Nuclear transport of fungal transcription factors mediated by Importin‐α and specificities of this interaction

Abstract

Importin‐α (Impα) is a protein that mediates nuclear import of macromolecules via recognition of nuclear localization sequences in the macromolecule that will be transported. Some proteins and transcription factors related with nitrogen and glycogen metabolism in fungus possess putative NLSs, suggesting that they can be transported into the nucleus via the classical nuclear import pathway, which is mediated by Impα and Importin‐β. This work presents biophysical and functional experiments that confirms the interaction between Impα from Neurospora crassa (NcImpα) and Mus musculus (MmImpα) complexed with potential NLSs of transcription factors NIT2; NCU1629 and PACC. Calorimetric analyses showed a high affinity of the NIT2_NLS, PACC_NLS and 1629_NLS peptides at the binding sites of NcImpα and a lower affinity with MmImpα. NcImpα/NIT2_NLS and MmImpα/NIT2_NLS complexes were cocrystallized and X‐ray diffraction data were collected with resolution of 2.5 and 1.5 Å, respectively. The final NcImpα/NIT2_NLS structure allowed unambiguously to observe that the NIT2_NLS peptide binds at major and minor binding sites of NcImpα. However, the final MmImpα/NIT2_NLS structure, showed the binding of the peptide just at the minor binding site. Nuclear import assays were performed with the potential NLS peptides conjugated with bovine serum albumin. Dextran was used to confirm the permeabilization of the cytoplasmatic membrane of cells by digitonin, without damaging the nuclear envelope. The nuclear import assays with NIT2‐NLS‐BSA‐Cy3 and 1629‐NLS‐BSA‐Cy3 indicated that a significant amount of these complexes were imported into the nucleus in digitonin‐permeabilized cells, incubated with cytosol from rabbit and an energy regenerating system. However the nuclear import was more efficient when a mammalian peptide was used, as confirmed by the quantification performed with Image J. The experiments confirmed that the peptides are functional NLSs, with high affinity to Impα and evidenced specificities of the interaction between NLS peptides with Impα from different organisms.Support or Funding InformationFAPESP ‐ São Paulo State Foundation