Chatterjee Mandovi, Paschal Bryce M
Center for Cell Signaling, University of Virginia, Charlottesville, Virginia Department of Cell Biology, University of Virginia, Charlottesville, Virginia.
Center for Cell Signaling, University of Virginia, Charlottesville, Virginia Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia
Mol Cell Biol. 2015 Feb;35(3):566-81. doi: 10.1128/MCB.01133-14. Epub 2014 Dec 1.
Transport regulation by the Ran GTPase requires its nuclear localization and GTP loading by the chromatin-associated exchange factor RCC1. These reactions generate Ran protein and Ran nucleotide gradients between the nucleus and the cytoplasm. Cellular stress disrupts the Ran gradients, but the specific mechanisms underlying this disruption have not been elucidated. We used biochemical approaches to determine how oxidative stress disrupts the Ran system. RCC1 exchange activity was reduced by diamide-induced oxidative stress and restored with dithiothreitol. Using mass spectrometry, we found that multiple solvent-exposed cysteines in RCC1 are oxidized in cells treated with diamide. The cysteines oxidized in RCC1 included Cys93, which is solvent exposed and unique because it becomes buried upon contact with Ran. A Cys93Ser substitution dramatically reduced exchange activity through an effect on RCC1 binding to RanGDP. Diamide treatment reduced the size of the mobile fraction of RCC1-green fluorescent protein in cells and inhibited nuclear import in digitonin-permeabilized cell assays. The Ran protein gradient was also disrupted by UV-induced stress but without affecting RCC1 exchange activity. Our data suggest that stress can disrupt the Ran gradients through RCC1-dependent and RCC1-independent mechanisms, possibly dependent on the particular stress condition.
Ran GTP酶介导的转运调控需要其在细胞核内定位，并通过与染色质相关的交换因子RCC1进行GTP加载。这些反应在细胞核和细胞质之间产生Ran蛋白和Ran核苷酸梯度。细胞应激会破坏Ran梯度，但这种破坏背后的具体机制尚未阐明。我们使用生化方法来确定氧化应激如何破坏Ran系统。二酰胺诱导的氧化应激会降低RCC1的交换活性，而二硫苏糖醇可使其恢复。通过质谱分析，我们发现用二酰胺处理的细胞中，RCC1中多个暴露于溶剂的半胱氨酸被氧化。RCC1中被氧化的半胱氨酸包括Cys93，它暴露于溶剂且具有独特性，因为它在与Ran接触时会被掩埋。Cys93Ser取代通过影响RCC1与RanGDP的结合，显著降低了交换活性。二酰胺处理减小了细胞中RCC1-绿色荧光蛋白的可移动部分的大小，并在洋地黄皂苷通透细胞试验中抑制了核输入。紫外线诱导的应激也会破坏Ran蛋白梯度，但不影响RCC1的交换活性。我们的数据表明，应激可能通过依赖RCC1和不依赖RCC1的机制破坏Ran梯度，这可能取决于特定的应激条件。
