Portal de Periódicos da CAPES

A Nuclear Localization Signal of Human Aryl Hydrocarbon Receptor Nuclear Translocator/Hypoxia-inducible Factor 1β Is a Novel Bipartite Type Recognized by the Two Components of Nuclear Pore-targeting Complex

1997; Elsevier BV; Volume: 272; Issue: 28 Linguagem: Inglês

10.1074/jbc.272.28.17640

1083-351X

Hidetaka Eguchi, Togo Ikuta, Taro Tachibana, Yoshihiro Yoneda, Kaname Kawajiri,

Toxic Organic Pollutants Impact

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 O2concentration, 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 β-galactosidase and microinjection of recombinant proteins containing various fragments of ARNT in the linker region of glutathioneS-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. 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 O2concentration, 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 β-galactosidase and microinjection of recombinant proteins containing various fragments of ARNT in the linker region of glutathioneS-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. Aryl hydrocarbon receptor (AhR) 1The 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; β-Gal, β-galactosidase; GST, glutathione S-transferase; GFP, green fluorescent protein; PCR, polymerase chain reaction. 1The 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; β-Gal, β-galactosidase; GST, glutathione S-transferase; GFP, green fluorescent protein; PCR, polymerase chain reaction. (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) (1Fujii-Kuriyama Y. Ema M. Sogawa K. Exp. Clin. Immunogenet. 1994; 11: 65-75PubMed Google Scholar, 2Poellinger L. Gottlicher M. Gustafsson J.-A. Trends Pharmacol. Sci. 1992; 13: 241-245Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 3Swanson H.I. Bradfield C.A. Pharmacogenetics. 1993; 3: 213-230Crossref PubMed Scopus (410) Google Scholar). 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 (4Kawajiri K. Hayashi S.-I. Ioannides C. Cytochrome P450: Metabolic and Toxicological Aspects. CRC Press, Boca Raton, FL1996: 77-97Google Scholar). 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 (5Landers J.P. Bunce N.J. Biochem. J. 1991; 276: 273-287Crossref PubMed Scopus (311) Google Scholar, 6Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2313) Google Scholar). 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 (7Hoffman E.C. Reyes H. Chu F.-F. Sander F. Conley L.H. Brooks B.A. Hankinson O. Science. 1991; 252: 954-958Crossref PubMed Scopus (829) Google Scholar). 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 (7Hoffman E.C. Reyes H. Chu F.-F. Sander F. Conley L.H. Brooks B.A. Hankinson O. Science. 1991; 252: 954-958Crossref PubMed Scopus (829) Google Scholar). Subsequent cloning of the ligand-binding subunit of AhR also showed structural similarities with ARNT in the bHLH and PAS domains (8Burbach K.M. Poland A. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8139-8185Crossref Scopus (711) Google Scholar, 9Ema M. Sogawa K. Watanabe N. Chujo Y. Matsusita N. Gotoh O. Funae Y. Fujii-Kuriyama Y. Biochem. Biophys. Res. Commun. 1992; 184: 246-253Crossref PubMed Scopus (349) Google Scholar). 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 (10Bacsi S.G. Hankinson O. J. Biol. Chem. 1996; 271: 8843-8850Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11Dong L. Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1996; 271: 7942-7948Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12Fukunaga B.N. Probst M.R. Reisz-Porszasz S. Hankinson O. J. Biol. Chem. 1995; 270: 29270-29278Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 13Lindebro M.C. Poellinger L. Whitelaw M.L. EMBO J. 1995; 14: 3528-3539Crossref PubMed Scopus (161) Google Scholar, 14Swanson H.I. Chan W.K. Bradfield C.A. J. Biol. Chem. 1995; 270: 26292-26302Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). 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-1α and HIF-1β (15Wang G.L. Semenza G.L. J. Biol. Chem. 1995; 270: 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (1678) Google Scholar); the former is a new member of the bHLH/PAS protein family, while the latter was found to be identical to ARNT (16Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4926) Google Scholar). Physiologically, HIF-1 responds to a low O2concentration and transactivates many genes, including erythropoietin (17Semenza G. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2137) Google Scholar) and vascular endothelial growth factor (18Forsythe J.A. Jiang B.-H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3135) Google Scholar). 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 (19Hord N.G. Perdew G.H. Mol. Pharmacol. 1994; 46: 618-626PubMed Google Scholar, 20Pollenz R.S. Sattler C.A. Poland A. Mol. Pharmacol. 1994; 45: 428-438PubMed Google Scholar). It has also been reported that overexpressed ARNT is present in both the cytoplasm and the nucleus in the insect cell system (21Chan W.K. Chu R. Jain S. Reddy J.K. Bradfield C.A. J. Biol. Chem. 1994; 269: 26464-26471Abstract Full Text PDF PubMed Google Scholar). 