The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells

Ekarat Hitakomate, Fiona E. Hood, Helen S. Sanderson, Paul R. Clarke

    Research output: Contribution to journalArticlepeer-review

    17 Citations (Scopus)

    Abstract

    Background: Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA.

    Results: We have investigated the mechanism of the dynamic interaction of the a isoform of human RCC1 (RCC1 alpha) with chromatin in live cells using fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) fusions. We show that the N-terminal tail stabilises the interaction of RCC1 alpha with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1 alpha with chromatin from being stabilised by Ran(T24N), a mutant that binds stably to RCC1 alpha. The interaction of RCC1 alpha with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes alpha-N-terminal methylation, and this interaction is no longer stabilised by Ran(T24N). However, alpha-N-terminal methylation of RCC1 alpha is not regulated by the binding of Ran(T24N). Conversely, the association of Ran with precipitated RCC1 alpha does not require the N-terminal tail of RCC1 alpha or its methylation. The mobility of RCC1 alpha on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1 alpha(D182A) can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by Ran(T24N).

    Conclusions: These results show that the stabilisation of the dynamic interaction of RCC1 alpha with chromatin by Ran in live cells requires the N-terminal tail of RCC1 alpha. alpha-N-methylation is not regulated by formation of the binary complex with Ran, but it promotes chromatin binding through the tail. This work supports a model in which the association of RCC1 alpha with chromatin is promoted by a conformational change in the a-N-terminal methylated tail that is induced allosterically in the binary complex with Ran.

    Original languageEnglish
    Article number43
    Pages (from-to)-
    Number of pages10
    JournalBMC Cell Biology
    Volume11
    DOIs
    Publication statusPublished - 21 Jun 2010

    Keywords

    • CHROMOSOME CONDENSATION RCC1
    • GUANINE-NUCLEOTIDE EXCHANGE
    • NUCLEAR IMPORT
    • PROTEIN
    • REGULATOR
    • MITOSIS
    • BINDING
    • PHOSPHORYLATION
    • GTPASE
    • LOCALIZATION

    Fingerprint

    Dive into the research topics of 'The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells'. Together they form a unique fingerprint.

    Cite this