLH domain. To identify which
segments are involved in the NLS activity of ARNT, we further divided
ARNT-(1-165) into two fragments, ARNT-(1-88) (Fig. 2F) and
ARNT-(89-165) (Fig. 2G). Fusion protein containing the
former fragment gave intense nuclear staining, while those containing the latter fragment did not. Finally, ARNT NLS was identified as a
region between the 39- and 61-amino acid residues (Figs. 2I
and 3A).
Fig. 2.
Identification of the region responsible for
the nuclear localization of ARNT. Various portions of ARNT were
synthesized using PCR, and the resulting fragments were fused to the
modified -Gal control vector. Subcellular localization of
transiently expressed fusion proteins was examined as described in the
legend of Fig. 1. A,
-Gal/ARNT-(1-789); B,
-Gal/ARNT-(1-485); C,
-Gal/ARNT-(1-165); D,
-Gal/ARNT-(166-485); E,
-Gal/ARNT-(486-789); F,
-Gal/ARNT-(1-88); G,
-Gal/ARNT-(89-165); H,
-Gal/ARNT-(1-61); I,
-Gal/ARNT-(39-61).
[View Larger Version of this Image (31K GIF file)]
Fig. 3.
Microinjection of recombinant GST-ARNT-GFP
proteins into HeLa cells. A, amino acid sequence of
ARNT-(39-61). Either the basic or acidic region is boxed.
The Ser residue, a candidate for the phosphorylation site, is
circled, and two basic amino acids in the C-terminal portion
are thickly underlined. B, microinjection of
recombinant proteins. The affinity-purified recombinant proteins were
microinjected into the cytoplasm of HeLa cells. After incubation at
37 °C for 30 min, the cells were fixed and the localization of
microinjected proteins was examined by fluorescent microscopy. a, GST-GFP; b, GST-NLSc-GFP; c,
GST-ARNT-(39-61)-GFP; d, GST-ARNT-(1-88;39-45)-GFP; e, GST-ARNT-(39-55)-GFP; f,
GST-ARNT-(39-45)-GFP.
[View Larger Version of this Image (83K GIF file)]
-D-galactopyranoside. When collected by
centrifugation, the bacteria showed a slightly greenish color
indicating the expression of GFP fusion proteins. The cells were
disrupted, and the cell lysates were subjected to affinity purification
using a glutathione-Sepharose resin. The bound protein was eluted by
addition of buffer containing glutathione. The fusion protein was
analyzed using 7.5% acrylamide gel and showed a major protein of
molecular mass 55 kDa (data not shown), which would be large enough to
prevent its diffusion into the nucleus. Actually, when microinjected
into the cytoplasm of HeLa cells, the GST-GFP protein was localized to
the cytoplasm even after incubation for 2 h (Fig. 3B,
a). Next, we constructed a plasmid by insertion of the SV40
NLSc fragment into the junction of the fusion gene (GST-NLSc-GFP), and
the gene product was prepared as above. Microinjected GST-NLSc-GFP
protein revealed the efficient nuclear import within 30 min of
incubation at 37 °C (Fig. 3B, b). Using this
system, various portions of the N-terminal region of ARNT were analyzed
(Fig. 3B, c-f). As was seen for the transient expression of
-Gal fusions (Fig. 2), only the GST-GFP fusion protein
that contains ARNT-(39-61) showed efficient nuclear localization (Fig.
3B, c), confirming that the fragment serves as an
NLS. As was expected from its similarity to the consensus sequence of SV40 large T-like NLS, deletion of the amino acid residues between 39 and 45 diminished its ability as an NLS, suggesting the absolute requirement of these basic amino acids for NLS activity (Fig. 3B, d). On the other hand, the single cluster of
basic amino acids was not sufficient for the full NLS activity, since
GST-ARNT-(39-55)-GFP (Fig. 3B, e) and
GST-ARNT-(39-45)-GFP (Fig. 3B, f) did not
translocate to the nucleus, implying the importance of the rest of the
amino acids between 46 and 61 for full NLS activity.
Fig. 4.
Effects of substitution of amino acids in the
C-terminal part of ARNT-(39-61) on the subcellular localization of
fusion proteins. The indicated mutants of ARNT-(39-61) were
analyzed by microinjection of GST-ARNT-GFP fusion proteins using HeLa
cells as described in the legend of Fig. 3. A, S57A;
B, S57T; C, K58A; D, R61A;
E, K58A/R61A.
[View Larger Version of this Image (30K GIF file)]
Fig. 5.
