Cross-talk between distinct nuclear import pathways enables efficient nuclear import of E47 in conjunction with its partner transcription factors.

Nuclear import of karyophilic proteins is carried out by a variety of mechanisms. We previously showed that two basic helix-loop-helix proteins, NeuroD1 and E47, synergistically affect each other's nuclear import. In this study, we dissected the molecular pathways underlying nuclear import of the NeuroD1/E47 heterodimer. In vitro nuclear import assays indicated that importin α family members are the major nuclear import receptors for E47. However, inhibition of importin α resulted in cytoplasmic retention of E47 that could be rescued by its binding partner, NeuroD1, through heterodimerization. In addition, nuclear import of NeuroD1 was importin α independent but importin β1 dependent. In primary neurons, localization of endogenous E47 was not affected by importin α inhibition, suggesting that neuronal E47 could be imported into the nucleus as a heterodimer with NeuroD1 by using importin β1 alone. We also found that E47 had similar nuclear import characteristics in C2C12 cells, where E47 heterodimerized with MyoD, another helix-loop-helix protein, suggesting functional conservation within the same family of transcription factors. Collectively, our data reveal that E47 is imported into the nucleus via multiple pathways, depending on the molecular binding mode, establishing a previously uncharacterized cross-talk between two distinct nuclear import pathways.