Intersectin-1s: a novel nucleo-cytoplasmic endocytic protein

A growing number of proteins involved in endocytosis are reported to undergo nucleocytoplasmic trafficking and interact with different nuclear factors. This mechanism, known as protein moonlighting, is emerging as a key factor in many biological pro- cesses, though little is known about the correlation between endocytosis and nuclear functions. Here we investigated the nuclear location of the multi-domain scaffold endocytic protein intersectin-1s, a protein involved in clathrin mediated endocytosis, cell signalling and actin cytoskele- ton rearrangements. A bioinformatic analysis of the protein sequence allowed the identification both of putative nuclear localization sequences (NLS) and nuclear export sequences (NES). Immunofluorescence studies and biochemical nuclei isolation experiments revealed the presence of small amounts of intersectin-1s in the nuclear com- partment, distributing between the nucleoplasm and the nuclear membrane, where it partially colocalizes with nuclear lamins. Despite the protein is mainly located in the cytoplasm at the steady state, cell treatment with Leptomycin B, a specific inhibi- tor of the chromosomal region maintenance 1 (CRM-1)-depen- dent nuclear export, resulted in its nuclear accumulation, suggesting that intersectin-1s is a moonlightining protein. Finally, by analyzing the subcellular localization of several Intersectin 1-s domains fused with YFP, or point mutants derivatives thereof impairing the putative NLS/NES, we could demonstrate that indeed intersectin-1s possesses several redundant signals mediat- ing its nucleocytoplasmic shuttling. In conclusion, our results show for the first time the existence of a nuclear pool of intersectin-1s, which shuttles between the cytosol and the nucleus and is localized in different intranuclear compartments.