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

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 α isoform of human RCC1 (RCC1α) 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 RCC1α with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1α with chromatin from being stabilised by Ran(T24N), a mutant that binds stably to RCC1α. The interaction of RCC1α with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes α-N-terminal methylation, and this interaction is no longer stabilised by Ran(T24N). However, α-N-terminal methylation of RCC1α is not regulated by the binding of Ran(T24N). Conversely, the association of Ran with precipitated RCC1α does not require the N-terminal tail of RCC1α or its methylation. The mobility of RCC1α on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1α(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 RCC1α with chromatin by Ran in live cells requires the N-terminal tail of RCC1α. α-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 RCC1α with chromatin is promoted by a conformational change in the α-N-terminal methylated tail that is induced allosterically in the binary complex with Ran.