DNA binding by the MAT?2 transcription factor controls its access to alternative ubiquitin-modification pathways.
ABSTRACT: Like many transcription factors, the yeast protein MATalpha2 (?2) undergoes rapid proteolysis via the ubiquitin-proteasome system (UPS). At least two ubiquitylation pathways regulate ?2 degradation: one pathway utilizes the ubiquitin ligase (E3) Doa10 and the other the heterodimeric E3 Slx5/Slx8. Doa10 is a transmembrane protein of the endoplasmic reticulum/inner nuclear membrane, whereas Slx5/Slx8 localizes to the nucleus and binds DNA nonspecifically. While a single protein can often be ubiquitylated by multiple pathways, the reasons for this "division of labor" are not well understood. Here we show that ?2 mutants with impaired DNA binding become inaccessible to the Slx5/Slx8 pathway but are still rapidly degraded through efficient shunting to the Doa10 pathway. These results are consistent with the distinct localization of these E3s. We also characterized a novel class of DNA binding-defective ?2 variants whose degradation is strongly impaired. Our genetic data suggest that this is due to a gain-of-function interaction that limits their access to Doa10. Together, these results suggest multiple ubiquitin-ligation mechanisms may have evolved to promote rapid destruction of a transcription factor that resides in distinct cellular subcompartments under different conditions. Moreover, gain-of-function mutations, which also occur with oncogenic forms of human transcription factors such as p53, may derail this fail-safe system.
Project description:The yeast transcription factor MAT?2 (?2) is a short-lived protein known to be ubiquitylated by two distinct pathways, one involving the ubiquitin-conjugating enzymes (E2s) Ubc6 and Ubc7 and the ubiquitin ligase (E3) Doa10 and the other operating with the E2 Ubc4 and the heterodimeric E3 Slx5/Slx8. Although Slx5/Slx8 is a small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL), it does not require SUMO to target ?2 but instead directly recognizes ?2. Little is known about the ?2 determinants required for its Ubc4- and STUbL-mediated degradation or how these determinants substitute for SUMO in recognition by the STUbL pathway. We describe two distinct degradation elements within ?2, both of which are necessary for ?2 recognition specifically by the Ubc4 pathway. Slx5/Slx8 can directly ubiquitylate a C-terminal fragment of ?2, and mutating one of the degradation elements impairs this ubiquitylation. Surprisingly, both degradation elements identified here overlap specific interaction sites for ?2 corepressors: the Mcm1 interaction site in the central ?2 linker and the Ssn6 (Cyc8) binding site in the ?2 homeodomain. We propose that competitive binding to ?2 by the ubiquitylation machinery and ?2 cofactors is balanced so that ?2 can function in transcription repression yet be short lived enough to allow cell-type switching.
Project description:Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post-translational modifications and the corresponding enzymatic machinery. Specifically, SUMO-targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance, and transcription. However, how STUbLs select specific substrates among myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co-localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term "ubiquitin hotspots". Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor-like Ymr111c/Euc1 to associate with these sites and to be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO-interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1-ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z and Rpd3L pathways in specific stress responses. Thus, our data suggest that STUbL-dependent ubiquitin hotspots shape chromatin during stress adaptation.
2019-01-01 | S-EPMC6545562 | BioStudies
Project description:Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post-translational modifications and the corresponding enzymatic machinery. Specifically, SUMO-targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance and transcription. However, how STUbLs select specific substrates amongst myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co-localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term 'ubiquitin hotspots'. Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor-like Ymr111c/Euc1 to associate with these sites and be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO-interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1-ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z- and Rpd3L-pathways in specific stress responses. Thus, our data suggest that STUbL-dependent ubiquitin hotspots shape chromatin during stress adaptation.
Project description:The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure-function analysis reveals a domain of Slx5 involved in nuclear localization and in the interaction with Slx5, SUMO, Slx8, and a novel interactor, the SUMO E3 ligase Siz1. We further analyzed the functional interaction of Slx5 and Siz1 in vitro and in vivo. We found that a recombinant Siz1 fragment is an in vitro ubiquitylation target of the Slx5/Slx8 STUbL. Furthermore, slx5 cells accumulate phosphorylated and sumoylated adducts of Siz1 in vivo. Specifically, we show that Siz1 can be ubiquitylated in vivo and is degraded in an Slx5-dependent manner when its nuclear egress is prevented in mitosis. In conclusion, our data provide a first look into the STUbL-mediated regulation of a SUMO E3 ligase.
Project description:Substrate discrimination in the ubiquitin-proteasome system is believed to be dictated by specific combinations of ubiquitin-protein ligases (E3s) and ubiquitin-conjugating enzymes (E2s). Here we identify Doa10/Ssm4 as a yeast E3 that is embedded in the endoplasmic reticulum (ER)/nuclear envelope yet can target the soluble transcription factor Matalpha2. Doa10 contains an unusual RING finger, which has ubiquitin-ligase activity in vitro and is essential in vivo for degradation of alpha2 via its Deg1 degradation signal. Doa10 functions with two E2s, Ubc6 and Ubc7, to ubiquitinate Deg1-bearing substrates, and it is also required for the degradation of at least one ER membrane protein. Interestingly, different short-lived ER proteins show distinct requirements for Doa10 and another ER-localized E3, Hrd1. Nevertheless, the two E3s overlap in function: A doa10Delta hrd1Delta mutant is far more sensitive to cadmium relative to either single mutant and displays strong constitutive induction of the unfolded protein response; this suggests a role for both E3s in eliminating aberrant ER proteins. The likely human ortholog of DOA10 is in the cri-du-chat syndrome critical region on chromosome 5p, suggesting that defective ubiquitin ligation might contribute to this common genetic disorder.
