Abstract
BackgroundRegulator 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.ResultsWe 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 RanT24N, 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 RanT24N. However, α-N-terminal methylation of RCC1α is not regulated by the binding of RanT24N. 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 RCC1αD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N.ConclusionsThese 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.
Highlights
Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase
The N-terminal tail of RCC1 is required for stable association with interphase chromatin To examine the role of the N-terminal region (NTR) or tail of RCC1α in its dynamic interaction with chromatin in live cells, we made N-terminal and C-terminal green fluorescent protein (GFP) fusion constructs of the RCC1α N-terminal tail, the RCC1 core domain (Δ27RCC1) and full-length RCC1α
We found that the co-expression of mCherry-RanWT or mCherry-RanT24N in cells did not affect the α-N-terminal methylation of RCC1α-GFP or endogenous RCC1 when compared to the co-expression of mCherry alone (Figure 4)
Summary
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. Near to the N-terminus is a short flexible region that contains a functional lysine-rich nuclear localisation signal (NLS) that associates with the import receptor dimer formed by importin-α3 and importin-β [5,15,16] In vitro, this basic N-terminal region (NTR) or tail can interact directly with DNA [13,17] and in cells it is involved in both the concentration of RCC1 in the nucleus and in its interaction with chromatin [5]. In the case of RCC1γ, a 17 amino acid insert stabilises its interaction with chromatin, reduces importin binding and alters its regulation by phosphorylation at serine 11 [21]
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