PREVIOUSLY IDENTIFIED PROTEIN OF UNCERTAIN FUNCTION IS KARYOPHERIN-ALPHA AND TOGETHER WITH KARYOPHERIN-BETA DOCKS IMPORT SUBSTRATE AT NUCLEAR-PORE COMPLEXES

Previously, we had purified a cytosolic protein complex, termed karyop herin, that functions in docking import substrate at the nuclear envel ope in digitonin-permeabilized cells and also had molecularly cloned a nd sequenced its 97-kDa beta subunit. We now report that the karyopher in alpha subunit is the previously identified protein NPI-1/SRP-1 of h itherto uncertain function. Using purified recombinant karyopherin alp ha or beta subunit, we showed that neither karyopherin alpha nor karyo pherin beta alone was sufficient for docking of import substrate at th e nuclear envelope. Docking occurred only when both subunits were pres ent. Moreover, docking of import substrate by the two recombinant kary opherin subunits was productive, as it led to nuclear internalization of the docked substrate in the presence of additional, previously char acterized cytosolic factors. In a binding assay using immobilized kary opherin alpha and beta subunits and import substrate as a ligand, we f ound that only karyopherin alpha bound ligand. We suggest that karyoph erin beta functions as an adaptor that binds both to karyopherin alpha and to any of a large number of docking sites that are represented by a repetitive peptide motif containing nucleoporins on both the cytopl asmic and nucleoplasmic side of the nuclear pore complex (NPC), bidire ctionally ferrying a complex of karyopherin alpha-substrate across the NPC.