Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme

Abstract

Although it is vital that cells detect and respond to oxidative stress to allow adaptation and repair damage, the underlying sensing and signaling mechanisms that control these responses are unclear. Protein ubiquitinylation plays an important role in controlling many biological processes, including cell division. In Saccharomyces cerevisiae, ubiquitinylation involves a single E1 enzyme, Uba1, with multiple E2s and E3s providing substrate specificity. For instance, the conserved E2 Cdc34 ubiquitinylates many substrates, including the cyclin-dependent kinase inhibitor Sic1, targeting it for degradation to allow cell cycle progression. Here we reveal that, in contrast to other ubiquitin pathway E2 enzymes, Cdc34 is particularly sensitive to oxidative inactivation, through sequestration of the catalytic cysteine in a disulfide complex with Uba1, by levels of oxidant that do not reduce global ubiquitinylation of proteins. This Cdc34 oxidation is associated with (i) reduced levels of Cdc34-ubiquitin thioester forms, (ii) increased stability of at least one Cdc34 substrate, Sic1, and (iii) Sic1-dependent delay in cell cycle progression. Together, these data reveal that the differential sensitivity of a ubiquitin pathway E2 enzyme to oxidation is utilized as a stress-sensing mechanism to respond to oxidative stress.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00559-12.

ACKNOWLEDGMENTS

We thank Elizabeth Veal for many discussions, Marc Flanagan, Janet Quinn, Elizabeth Veal, and Caroline Wilkinson for comments on the manuscript, Chris Grant, David Lydall, and Caroline Wilkinson for reagents and strains, and Michelle Wray for technical assistance.

The research was supported by the Medical Research Council (K.S.D. and E.L.R.), the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre based at Newcastle upon Tyne Hospitals NHS Foundation Trust and Newcastle University (E.L.R.), and the Faculty of Medical Sciences Newcastle University (K.S.D. and B.A.M.).

The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health.