Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex

The interactions between transport receptors and proteins of the nuclear po re complex (NPC) are fundamental to understanding nucleocytoplasmic transpo rt. In order to delineate the path that a particular transport receptor tak es through the NPC, we have employed fluorescence resonance energy transfer (FRET) between enhanced cyan and yellow fluorescent proteins (ECFP, EYFP) in living cells. A panel of yeast strains expressing functional receptor-EC FP and nucleoporin-EYFP fusions has been analyzed with a FRET assay. With t his approach, we define points of contact in the NPC for the related import in Pse1/Kap121 and exportin Msn5. These data demonstrate the utility of FRE T in mapping dynamic protein interactions in a genetic system. Furthermore, the data indicate that an importin and exportin have overlapping pathways through the NPC.