Alhebshi, Alawiah and Sideri, Theodora C. and Holland, Sara L. and Avery, Simon V. (2012) The essential iron- sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species

Oxidative stress mediated by reactive oxygen species (ROS) is linked to degenerative conditions in humans and damage to an array of cellular components. However, it is unclear which molecular target(s) may be the primary “Achilles’ heel” of organisms, accounting for the inhibitory action of ROS. Rli1p (ABCE1) is an essential and highly conserved protein of eukaryotes and archaea that requires notoriously ROS-labile cofactors (Fe-S clusters) for its functions in protein synthesis. In this study, we tested the hypothesis that ROS toxicity is caused by Rli1p dysfunction. In addition to being essential, Rli1p activity (in nuclear ribosomal-subunit export) was shown to be impaired by mild oxidative stress in yeast. Furthermore, prooxidant resistance was decreased by RLI1 repression and increased by RLI1 overexpression. This Rlip1 dependency was abolished during anaerobicity and accentuated in cells expressing a FeS cluster–defective Rli1p construct. The protein’s FeS clusters appeared ROS labile during in vitro incubations, but less so in vivo. Instead, it was primarily 55FeS-cluster supply to Rli1p that was defective in prooxidant-exposed cells. The data indicate that, owing to its essential nature but dependency on ROS-labile FeS clusters, Rli1p function is a primary target of ROS action. Such insight could help inform new approaches for combating oxidative stress–related disease. INTRODUCTION Reactive oxygen species (ROS) are a necessary evil of aerobic life, generated continuously during respiration with the potential to cause oxidative deterioration of proteins, lipids, and DNA. ROS damage is linked to serious diseases in humans, including amyotrophic lateral sclerosis, Alzheimer’s disease, Friedreich’s ataxia, and cancer (Roberts et al., 2009). Furthermore, ROS generation is elevated by environmental perturbation, with oxidative stress being common to the effects of diverse natural (e.g., radiation) and anthropogenic (e.g., chemical pollutant) stresses (Avery, 2001; Limon-Pacheco and Gonsebatt, 2009). The responses invoked by organisms to counter oxidative stress have received considerable research attention over the last two decades. These include the up-regulation of ROS-scavenging proteins, such as peroxidases and superoxide dismutases, or enzymes that reverse oxidative damage, such as methionine sulfoxide reductases. Oxidative stress responses are now well characterized in a diverse range of organisms (Imlay, 2008). However, when such defenses are overwhelmed, a key question remains: What is the principal cellular function(s) targeted by ROS that accounts for their toxicity? Whereas oxidative damage to cellular macromolecules is very widely reported, just two types of effect are thought potentially to cause ROS toxicity: gain of toxic function or loss of essential cellular function (Avery, 2011). Gain-of-function mechanisms could include accumulation of toxic oxidized-protein aggregates (Holland et al., 2007) or apoptotic cell death (Circu and Aw, 2010). Essential targets may include membrane lipid integrity, via lipid peroxidation, and certain ROS-susceptible proteins (Avery, 2011; Daly, 2012). Recent studies indicate that essential protein targets of ROS include functions required for faithful mRNA translation (Holland et al., 2007; Monitoring Editor Thomas D. Fox Cornell University Received: May 30, 2012 Revised: Jul 19, 2012 Accepted: Jul 24, 2012 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E12-05-0413) on August 1, 2012. *Present address: Division of Biosciences, University College London, London WC1E 6BT, United Kingdom. Address correspondence to: Simon V. Avery (Simon.Avery@nottingham.ac.uk). © 2012 Alhebshi et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. Abbreviations used: DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; HA, hemagglutinin; HRP, horseradish peroxidase; MSR, methionine sulfoxide reductase; ORF, open reading frame; qRT-PCR, quantitative RT-PCR; ROS, reactive oxygen species; YNB, yeast–nitrogen base; YPD, yeast–peptone– dextrose. http://www.molbiolcell.org/content/suppl/2012/08/01/mbc.E12-05-0413v1.DC1. html Supplemental Material can be found at: