Activation of the Slx5-Slx8 ubiquitin ligase by poly-small ubiquitin-like modifier conjugates
ABSTRACT Protein sumoylation is a regulated process that is important for the health of human and yeast cells. In budding yeast, a subset of sumoylated proteins is targeted for ubiquitination by a conserved heterodimeric ubiquitin (Ub) ligase, Slx5-Slx8, which is needed to suppress the accumulation of high molecular weight small ubiquitin-like modifier (SUMO) conjugates. Structure-function analysis indicates that the Slx5-Slx8 complex contains multiple SUMO-binding domains that are collectively required for in vivo function. To determine the specificity of Slx5-Slx8, we assayed its Ub ligase activity using sumoylated Siz2 as an in vitro substrate. In contrast to unsumoylated or multisumoylated Siz2, substrates containing poly-SUMO conjugates were efficiently ubiquitinated by Slx5-Slx8. Although Siz2 itself was ubiquitinated, the bulk of the Ub was conjugated to SUMO residues. Slx5-Slx8 primarily mono-ubiquitinated the N-terminal SUMO moiety of the chain. These data indicate that the Slx5-Slx8 Ub ligase is stimulated by poly-SUMO conjugates and that it can ubiquitinate a poly-SUMO chain.
SourceAvailable from: Karol Krzysztof Kramarz[Show abstract] [Hide abstract]
ABSTRACT: The Saccharomyces cerevisiae Uls1 belongs to the Swi2/Snf2 family of DNA-dependent ATPases and a new protein family of SUMO-targeted ubiquitin ligases. Here we show that Uls1 is implicated in DNA repair independently of the replication stress response pathways mediated by the endonucleases Mus81 and Yen1 and the helicases Mph1 and Srs2. Uls1 works together with Sgs1 and we demonstrate that the attenuation of replication stress-related defects in sgs1Δ by deletion of ULS1 depends on a functional of Rad51 recombinase and post-replication repair pathway mediated by Rad18 and Rad5, but not on the translesion polymerase, Rev3. The higher resistance of sgs1Δ uls1Δ mutants to genotoxic stress compared to single sgs1Δ cells is not the result of decreased formation or accelerated resolution of recombination-dependent DNA structures. Instead, deletion of ULS1 restores stability of the rDNA region in sgs1Δ cells. Our data suggest that Uls1 may contribute to genomic stability during DNA synthesis and channel the repair of replication lesions into the Sgs1-dependent pathway, with DNA translocase and SUMO binding activities of Uls1 as well as a RING domain being essential for its functions in replication stress response.DNA Repair 09/2014; 21:24–35. DOI:10.1016/j.dnarep.2014.05.008 · 3.36 Impact Factor
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ABSTRACT: The establishment, maintenance, and dissolution of sister chromatid cohesion are sequentially coordinated during the cell cycle to ensure faithful chromosome transmission. This cell-cycle-dependent regulation of cohesion is mediated, in part, by distinct posttranslational modifications of cohesin, a protein complex consisting of the Smc1-Smc3 ATPase, the Mcd1/Scc1 α-kleisin, and Scc3. Although cohesion is established in S phase, cohesins are not sufficient to maintain cohesion as cells progress from G2 to the metaphase-to-anaphase transition. Rather, the cohesin-associated factor Pds5 is also required to keep sisters paired until anaphase onset. How Pds5 maintains cohesion at the molecular level and whether this maintenance involves the regulation of cohesin modifications remains to be defined. In pds5 mutants, we find that Mcd1 is extensively SUMOylated and that premature sister separation requires Siz2-dependent polySUMOylation. Moreover, abrogation of Pds5 function promotes the proteasome-dependent degradation of Mcd1 and a significant loss of cohesin from chromatin independently of anaphase onset. We further demonstrate that inactivation of the Slx5-Slx8 SUMO-targeted ubiquitin ligase, required for targeting polySUMOylated factors for proteasome-mediated destruction, limits Mcd1 turnover and restores both cell growth and cohesion in metaphase cells defective for Pds5 function. We propose that Pds5 maintains cohesion, at least in part, by antagonizing the polySUMO-dependent degradation of cohesin.Current biology: CB 01/2014; DOI:10.1016/j.cub.2013.12.038 · 9.92 Impact Factor
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ABSTRACT: Sumoylation, a post-translational protein modification by small ubiquitin-like modifier (SUMO), has been implicated in many stress responses. Here we analyzed the potential role of sumoylation in osmo-response in yeast. We find that osmotic stress induces rapid accumulation of sumoylated species in normal yeast cells. Interestingly, disruption of MAP kinase Hog1 leads to a much higher level of accumulation of sumoylated conjugates that are independent of new protein synthesis. We also find that the accumulation of sumoylated species is dependent on a SUMO ligase Siz1. Notably, overexpression of SIZ1 in HOG1-disruption mutants (hog1Δ) but not in wild type cells leads to a markedly increased and prolonged accumulation of sumoylated species. Examination of osmo-tolerance of yeast mutants that display either an increase or a decrease in the global sumoylation level revealed an inverse relationship between accumulation of sumoylated conjugates and osmo-tolerance. Further investigation has shown that many of the sumoylated species induced by hyperosmotic stress are actually poly-sumoylated. Together, these findings indicate that abnormal accumulation of poly-sumoylated conjugates is harmful for osmo-tolerance in yeast, and suggest that Hog1 promotes adaptation to hyperosmotic stress partially via regulation of global sumoylation level.PLoS ONE 02/2014; 9(2):e87306. DOI:10.1371/journal.pone.0087306 · 3.53 Impact Factor