title: Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope.
abstract: BACKGROUND
  Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide.
  
  
  RESULTS
  We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic 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 its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found 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 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.
authors.affiliation: Wellcome/CRC Institute, Cambridge, UK.
authors.forename: D
authors.initials: D
authors.lastname: Görlich
authors.forename: S
authors.initials: S
authors.lastname: Kostka
authors.forename: R
authors.initials: R
authors.lastname: Kraft
authors.forename: C
authors.initials: C
authors.lastname: Dingwall
authors.forename: R A
authors.initials: RA
authors.lastname: Laskey
authors.forename: E
authors.initials: E
authors.lastname: Hartmann
authors.forename: S
authors.initials: S
authors.lastname: Prehn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005075:Biological Evolution; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D028901:alpha Karyopherins
chemical_list: D002352:Carrier Proteins; D028884:Karyopherins; D009687:Nuclear Proteins; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear; D028901:alpha Karyopherins; C087978:karyopherin alpha 2