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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