Molecular interactions between the importin α/β heterodimer and proteins involved in vertebrate nuclear protein import

Piergiorgio Percipalle, David W. Clarkson, Helen M. Kent, Daniela Rhodes, Murray Stewart

Research output: Contribution to journalArticle

Abstract

We have used in vitro binding assays to examine specific interactions between a number of cytoplasmic and nuclear pore proteins involved in nuclear protein import in vertebrates. We demonstrate that nuclear transport factor 2 (NTF2), nucleoporin p62 and the Ras-like GTPase Ran bind to the importin heterodimer via its β subunit. The binding behaviour of p62 truncation mutants indicated that importin-β interacts primarily with the α-helical coiled-coil rod domain of nucleoporin p62 and not with the N-terminal domain that contains a number of degenerate repeats based on the xFxFG sequence motif. The binding of Ran to importin-β was sensitive to its nucleotide state, with RanGTP binding strongly, whereas RanGDP binding could not be detected using our assay conditions. RanGTP, but not RanGDP, was able to displace p62 bound to the importin α/β complex, suggesting that the binding sites for p62 and RanGTP on importin-β overlap. Moreover, RanGTP, but not RanGDP, weakened the interaction between importin-α and importin-β in a concentration-dependent manner. NTF2 bound to the importin heterodimer but did not displace p62, suggesting that the NTF2 and p62 binding sites on importin-β do not overlap. The set of interactions we observed was not altered by the binding of NLS-containing substrates such as transcription factor IIIA to the importin heterodimer. Our results are consistent with models for nuclear protein import in which Ran nucleotide exchange modulates the binding of the importin-substrate complexes during translocation through nuclear pore complexes.

Original languageEnglish (US)
Pages (from-to)722-732
Number of pages11
JournalJournal of Molecular Biology
Volume266
Issue number4
DOIs
StatePublished - Mar 7 1997

Fingerprint

Karyopherins
Cell Nucleus Active Transport
Nuclear Proteins
Vertebrates
Proteins
Nuclear Pore
Transcription Factor TFIIIA
Nucleotides
Binding Sites
ras Proteins
Porins

Keywords

  • Importin heterodimer
  • NTF2
  • Nuclear transport
  • Nucleoporin p62
  • Ran

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Molecular interactions between the importin α/β heterodimer and proteins involved in vertebrate nuclear protein import. / Percipalle, Piergiorgio; Clarkson, David W.; Kent, Helen M.; Rhodes, Daniela; Stewart, Murray.

In: Journal of Molecular Biology, Vol. 266, No. 4, 07.03.1997, p. 722-732.

Research output: Contribution to journalArticle

Percipalle, Piergiorgio ; Clarkson, David W. ; Kent, Helen M. ; Rhodes, Daniela ; Stewart, Murray. / Molecular interactions between the importin α/β heterodimer and proteins involved in vertebrate nuclear protein import. In: Journal of Molecular Biology. 1997 ; Vol. 266, No. 4. pp. 722-732.
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