Single-molecule measurements of importin alpha/cargo complex dissociation at the nuclear pore.

Macromolecules are transported between the cytoplasm and the nucleoplasm of eukaryotic cells through nuclear pore complexes (NPCs). Large (more than approximately 40 kDa) transport cargoes imported into the nucleus typically form a complex with at least one soluble transport cofactor of the importin (Imp) beta superfamily. Many cargoes require an accessory cofactor, Imp alpha, which binds to Imp beta and to the nuclear localization sequence on the cargo. We previously reported the use of narrow-field epifluorescence microscopy to directly monitor cargoes in transit through NPCs in permeabilized cells. We now report an expanded approach in which single-molecule fluorescence resonance energy transfer (FRET) is used to detect the disassembly of Imp alpha/cargo complexes as they transit through NPCs. We found that CAS, the recycling cofactor for Imp alpha, and RanGTP are essential for this dissociation process. After Imp alpha/cargo complex dissociation, most Imp alpha and cargo molecules entered the nucleoplasm. In contrast, the majority of Imp alpha/cargo complexes that did not dissociate at the NPC in the presence of CAS and RanGTP returned to the cytoplasm. These data are consistent with a model in which Imp alpha/cargo complexes are dissociated on the nucleoplasmic side of the NPC, and this dissociation requires both CAS and RanGTP.