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 (22Wood S.W. Gleadle J.M. Pugh C.W. Hankinson O. Ratcliffe P.J. J. Biol. Chem. 1996; 271: 15117-15123Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), 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 (23Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1842) Google Scholar). 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 (24Görlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1060) Google Scholar). 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 (24Görlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1060) Google Scholar, 25Powers M.A. Forbes D.J. Cell. 1994; 79: 931-934Abstract Full Text PDF PubMed Scopus (115) Google Scholar): importin-α (26Adam S.A. Gerace L. Cell. 1991; 66: 837-847Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 27Görlich D. Prehn S. Laskey R.A. Hartmann E. Cell. 1994; 79: 767-778Abstract Full Text PDF PubMed Scopus (597) Google Scholar, 28Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (269) Google Scholar, 29Moroianu J. Blobel G. Radu A. Pro. Natl. Acad. Sci. U. S. A. 1995; 92: 2008-2011Crossref PubMed Scopus (249) Google Scholar, 30Weis K. Mattaj I.W. Lamond A.I. Science. 1995; 268: 1049-1053Crossref PubMed Scopus (306) Google Scholar), importin-β (31Adam E.J. Adam S.A. J. Cell Biol. 1994; 125: 547-555Crossref PubMed Scopus (256) Google Scholar, 32Chi N.C. Adam E.J.H. Adam S.A. J. Cell Biol. 1995; 130: 265-274Crossref PubMed Scopus (246) Google Scholar, 33Görlich D. Kostka S. Kraft R. Dingwall C. Laskey R.A. Hartmann E. Prehn S. Curr. Biol. 1995; 5: 383-392Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 34Imamoto N. Shimamoto T. Kose S. Takao T. Tachibana T. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. FEBS Lett. 1995; 368: 415-419Crossref PubMed Scopus (154) Google Scholar, 35Radu A. Blobel G. Moore M.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1769-1773Crossref PubMed Scopus (381) Google Scholar), the GTPase Ran/TC4 (36Melchior F. Paschal B. Evans J. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (472) Google Scholar, 37Moore M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (637) Google Scholar), and pp15 (38Moore M.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10212-10216Crossref PubMed Scopus (290) Google Scholar). 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-α, which associates with the 97-kDa cellular factor importin β (39Görlich D. Henklein P. Laskey R.A. Hartmann E. EMBO J. 1996; 15: 1810-1817Crossref PubMed Scopus (360) Google Scholar, 40Imamoto N. Tachibana T. Matsubae M. Yoneda Y. J. Biol. Chem. 1995; 270: 8559-8565Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This NLS recognition complex docks to the nuclear pore complex via importin-β (41Görlich D. Vogel F. Mills A.D. Hartmann E. Laskey R.A. Nature. 1995; 377: 246-248Crossref PubMed Scopus (405) Google Scholar) and subsequently is translocated through the pore by an energy-dependent, Ran-dependent mechanism (37Moore M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (637) Google Scholar). Although the association of importin-α (PTAC58) and importin-β (PTAC97) with SV40 T NLS has been investigated extensively, not much is known about other NLSs of nucleoproteins, including transcription factors (42Vandromme M. Gauther-Rouviére C. Lamb N. Fernandez A. Trends Biochem. Sci. 1996; 21: 59-64Abstract Full Text PDF PubMed Scopus (158) Google Scholar). In the present study, we investigated the subcellular localization of ARNT using a transient expression of chimeric constructs of ARNT and β-galactosidase (β-Gal), clarifying nuclear localization of the ARNT protein. Subsequent analysis of various portions of ARNT using β-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 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 β-Gal expression vector, theNcoI 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 β-Gal fusion proteins with various portions of ARNT, an artificialBglII site was created in front of the stop codon of β-galactosidase gene of pSVβ-Gal vector (Promega) by PCR-mediated mutation. The BglII-BglII fragment of the ARNT cDNA was ligated to the BglII site of the modified β-Gal control vector to generate β-Gal/ARNT-(1–789) vector. Various portions of ARNT cDNA were amplified by PCR using β-Gal/ARNT-(1–789) vector as a template and Pfu DNA polymerase with specific sets of primers to generate artificialBglII 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 TCTACC TAG TTG TGG CCT CTG GAT) for ARNT-(1–485); F1 and R1 (TTCAGA 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 TTAAGA 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 (TTCAGA 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 (TTCAGA 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 β-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 β-Gal control vector. To generate the fragment ARNT-(1–88;Δ39–45), 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;Δ39–45). For the preparation of ARNT-(39–61;Δ48–54), 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. 