Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-α
- 6 December 2017
- journal article
- research article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 474 (24), 4091-4104
- https://doi.org/10.1042/bcj20170654
Abstract
The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP–NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus. Nuclear translocation was also investigated using HeLa cells, which showed that the putative NIT-2 nuclear localization sequence (NLS; 915TISSKRQRRHSKS927) was recognized by importin-α and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-α (NcImpα) and the NIT-2 NLS was quantified with calorimetric assays, leading to the observation that the peptide bound to two sites with different affinities, which is typical of a monopartite NLS sequence. The crystal structure of the NcImpα/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpα with the major NLS-binding site playing a primary role. This result contrasts other recent studies that suggested a major role for the minor NLS-binding site in importin-α from the α2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements.This publication has 54 references indexed in Scilit:
- A Genome-wide Screen for Neurospora crassa Transcription Factors Regulating Glycogen MetabolismMolecular & Cellular Proteomics, 2011
- Nuclear transporters in a multinucleated organism: functional and localization analyses in Aspergillus nidulansMolecular Biology of the Cell, 2011
- Importin-α Protein Binding to a Nuclear Localization Signal of Carbohydrate Response Element-Binding Protein (ChREBP)Online Journal of Public Health Informatics, 2011
- A Minimal Nuclear Localization Signal (NLS) in Human Phospholipid Scramblase 4 That Binds Only the Minor NLS-binding Site of Importin α1Online Journal of Public Health Informatics, 2011
- Molecular Basis for the Recognition of Phosphorylated STAT1 by Importin α5Journal of Molecular Biology, 2010
- PHENIX: a comprehensive Python-based system for macromolecular structure solutionActa crystallographica. Section D, Structural biology, 2010
- MolProbity: all-atom structure validation for macromolecular crystallographyActa crystallographica. Section D, Structural biology, 2009
- Six Classes of Nuclear Localization Signals Specific to Different Binding Grooves of Importin αOnline Journal of Public Health Informatics, 2009
- [20] Processing of X-ray diffraction data collected in oscillation modeMethods in Enzymology, 1997
- AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMRJournal of Biomolecular NMR, 1996