title: Nuclear Respiratory Factor 2β (NRF-2β) recruits NRF-2α to the nucleus by binding to importin-α:β via an unusual monopartite-type nuclear localization signal.
abstract: Nuclear respiratory factor 2 (NRF-2) is a mammalian transcription factor composed of two distinct and unrelated proteins: NRF-2α, which binds to DNA through its Ets domain, and NRF-2β, which contains the transcription activation domain. The activity of NRF-2 in neurons is regulated by nuclear localization; however, the mechanism by which NRF-2 is imported into the nucleus remains unknown. By using in vitro nuclear import assays and immuno-cytofluorescence, we dissect the nuclear import pathways of NRF-2. We show that both NRF-2α and NRF-2β contain intrinsic nuclear localization signals (NLSs): the Ets domain within NRF-2α and the NLS within NRF-2β (amino acids 311/321: EEPPAKRQCIE) that is recognized by importin-α:β. When NRF-2α and NRF-2β form a complex, the nuclear import of NRF-2αβ becomes strictly dependent on the NLS within NRF-2β. Therefore, the nuclear import mechanism of NRF-2 is unique among Ets factors. The NRF-2β NLS contains only two lysine/arginine residues, unlike other known importin-α:β-dependent NLSs. Using ELISA-based binding assays, we show that it is bound by importin-α in almost the same manner and with similar affinity to that of the classical monopartite NLSs, such as c-myc and SV40 T-antigen NLSs. However, the part of the tryptophan array of importin-α that is essential for the recognition of classical monopartite NLSs by generating apolar pockets for the P3 and the P5 lysine/arginine side chains is not required for the recognition of the NRF-2β NLS. We conclude that the NRF-2β NLS is an unusual but is, nevertheless, a bona fide monopartite-type NLS.
authors.affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa, Chiba Prefecture 277-8562, Japan. rippei.hayashi@cancer.org.uk
authors.forename: Rippei
authors.initials: R
authors.lastname: Hayashi
authors.forename: Nono
authors.initials: N
authors.lastname: Takeuchi
authors.forename: Takuya
authors.initials: T
authors.lastname: Ueda
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D051268:GA-Binding Protein Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D028901:alpha Karyopherins / Q000737:chemistry / Q000378:metabolism*; D028961:beta Karyopherins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D051268:GA-Binding Protein Transcription Factor; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D028901:alpha Karyopherins; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins
keywords: BSA; DNA helicase Q1-interacting protein 1; Ets transcription factor; GFP; IBB; NLS; NPI-1; NRF-2; ORF; PBS; Qip1; RAG cohort 1; Rch1; TB; bovine serum albumin; green fluorescent protein; importin-β binding; in vitro nuclear import assay; native gel protein binding assay; nuclear import; nuclear localization signal; nuclear respiratory factor 2; nucleoprotein interactor 1; open reading frame; phosphate-buffered saline; protein binding via cysteine; transport buffer