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′ 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 withXmaI, 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. TheBamHI-EcoRI fragment of the resultant was ligated to the pGEX 2T (Pharmacia) vector to give the GST-NLSc-GFP vector. Electroporation was carried out using 15 μg each of β-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 β-Gal was carried out as follows; the cells were washed twice with PBS, followed by fixation with 0.2% glutaraldehyde in 0.1m sodium phosphate buffer (pH 7.0), 1 mmMgCl2 for 15 min; they were stained with 0.2% 5-bromo-4-chloro-3-indolyl β-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. The GST-ARNT-GFP vectors described above were introduced into theEscherichia coli strain BL21. A single colony was picked and cultured in LB broth/Amp until A 600 reached 1.2, then isopropyl-1-thio-β-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 mmNaCl, 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 mmpotassium acetate, and 2 mm dithiothreitol. Microinjection experiments were performed essentially as described previously (43Yoneda Y. Imamoto-Sonobe N. Yamaizumi M. Uchida T. Exp. Cell. Res. 1987; 173: 586-595Crossref PubMed Scopus (174) Google Scholar). 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 (28Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (269) Google Scholar). 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 (40Imamoto N. Tachibana T. Matsubae M. Yoneda Y. J. Biol. Chem. 1995; 270: 8559-8565Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Digitonin-permeabilized MDBK cells were prepared based on the method of Adam et al. (44Adam S.A. Sterne-Marr R. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (763) Google Scholar) as described previously (45Okuno Y. Imamoto N. Yoneda Y. Exp. Cell. Res. 1993; 206: 134-142Crossref PubMed Scopus (66) Google Scholar). 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 β-Gal and ARNT under the control of the SV40 enhancer/promoter. Since the molecular mass of the β-Gal is large enough (120 kDa) to prevent passage through the nuclear pore by diffusion, bacterial β-Gal has been widely used as a reporter gene for the determination of subcellular localization of expressed protein. The expression vector of β-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 β-Gal with 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside are shown in Fig. 1. As expected, no staining of the nucleus was observed for the expressed β-Gal alone (Fig. 1 A), while the fusion of β-Gal with the NLS of SV40 large T antigen (β-Gal/SV40 NLS) gave strong nuclear localization in transfected cells (Fig. 1 B). When the chimeric gene of β-Gal/ARNT-(1–789) was expressed, the fused product was clearly localized in the nucleus of all three cell lines tested (Fig. 1 C), which agreed well with the results obtained by immunohistochemical analysis (19Hord N.G. Perdew G.H. Mol. Pharmacol. 1994; 46: 618-626PubMed Google Scholar, 20Pollenz R.S. Sattler C.A. Poland A. Mol. Pharmacol. 1994; 45: 428-438PubMed Google Scholar). 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 β-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.2 B) and/or PAS domain (Fig. 2 C) did not affect the nuclear localization of β-Gal/ARNT fusion proteins. In contrast, fusion proteins of either β-Gal/ARNT-(166–485) (Fig. 2 D) or β-Gal/ARNT-(486–789) (Fig. 2 E) 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. 2 C). 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. 2 F) and ARNT-(89–165) (Fig. 2 G). 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. 2 Iand 3 A).Figure 1Subcellular localization of β-Gal/ARNT-(1–789) fusion protein in HeLa, Hepa-1, and Hepa-1 c4 mutant cells. An expression vector of β-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 β-d-galactopyranoside solution. The subcellular localization of the fusion proteins were examined by microscopy.A, β-Gal control vector; B, β-Gal/SV40 NLS, a fusion of β-Gal with the NLS of SV40 large T antigen; C, β-Gal/ARNT-(1–789). The hatched boxes in the PAS region represent PAS A and PAS B direct repeat. The dotted andsolid boxes represent bHLH and clusters of basic amino acids, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Identification 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 β-Gal control vector. Subcellular localization of transiently expressed fusion proteins was examined as described in the legend of Fig. 1. A, β-Gal/ARNT-(1–789); B, β-Gal/ARNT-(1–485); C, β-Gal/ARNT-(1–165);D, β-Gal/ARNT-(166–485); E, β-Gal/ARNT-(486–789); F, β-Gal/ARNT-(1–88);G, β-Gal/ARNT-(89–165); H, β-Gal/ARNT-(1–61); I, β-Gal/ARNT-(39–61).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (46Cormack B.P. Valdivia R. Falkow S. Gene ( Amst. ). 1996; 173: 33-38Crossref PubMed Scopus (2474) Google Scholar), 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-β-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 mole