Analysis of the central region of
ARNT-(39-61). A, effect of alteration of negative charges
in the central part of ARNT-(39-61) on the subcellular localization of
fusion proteins. The central part of ARNT-(39-61) was mutated as
described under "Experimental Procedures." The resultant fragments
were analyzed by transient expression of -Gal/ARNT fusion proteins
(left), and microinjection of mutated ARNT-(39-61) as a
GST-ARNT-GFP fusion proteins (right). B, effects
of deletion of the central part of ARNT-(39-61) on the subcellular
localization of fusion proteins. Wild type ARNT-(39-61) as well as
ARNT-(39-61;
48-54) were analyzed by transient expression of
-Gal/ARNT fusion proteins.
[View Larger Version of this Image (39K GIF file)]
Fig. 6.
Analysis of ARNT-(39-61) using in
vitro nuclear transport assay. GST-NLSc-GFP
(A-C), GST-ARNT-(39-61)-GFP (D-F), and GST-ARNT-(39-61)K58A/R61A-GFP (G and H) were
incubated with buffer only (A and D), cytosol
with ATP (B, E, and G), or with PTAC58 and PTAC97 (C, F, and H) as described
under "Experimental Procedures."
[View Larger Version of this Image (71K GIF file)]
-galactosidase gene. Our observation confirmed the previous reports using immunohistochemical techniques as
shown by Pollenz et al. and Hord and Perdew (19, 20). Since the heterodimeric partner AhR presents in the cytoplasm in the absence
of ligands and translocates to the nucleus upon binding of
ligands in the ARNT deficient cell line, Hepa-1 c4 (20), these two
subunits may translocate independently to the nucleus, where they may
form a heterodimer to bind to the cognate DNA sequence. On the other
hand, Reisz-Porszasz et al. (50) reported that the mouse
ARNT deletion mutant named bHLHAB, which contains amino acids 70-474,
can transactivate the CAT reporter construct under the control of the
XRE-containing promoter. Apparently, the bHLHAB lacks the corresponding
region of the NLS identified for human ARNT in this study, thus the
bHLHAB would not translocate to the nucleus independently. Since they
have shown that the bHLHAB forms a heterodimer with AhR in the presence
of TCDD in vitro (50), it is likely that the formation of
heterodimer of bHLHAB and AhR occurs in the cytoplasm and then the
bHLHAB-AhR complex may utilize the NLS of AhR to translocate from the
cytoplasm to the nuclei. Wood et al. (22) have recently
shown that the bHLHAB is also capable of transactivating HIF-1 activity
in the Hepa-1 c4 mutant cell lines, suggesting that the
heterodimerization of bHLHAB with another partner of HIF-1
could
also occur in the cytoplasm. Taken together, ARNT/HIF-1
might
heterodimerize with either AhR or HIF-1
in both the cytoplasm and
the nucleus, depending on the subcellular distribution of ARNT/HIF-1
at the preactivated stage.
Fig. 7.
Comparison of determined NLS of human ARNT
with the corresponding region of mouse ARNT and ARNT2. Bars
indicate gaps to give maximum matching among these three
proteins.
[View Larger Version of this Image (17K GIF file)]
) can
recognize various types of NLSs, including a variant bipartite NLS of
ARNT, to target the nuclear pore. Of course, we can not exclude the
possibility that NLS of ARNT may be recognized by an unknown specific
receptor resulting in efficient targeting of the nuclear pore. Further
investigation will be required to clarify the contribution of PTAC58 to
the nuclear pore targeting of ARNT in the cells.
to
consist of 23 amino acid residues spanning amino acids 39-61, which
was shown to be a novel bipartite type of NLS. Our observations combined with others led to the notion that either AhR or HIF-1
might form a heterodimer with ARNT/HIF-1
in both the nucleus and
cytoplasm, depending on the subcellular localization of ARNT/HIF-1
in the preactivated state.
*
This work was supported in part by grants for advanced
research on cancer from the Ministry of Education, Science, Sports and
Culture of Japan and a research grant from the Ministry of Health and
Welfare of Japan for the 2nd Term Comprehensive 10-Year Strategy for
Cancer Control.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: Dept. of
Biochemistry, Saitama Cancer Center Research Institute, Ina-machi,
Kitaadachi-gun, Saitama 362, Japan. Tel.: 81-48-722-1111 (ext. 251);
Fax: 81-48-722-1739; E-mail: kawajiri{at}saitama-cc.go.jp.
1
The abbreviations used are: AhR, aryl
hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear
translocator; HIF-1, hypoxia-inducible factor 1; NLS, nuclear
localization signal; bHLH, basic-helix-loop-helix; PAS, PER-ARNT-SIM
homology region; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PTAC, nuclear pore-targeting complex; -Gal,
-galactosidase; GST,
glutathione S-transferase; GFP, green fluorescent protein; PCR, polymerase chain reaction.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.