Interplays between Sumoylation, SUMO-Targeted Ubiquitin Ligases, and the Ubiquitin-Adaptor Protein Ufd1 in Fission Yeast

In eukaryotes many players in the DNA-damage response (DDR) catalyze protein sumoylation or ubiquitylation. Emphasis has been placed on how these modifications orchestrate the sequential recruitment of repair factors to sites of DNA damage or stalled replication forks. Here, we shed light on a pathway in which sumoylated factors are eliminated through the coupled action of Sumo-targeted ubiquitin ligases (STUbLs) and the ubiquitin-fusion degradation protein 1 (Ufd1). Ufd1 is a subunit of the Cdc48-Ufd1-Npl4 complex implicated in the sorting of ubiquitylated substrates for degradation by the proteasome. We find that in fission yeast, Ufd1 interacts physically and functionally with the Sumo-targeted ubiquitin ligase (STUbL) Rfp1, homologous to human RNF4, and with the Sumo E3 ligase Pli1, homologous to human PIAS1. Deleting a C-terminal domain of Ufd1 that mediates the interaction of Ufd1 with Rfp1, Pli1, and Sumo (ufd1ΔCt213-342) lead to an accumulation of high-molecular-weight Sumo conjugates and caused severe genomic instabilities. The spectrum of sensitivity of ufd1ΔCt213-342 cells to genotoxins, the epistatic relationships of ufd1ΔCt213-342 with mutations in DNA repair factors, and the localization of the repair factor Rad22 in ufd1ΔCt213-342 cells point to ufd1ΔCt213-342 cells accumulating aberrant structures during replication that require homologous recombination (HR) for their repair. We present evidence that HR is however often not successful in ufd1ΔCt213-342 cells and we identify Rad22 as one of the high-molecular-weight conjugates accumulating in the ufd1ΔCt213-342 mutant consistent with Rad22 being a STUbL/Ufd1 substrate. Suggesting a direct role of Ufd1 in the processing of Sumo-conjugates, Ufd1 formed nuclear foci colocalizing with Sumo during the DDR, and Sumoconjugates accumulated in foci in the ufd1ΔCt213-342 mutant. Broader functional relationships between Ufd1 and STUbLs conceivably affect numerous cellular processes beyond the DDR. Citation: Køhler JB, Jørgensen MLM, Beinoraité G, Thorsen M, Thon G (2013) Concerted Action of the Ubiquitin-Fusion Degradation Protein 1 (Ufd1) and Sumo-Targeted Ubiquitin Ligases (STUbLs) in the DNA-Damage Response. PLoS ONE 8(11): e80442. doi:10.1371/journal.pone. 0080442 Editor: Anja-Katrin Bielinsky, University of Minnesota, United States of America Received June 22, 2013; Accepted October 2, 2013; Published November 12, 2013 Copyright: © 2013 Køhler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors' research was supported by the Danish Research Council (http://fivu.dk/en/; grant 09-064284 to GT), the Novo Nordisk Foundation (http://www.novonordiskfonden.dk/en) and the University of Copenhagen Center of Excellence MolPhysX (http://www.ku.dk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: gen@bio.ku. dk