Cushman Ian, Stenoien David, Moore Mary Shannon
Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Mol Biol Cell. 2004 Jan;15(1):245-55. doi: 10.1091/mbc.e03-06-0409. Epub 2003 Oct 17.
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 Crm1/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 alpha-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.
通过对稳定转染的tsBN2细胞中GFP-RCC1进行光漂白后的荧光恢复分析确定，染色体凝聚调节因子（RCC1）与染色质的结合具有高度动态性。显微注射野生型或Q69L Ran显著减慢了GFP-RCC1的移动速度，而T24N Ran（核苷酸加载有缺陷）则进一步降低了其移动速度。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-RCC1的移动性发生了显著改变。抑制Crm1/RanGTP依赖性核输出的雷帕霉素B显著增加了RCC1的移动性，高浓度放线菌素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）以及能量耗竭也有同样效果。然而，仅抑制mRNA转录对GFP-RCC1的移动性没有影响，这与mRNA输出是一个Ran非依赖性过程一致。在通透细胞中，从染色质高效释放GFP-RCC1需要胞质溶胶和GTP。重组Ran不能替代胞质溶胶，高浓度补充Ran会抑制胞质溶胶刺激的释放。因此，体外RCC1从染色质的释放需要一种不同于Ran或除Ran之外的因子，并且在体内似乎与Ran依赖性运输货物的可用性相关。