Project description:High-resolution imaging shows that persistent DNA damage in budding yeast localizes in distinct perinuclear foci for repair. The signals that trigger DNA double-strand break (DSB) relocation or determine their destination are unknown. We show here that DSB relocation to the nuclear envelope depends on SUMOylation mediated by the E3 ligases Siz2 and Mms21. In G1, a polySUMOylation signal deposited coordinately by Mms21 and Siz2 recruits the SUMO targeted ubiquitin ligase Slx5/Slx8 to persistent breaks. Both Slx5 and Slx8 are necessary for damage relocation to nuclear pores. When targeted to an undamaged locus, however, Slx5 alone can mediate relocation in G1-phase cells, bypassing the requirement for polySUMOylation. In contrast, in S-phase cells, monoSUMOylation mediated by the Rtt107-stabilized SMC5/6-Mms21 E3 complex drives DSBs to the SUN domain protein Mps3 in a manner independent of Slx5. Slx5/Slx8 and binding to pores favor repair by ectopic break-induced replication and imprecise end-joining.
Project description:The Slx5-Slx8 complex is a ubiquitin ligase that preferentially ubiquitylates SUMOylated substrates, targeting them for proteolysis. Mutations in SLX5, SLX8, and other SUMO pathway genes were previously identified in our laboratory as genomic suppressors of a point mutation (mot1-301) in the transcriptional regulator MOT1 To further understand the links between the SUMO and ubiquitin pathways, a screen was performed for high-copy suppressors of mot1-301, yielding three genes (MOT3, MIT1, and ULS1). MOT3 and MIT1 have characteristics of prions, and ULS1 is believed to encode another SUMO-targeted ubiquitin ligase (STUbL) that functionally overlaps with Slx5-Slx8. Here we focus on ULS1, obtaining results suggesting that the relationship between ULS1 and SLX5 is more complex than expected. Uls1 interacted with Slx5 physically in to yeast two-hybrid and co-immunoprecipitation assays, a uls1 mutation that blocked the interaction between Uls1 and Slx5 interfered with ULS1 function, and genetic analyses indicated an antagonistic relationship between ULS1 and SLX5 Combined, our results challenge the assumption that Uls1 and Slx5 are simply partially overlapping STUbLs and begin to illuminate a regulatory relationship between these two proteins.
Project description:The Slx5/Slx8 protein complex, a heterodimeric SUMO-targeted ubiquitin ligase, plays an important role in genomic integrity. Slx5/Slx8 is believed to interact with sumoylated proteins that reside in the nuclei of budding yeast cells. In this complex, Slx5, owing to at least two SUMO interacting motifs (SIMs), has been proposed to be the targeting subunit of the Slx8 ubiquitin ligase. However, little is known about the exact subnuclear localization and targets of Slx5/Slx8. In this study we show that Slx5, but not Slx8, forms prominent nuclear foci. The formation of these foci depends on SUMO and a SIM in Slx5. Therefore, we investigated the subnuclear localization and potential chromatin association of Slx5. Using co-localization studies in live cells and fixed chromatin, we were able to localize Slx5 to DNA damage induced foci of Rad52 and Rad9, two proteins involved in the cellular response to DNA damage. Subsequent chromatin immunoprecipitation (ChIP) studies revealed that Slx5 is associated with HO endonuclease induced chromosome breaks. Surprisingly, real-time PCR analysis of Slx5 ChIPs revealed that the level of Slx5 at HO breaks in an slx8 deletion background is reduced about 4-fold. These results indicate that the DNA-damage targeting of Slx5/Slx8 depends on formation of the heterodimer and that this occurs at a subset of nuclear foci also containing DNA damage repair and checkpoint factors.
Project description:Recent findings suggest important roles for nuclear organization in gene expression. In contrast, little is known about how nuclear organization contributes to genome stability. Epistasis analysis (E-MAP) using DNA repair factors in yeast indicated a functional relationship between a nuclear pore subcomplex and Slx5/Slx8, a small ubiquitin-like modifier (SUMO)-dependent ubiquitin ligase, which we show physically interact. Real-time imaging and chromatin immunoprecipitation confirmed stable recruitment of damaged DNA to nuclear pores. Relocation required the Nup84 complex and Mec1/Tel1 kinases. Spontaneous gene conversion can be enhanced in a Slx8- and Nup84-dependent manner by tethering donor sites at the nuclear periphery. This suggests that strand breaks are shunted to nuclear pores for a repair pathway controlled by a conserved SUMO-dependent E3 ligase.
Project description: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.