期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 425, 期 18, 页码 3536-3548出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2013.07.007
关键词
nuclear import; Ets transcription factor; native gel protein binding assay; protein binding via cysteine; in vitro nuclear import assay
资金
- Japan Society Promotion of Science Fellowship for Japanese Junior Scientists
- Ministry of Education, Culture, Sports, Science and Technology of Japan
- Grants-in-Aid for Scientific Research [23247013, 25440004] Funding Source: KAKEN
Nuclear respiratory factor 2 (NRF-2) is a mammalian transcription factor composed of two distinct and unrelated proteins: NRF-2 alpha, which binds to DNA through its Ets domain, and NRF-2 beta, 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-2 alpha and NRF-2 beta contain intrinsic nuclear localization signals (NLSs): the Ets domain within NRF-2 alpha and the NLS within NRF-2 beta (amino acids 311/321: EEPPAKRQCIE) that is recognized by importin-alpha:beta. When NRF-2 alpha and NRF-2 beta form a complex, the nuclear import of NRF-2 alpha beta becomes strictly dependent on the NLS within NRF-2 beta. Therefore, the nuclear import mechanism of NRF-2 is unique among Ets factors. The NRF-2 beta NLS contains only two lysine/arginine residues, unlike other known importin-alpha:beta-dependent NLSs. Using ELISA-based binding assays, we show that it is bound by importin-a 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-alpha that is essential for the recognition of classical monopartite NLSs by generating apolar pockets for the P-3 and the P-5 lysine/arginine side chains is not required for the recognition of the NRF-2 beta NLS. We conclude that the NRF-2 beta NLS is an unusual but is, nevertheless, a bona fide monopartite-type NLS. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
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