2 DIFFERENT SUBUNITS OF IMPORTIN COOPERATE TO RECOGNIZE NUCLEAR-LOCALIZATION SIGNALS AND BIND THEM TO THE NUCLEAR-ENVELOPE

Background: Selective protein import into the cell nucleus occurs in t wo steps: binding to the nuclear envelope, followed by energy-dependen t transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G prote in Ran/TC4 is essential for the second. We have previously purified th e 60 kD importin protein (importin 60) as a single polypeptide. Result s: We have identified importin 90, a 90 kD second subunit that dissoci ates from importin 60 during affinity chromatography on nickel (II)-ni trolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence ia human homologue; the amino-aci d sequence of importin 90 is strikingly conserved between the two spec ies. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 o n nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear l ocalization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bov ine serum albumin was allowed to form complexes with cytosolic protein s in Xenopus egg extracts; the complexes were then retrieved with stre ptavidin-agarose. The pattern oi bound proteins was surprisingly simpl e and showed only two predominant bands: those of the importin complex . We also expressed the human homologue of importin 60, Rch1p, and fou nd that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. Conclusions: Importin consists of a 60 a nd a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind t o the nuclear envelope.