Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-alpha

Autor(es)

Bernardes, Natalia E. ; Takeda, Agnes A. S. ; Dreyer, Thiago R. ; Cupertino, Fernanda B. ; Virgilio, Stela ; Pante, Nelly ; Bertolini, Maria Celia ; Fontes, Marcos R. M.

• Tipo de documento: Artigo Científico
• Fonte: Biochemical Journal; v. 474, p. 14-pg., 2017-12-15.

Resumo

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; (915)TISSKRQRRHSKS(927)) was recognized by importin-a and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-alpha (NcImp alpha) 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 NcImp alpha/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpa 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-alpha from the alpha 2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements. (AU)

• Processo FAPESP: 14/10289-0 - Estudos estruturais com a importina-± do fungo Neurospora crassa e peptídeos de sequências de localização nuclear (NLS) de proteínas relacionadas ao metabolismo de fungos
• Beneficiário: Natália Elisa Bernardes
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/24705-3 - O fungo filamentoso Neurospora crassa como um organismo modelo para a caracterização funcional de proteínas/fatores de transcrição que regulam o metabolismo de carboidratos
• Beneficiário: Maria Celia Bertolini
• Modalidade de apoio: Auxílio à Pesquisa - Temático