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PDBsum entry 2c1t
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Protein transport/membrane protein
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PDB id
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2c1t
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References listed in PDB file
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Key reference
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Title
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Nup50/npap60 function in nuclear protein import complex disassembly and importin recycling.
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Authors
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Y.Matsuura,
M.Stewart.
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Ref.
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EMBO J, 2005,
24,
3681-3689.
[DOI no: ]
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PubMed id
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Abstract
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Nuclear import of proteins containing classical nuclear localization signals
(NLS) is mediated by the importin-alpha:beta complex that binds cargo in the
cytoplasm and facilitates its passage through nuclear pores, after which nuclear
RanGTP dissociates the import complex and the importins are recycled. In
vertebrates, import is stimulated by nucleoporin Nup50, which has been proposed
to accompany the import complex through nuclear pores. However, we show here
that the Nup50 N-terminal domain actively displaces NLSs from importin-alpha,
which would be more consistent with Nup50 functioning to coordinate import
complex disassembly and importin recycling. The crystal structure of the
importin-alpha:Nup50 complex shows that Nup50 binds at two sites on
importin-alpha. One site overlaps the secondary NLS-binding site, whereas the
second extends along the importin-alpha C-terminus. Mutagenesis indicates that
interaction at both sites is required for Nup50 to displace NLSs. The
Cse1p:Kap60p:RanGTP complex structure suggests how Nup50 is then displaced on
formation of the importin-alpha export complex. These results provide a
rationale for understanding the series of interactions that orchestrate the
terminal steps of nuclear protein import.
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Figure 2.
Figure 2 Crystal structure of the Nup50:importin-  complex
and competition assays using structure-based mutants showed that
Nup50 binds at two sites on importin- ,
and the NLS displacement by Nup50 requires interaction with both
sites on importin- .
(A) Overview of the Nup50:importin- complex
with the 2F[o]-F[c] map around Nup50 contoured at 1  superimposed.
Nup50 residues are shown in ball-and-stick format. (B)
Electrostatic potential on importin- ,
with Nup50 removed, shaded from -9 kT/e (red) to +9 kT/e (blue)
calculated using GRASP (Nicholls et al, 1991). (C) FRET-based
competition assay. Emission profiles of 0.2  M
BFP-  IBB
importin- and
0.2 M
SV40 NLS-GFP without (red) 1 M
Nup50 (residues 1-109) or with (black) 1 M
Nup50 (residues 1-109, wild type) or with (blue) 1 M
Nup50 (residues 1-109, K3E/R4D), or with (green) 1 M
Nup50 (residues 1-109, R38A/R45D). (D) FRET-based competition
assay. Emission profiles of 0.2 M
BFP- IBB
importin- and
0.2 M
NP NLS-GFP without (red) 1 M
Nup50 (residues 1-109) or with (black) 1 M
Nup50 (residues 1-109, wild type) or with (blue) 1 M
Nup50 (residues 1-109, K3E/R4D) or with (green) 1 M
Nup50 (residues 1-109, R38A/R45D).
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Figure 5.
Figure 5 Nup50 (Nup2p) binds to sites crucial for the formation
of nuclear export complex of importin- (Kap60p)
with CAS (Cse1p) and RanGTP. (A) The Cse1p:Kap60p:RanGTP complex
(Matsuura and Stewart, 2004). (B) Nup50 (red) and Nup2p (blue)
are superimposed on the Kap60p ARM repeats in the
Cse1p:Kap60p:RanGTP complex. (C) (A) and (B) superimposed.
Cse1p, yellow; Ran, light blue; Kap60p IBB, pink; Kap60p ARM
repeats, green. GTP is shown as space-filling model.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
EMBO J
(2005,
24,
3681-3689)
copyright 2005.
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