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Title
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Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope.
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Authors
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D.Görlich,
S.Kostka,
R.Kraft,
C.Dingwall,
R.A.Laskey,
E.Hartmann,
S.Prehn.
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Ref.
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Curr Biol, 1995,
5,
383-392.
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PubMed id
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Abstract
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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.
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