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 alpha isoform of human RCC1 (RCC1alpha) 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 RCC1alpha with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1alpha with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1alpha. The interaction of RCC1alpha with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes alpha-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, alpha-N-terminal methylation of RCC1alpha is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1alpha does not require the N-terminal tail of RCC1alpha or its methylation. The mobility of RCC1alpha on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1alphaD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N. CONCLUSIONS: These results show that the stabilisation of the dynamic interaction of RCC1alpha with chromatin by Ran in live cells requires the N-terminal tail of RCC1alpha. 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 RCC1alpha with chromatin is promoted by a conformational change in the alpha-N-terminal methylated tail that is induced allosterically in the binary complex with Ran.
Pubmed ID: 20565941 RIS Download
Active Transport, Cell Nucleus | Allosteric Regulation | Cell Cycle Proteins | Cell Nucleus | Chromatin | Cloning, Molecular | Guanine Nucleotide Exchange Factors | HeLa Cells | Humans | Methylation | Mutation | Nuclear Proteins | Protein Binding | Protein Isoforms | Protein Stability | Protein Structure, Tertiary | ran GTP-Binding Protein