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.

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.

Hayashi R ，Takeuchi N ，Ueda T 《-》

An importin alpha/beta-recognized bipartite nuclear localization signal mediates targeting of the human herpes simplex virus type 1 DNA polymerase catalytic subunit pUL30 to the nucleus.

Alvisi G ，Musiani D ，Jans DA ，Ripalti A ... -  《BIOCHEMISTRY》

Identification of nuclear import and export signals within Fli-1: roles of the nuclear import signals in Fli-1-dependent activation of megakaryocyte-specific promoters.

Hu W ，Philips AS ，Kwok JC ，Eisbacher M ，Chong BH ... -  《-》

A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin alpha/beta pathway.

Lischka P ，Sorg G ，Kann M ，Winkler M ，Stamminger T ... -  《-》

Interactions between HIV Rev and nuclear import and export factors: the Rev nuclear localisation signal mediates specific binding to human importin-beta.

The human immunodeficiency virus type 1 (HIV-1) Rev protein binds to unspliced HIV-1 pre-mRNA and exports it from the nucleus. Rev itself can "shuttle" between the nucleus and cytoplasm. This bi-directional transport is mediated by two specific Rev sequences: a nuclear localisation signal (NLS), which overlaps the RNA-binding domain, and a distinct nuclear export signal (NES). In this study we characterised new monoclonal antibodies that bind different epitopes of Rev, including the import and export sequences. In RNA bandshift assays, we observed that formation of a multimeric complex between Rev and its target RNA completely masks the Rev NLS, whereas the NES remains readily accessible. We then tested for signal-mediated interactions between Rev and different nuclear transport receptors, using mutations in the Rev NES or NLS to control for specificity. Extensive biochemical analyses did not reveal any direct NES-dependent interaction between Rev (free or RNA-bound) and the previously proposed export co-factors, human RIP/Rab and eIF-5A. By contrast, similar tests showed that Rev binds directly via its arginine-rich NLS to the human nuclear import receptor, importin-beta. This interaction was highly specific and was abolished by mutation in the Rev NLS. Importin-beta did not bind to the RNA-bound form of Rev, providing a mechanism to ensure that Rev is imported only following release of its RNA cargo. Unlike many NLS-containing proteins that bind stably to an importin-alpha/beta heterodimer, the binding of Rev to importin-beta was actually blocked by importin-alpha receptor. Our findings suggest that Rev and importin-alpha bind (via an arginine-rich sequence) to a similar region on importin-beta. In addition, we show that the complex between Rev and importin-beta can be dissociated by the nuclear Ran GTPase, but only when Ran is in the GTP-bound form. The series of interactions we describe provide a novel pathway for the import of Rev across the nuclear pore complex, and a mechanism for its release into the nucleoplasm.

Henderson BR ，Percipalle P 《JOURNAL OF MOLECULAR BIOLOGY》