Disruption of the Ran System by Cysteine Oxidation of the Nucleotide Exchange Factor RCC1

Abstract

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.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01133-14.

ACKNOWLEDGMENTS

We thank Zygmunt Derewenda for his help with interpreting crystal structures, Nicholas Sherman for performing the MS (W. M. Keck Biomedical Mass Spectrometry Laboratory at the University of Virginia), and Mark Conaway for guidance with statistical analyses. We also thank Dennis Templeton for suggesting the use of diamide as a thiol oxidant in these studies. We thank Adam Spencer for technical assistance and Chelsi J. Snow and Chun-Song Yang for reading the manuscript.

This work was supported by National Institutes of Health award 1RO1AG040162 (to B. M. Paschal).