Hitakomate Ekarat, Hood Fiona E, Sanderson Helen S, Clarke Paul R
Biomedical Research Institute, School of Medicine, College of Medicine, Dentistry and Nursing, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK.
BMC Cell Biol. 2010 Jun 21;11:43. doi: 10.1186/1471-2121-11-43.
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 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.
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.
染色体凝聚调节因子1（RCC1）是Ran GTP酶的鸟嘌呤核苷酸交换因子。RCC1在染色质上局部生成Ran-GTP对于核质运输、有丝分裂纺锤体组装和核膜形成至关重要。RCC1的N端尾巴及其与Ran的结合对于其在细胞中与染色质的相互作用都很重要。在体外，Ran与RCC1的结合会诱导N端尾巴发生构象变化，从而促进其与DNA的相互作用。
我们使用绿色荧光蛋白（GFP）融合蛋白的光漂白后荧光恢复（FRAP）技术，研究了人RCC1的α异构体（RCC1α）在活细胞中与染色质动态相互作用的机制。我们发现N端尾巴稳定了RCC1α与染色质的相互作用，并且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。去除尾巴会阻止RCC1α与染色质的相互作用被RanT24N（一种与RCC1α稳定结合的突变体）稳定。赖氨酸4（K4Q）突变会破坏α-N端甲基化，从而使RCC1α与染色质的相互作用不稳定，并且这种相互作用不再被RanT24N稳定。然而，RCC1α的α-N端甲基化不受RanT24N结合的调节。相反，Ran与沉淀的RCC1α的结合不需要RCC1α的N端尾巴或其甲基化。天冬氨酸182（D182A）突变会增加RCC1α在染色质上的流动性，该突变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，并且其与染色质的相互作用被RanT24N稳定。
这些结果表明，在活细胞中，Ran稳定RCC1α与染色质的动态相互作用需要RCC1α的N端尾巴。α-N甲基化不受与Ran形成二元复合物的调节，但它通过尾巴促进染色质结合。这项工作支持了一个模型，即RCC1α与染色质的结合是由α-N端甲基化尾巴的构象变化促进的，这种变化是在与Ran的二元复合物中通过变构诱导产生的。
