The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells.
The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells.
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DOI：
10.1186/1471-2121-11-43
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发表时间：
2010-06-21
期刊：
影响因子：
--
通讯作者：
Clarke PR
中科院分区：
文献类型：
--
作者：
Hitakomate E;Hood FE;Sanderson HS;Clarke PR
染色体凝聚1调节因子（RCC1）是Ran GTPase的鸟嘌呤核苷酸交换因子。RCC1在染色质上局部生成Ran-GTP对核细胞质运输、有丝分裂纺锤体组装和核膜形成至关重要。RCC1的n端尾部及其与Ran的关联对于其与细胞中染色质的相互作用都很重要。在体外，Ran与RCC1的结合诱导n端尾部的构象变化，促进其与DNA的相互作用。我们利用绿色荧光蛋白（GFP）融合体的光漂白后荧光恢复（FRAP）研究了人RCC1α亚型（RCC1α）与活细胞染色质的动态相互作用机制。我们发现n端尾部稳定了RCC1α与染色质的相互作用，这一功能可以部分被另一个富含赖氨酸的核定位信号所取代。去除尾部会阻止RCC1α与染色质的相互作用被RanT24N稳定，RanT24N是一个稳定结合RCC1α的突变体。RCC1α与染色质的相互作用因赖氨酸4 （K4Q）突变而不稳定，赖氨酸4会消除α- n端甲基化，RanT24N不再稳定RCC1α与染色质的相互作用。然而，RCC1α α- n端甲基化不受RanT24N结合的调节。相反，Ran与沉淀的RCC1α的关联不需要RCC1α的n端尾部或其甲基化。RCC1α在染色质上的迁移率通过天冬氨酸182 （D182A）突变而增加，从而抑制鸟嘌呤-核苷酸交换活性，但RCC1αD182A仍然可以结合无核苷酸的Ran，并通过RanT24N稳定其与染色质的相互作用。这些结果表明，Ran在活细胞中稳定RCC1α与染色质的动态相互作用需要RCC1α的n端尾部。α- n -甲基化不受与Ran形成二元复合物的调节，但它通过尾部促进染色质结合。这项工作支持了RCC1α与染色质的结合是通过α- n末端甲基化尾部的构象变化促进的模型，这种变化是在与Ran的二元配合物中诱导的变构变化。
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. 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 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. 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.
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影响因子：
5.6
作者：
England JR;Huang J;Jennings MJ;Makde RD;Tan S
通讯作者：
Tan S
影响因子：
--
作者：
Hutchins JR;Moore WJ;Clarke PR
通讯作者：
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影响因子：
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作者：
OHTSUBO, M;KAI, R;NISHIMOTO, T
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RCC1蛋白是染色体凝结发作的调节剂，位于细胞核中并与DNA结合。
影响因子：
7.8
作者：
Ohtsubo, M;Okazaki, H;Nishimoto, T
通讯作者：
Nishimoto, T
RAN交换因子RCC1的核进口至少由两种不同的机制介导。
影响因子：
7.8
作者：
Nemergut, M E;Macara, I G
通讯作者：
Macara, I G