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

doi:10.1074/jbc.272.28.17640

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*

From the ^‡Department of Biochemistry, Saitama Cancer Center Research Institute, 818 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362 and the ^§Department of Anatomy and Cell Biology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan

Abstract

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.

Footnotes

↵* 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; β-Gal, β-galactosidase; GST, glutathione S-transferase; GFP, green fluorescent protein; PCR, polymerase chain reaction.
- Received March 17, 1997.