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 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.