Analysis of Nuclear Protein Import and Export In Vitro Using Fluorescent Cargoes

Nucleocytoplasmic transport of protein is central to the regulation of many cellular functions. Nuclear import and export takes place through nuclear pore complexes (NPCs; reviewed in refs. 1 and 2), large supramolecular structures that span the nuclear envelope. Cargo proteins contain specific signals for nuclear transport, which are often short stretches of amino acids. Nuclear localization signals (NLSs) direct the nuclear import of proteins, whereas nuclear export sequences (NESs) specify protein export from the nucleus. Both types of signals are recognized by nucleocytoplasmic shuttling receptor proteins belonging to the importin/karyopherin β family (reviewed in refs. 3 and 4 ). After binding to cargo, these receptors interact with proteins of the NPC to mediate cargo translocation into or out of the nucleus. The best-characterized nuclear-import pathway utilizes the major import-receptor importin β, which mediates the transport of proteins with basic amino-acid-rich NLSs. Importin (3 interacts with cargo molecules either directly (5) or via an adapter protein, importin α (3 ). Export of many proteins from the nucleus is mediated by the export receptor chromosome region maintenance 1 (CRM1), which recognizes leucine-rich NESs (6 -10 ).