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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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^D182Acan still bind nucleotide-free Ran and its interaction with chromatin is stabilised by Ran^T24N.</p> <p>Conclusions</p> <p>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.</p

Three-dimensional context rather than NLS amino acid sequence determines importin α subtype specificity for RCC1

Importin α3 facilitates the nuclear transport of the Ran guanine nucleotide exchange factor RCC1. Here the authors reveal the molecular basis for the selectivity of RCC1 for importin α3 vs the generic importin α1 and discuss the evolution of importin α isoforms