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 solventexposed 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. R egulation of nuclear transport by the Ran GTPase involves two integrated cycles, a nucleotide cycle and a nucleocytoplasmic shuttling cycle. The nucleotide cycle, which is a general feature of GTPases, depends on GTP loading onto Ran and a subsequent step of GTP hydrolysis (1, 2). GTP loading onto Ran occurs in the nucleus through the action of the nucleotide exchange factor RCC1, which, upon binding Ran, promotes GDP release and GTP binding (3,4). GTP binding to Ran is favored over GDP rebinding because of the GTP/GDP ratio (ϳ10:1) in cells (4, 5). The nuclear export phase of the nucleocytoplasmic shuttling cycle occurs as a result of high-affinity binding of RanGTP to nuclear transport receptors that translocate from the nucleus to the cytoplasm. In the cytoplasm, GTP hydrolysis by Ran, which promotes release from the transport receptors, occurs through association with the Ran GTPase-activating protein (GAP) (6). RanGDP can then engage with a dedicated import receptor, NTF2, and undergo reimport (7-10). Thus, Ran undergoes import, nucleotide exchange, export, and nucleotide hydrolysis. These reactions generate two nuclear/cytoplasmic (N/C) Ran gradients, a Ran protein gradient (ϳ3:1) and a RanGTP gradient (ϳ100:1) (11, 12). Disruption of the Ran protein gradient by depletion of NTF2 reduces Ran GTPdependent import (13,14), and inhibition of GTP loading with a temperature-sensitive allele of RCC1 disrupts the Ran protein gradient (15). Because disruption of the Ran protein gradient perturbs the nucleotide cycle and vice versa, the two cycles appear to be linked in the cell.RCC1 is the only known nucleotide exchange factor for Ran, and the exclusive nuclea...

Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites

“…3C). In human, cellular stress and elevated H 2 O 2 levels disrupt the Ran system, and this was partly attributed to cysteine oxidation of the nucleotide exchange factor RCC1, although independent mechanisms were suggested (48). Cys-SOH in the active sites of AtLSF2 and AtSEX4 (SI Appendix, Table S2) align with the nucleophilic Cys of 2 human Protein Tyr Phosphatases (PTPs), VACCINIA H1-RELATED PHOSPHATASE (HsVHR) and SLINGSHOT HOMOLOG3 (HsSSH3) (Fig.…”

Inside out: the role of nucleocytoplasmic transport in ALS and FTLD

“…Postmitotic cells are unable to repair damaged nuclear pores, which become leaky during aging. Oxidative stress, a well-known hallmark of aging and implicated in several neurodegenerative disorders [ 65 ], is known to cause such damage to the nuclear pore [ 23 , 140 ], but also to other components of the nuclear transport machinery [ 18 , 59 ]. Several nuclear transport factors themselves have already been found misregulated in post - mortem ALS and FTLD patient material [ 58 , 84 , 92 , 126 , 144 ] (Fig.…”

Dependence of the structure and mechanics of metaphase chromosomes on oxidized cysteines

“…However, disulfide bonding of the condensin subunit CAP-G (Kuga et al, 2007), RCC1 (Chatterjee and Paschal, 2015), and NONO/p54NRB (Hwang et al, 2009) have been observed in other studies.…”