The dynamic association of RCC1 with chromatin is modulated by Ran-dependent nuclear transport
The dynamic association of RCC1 with chromatin is modulated by Ran-dependent nuclear transport
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DOI：
10.1091/mbc.e03-06-0409
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发表时间：
2004-01-01
影响因子：
3.3
通讯作者：
Moore, MS
中科院分区：
文献类型：
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作者：
Cushman, I;Stenoien, D;Moore, MS
染色体凝集调节因子（RCC 1）与染色质的结合是高度动态的，如通过稳定转染的tsBN 2细胞中GFP-RCC 1的光漂白分析后的荧光恢复所确定的。显微注射野生型或Q69 L Ran显著减慢GFP-RCC 1的迁移率，而T24 N Ran（核苷酸加载缺陷）进一步降低GFP-RCC 1的迁移率。我们发现，在用破坏不同RAN依赖性核输出途径的试剂处理后，核内GFP-RCC 1的流动性发生了显着变化。抑制Crml/RanGTP依赖性核输出的瘦霉素B显著增加了RCC 1的迁移率，高水平的放线菌素D（抑制RNA聚合酶I、II和III）或α-鹅膏蕈碱（抑制RNA聚合酶II和III）以及能量消耗也是如此。然而，仅抑制mRNA转录对GFP-RCC 1迁移率没有影响，这与mRNA输出是一个Ran独立的过程一致。在透化细胞中，GFP-RCC 1从染色质中有效释放需要胞质溶胶和GTP。重组RAN不能替代胞质溶胶，高水平的补充RAN抑制胞质溶胶刺激的释放。因此，体外从染色质释放RCC 1需要不同于Ran或除了Ran之外的因子，并且似乎在体内与Ran依赖性转运货物的可用性相关。
Regulator of chromosome condensation (RCC1) binding to chromatin is highly dynamic, as determined by fluorescence recovery after photobleaching analysis of GFP-RCC1 in stably transfected tsBN2 cells. Microinjection of wild-type or Q69L Ran markedly slowed the mobility of GFP-RCC1, whereas T24N Ran (defective in nucleotide loading) decreased it further still. We found significant alterations in the mobility of intranuclear GFP-RCC1 after treatment with agents that disrupt different Ran-dependent nuclear export pathways. Leptomycin B, which inhibits Crml/RanGTP-dependent nuclear export, significantly increased the mobility of RCC1 as did high levels of actinomycin D (to inhibit RNA polymerases I, II, and III) or a-amanitin (to inhibit RNA polymerases II and III) as well as energy depletion. Inhibition of just mRNA transcription, however, had no affect on GFP-RCC1 mobility consistent with mRNA export being a Ran-independent process. In permeabilized cells, cytosol and GTP were required for the efficient release of GFP-RCC1 from chromatin. Recombinant Ran would not substitute for cytosol, and high levels of supplemental Ran inhibited the cytosol-stimulated release. Thus, RCC1 release from chromatin in vitro requires a factor(s) distinct from, or in addition to, Ran and seems linked in vivo to the availability of Ran-dependent transport cargo.