Activation of the Slx5-Slx8 ubiquitin ligase by poly-small ubiquitin-like modifier conjugates

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.     

Distinct SUMO Ligases Cooperate with Esc2 and Slx5 to Suppress Duplication-Mediated Genome Rearrangements

Suppression of duplication-mediated gross chromosomal rearrangements (GCRs) is essential to maintain genome integrity in eukaryotes. Here we report that SUMO ligase Mms21 has a strong role in suppressing GCRs in Saccharomyces cerevisiae, while Siz1 and Siz2 have weaker and partially redundant roles. Understanding the functions of these enzymes has been hampered by a paucity of knowledge of their substrate specificity in vivo. Using a new quantitative SUMO-proteomics technology, we found that Siz1 and Siz2 redundantly control the abundances of most sumoylated substrates, while Mms21 more specifically regulates sumoylation of RNA polymerase-I and the SMC-family proteins. Interestingly, Esc2, a SUMO-like domain-containing protein, specifically promotes the accumulation of sumoylated Mms21-specific substrates and functions with Mms21 to suppress GCRs. On the other hand, the Slx5-Slx8 complex, a SUMO-targeted ubiquitin ligase, suppresses the accumulation of sumoylated Mms21-specific substrates. Thus, distinct SUMO ligases work in concert with Esc2 and Slx5-Slx8 to control substrate specificity and sumoylation homeostasis to prevent GCRs.     

SUMO-targeted ubiquitin ligases in genome stability

We identify the SUMO-Targeted Ubiquitin Ligase (STUbL) family of proteins and propose that STUbLs selectively ubiquitinate sumoylated proteins and proteins that contain SUMO-like domains (SLDs). STUbL recruitment to sumoylated/SLD proteins is mediated by tandem SUMO interaction motifs (SIMs) within the STUbLs N-terminus. STUbL-mediated ubiquitination maintains sumoylation pathway homeostasis by promoting target protein desumoylation and/or degradation. Thus, STUbLs establish a novel mode of communication between the sumoylation and ubiquitination pathways. STUbLs are evolutionarily conserved and include: Schizosaccharomyces pombe Slx8-Rfp (founding member), Homo sapiens RNF4, Dictyostelium discoideum MIP1 and Saccharomyces cerevisiae Slx5-Slx8. Cells lacking Slx8-Rfp accumulate sumoylated proteins, display genomic instability, and are hypersensitive to genotoxic stress. These phenotypes are suppressed by deletion of the major SUMO ligase Pli1, demonstrating the specificity of STUbLs as regulators of sumoylated proteins. Notably, human RNF4 expression restores SUMO pathway homeostasis in fission yeast lacking Slx8-Rfp, underscoring the evolutionary functional conservation of STUbLs. The DNA repair factor Rad60 and its human homolog NIP45, which contain SLDs, are candidate STUbL targets. Consistently, Rad60 and Slx8-Rfp mutants have similar DNA repair defects.     

Genetic evidence that polysumoylation bypasses the need for a SUMO-targeted Ub ligase

Saccharomyces cerevisiae cells lacking the Slx5-Slx8 SUMO-targeted Ub ligase display increased levels of sumoylated and polysumoylated proteins, and they are inviable in the absence of the Sgs1 DNA helicase. One explanation for this inviability is that one or more sumoylated proteins accumulate to toxic levels in sgs1Δ slx5Δ cells. To address this possibility, we isolated a second-site suppressor of sgs1Δ slx5Δ synthetic lethality and identified it as an allele of the ULP2 SUMO isopeptidase. The suppressor, ulp2-D623H, behaved like the ulp2Δ allele in its sensitivity to heat, DNA replication stress, and DNA damage. Surprisingly, deletion of ULP2, which is known to promote the accumulation of poly-SUMO chains, suppressed sgs1Δ slx5Δ synthetic lethality and the slx5Δ sporulation defect. Further, ulp2Δ's growth sensitivities were found to be suppressed in ulp2Δ slx5Δ double mutants. This mutual suppression indicates that SLX5-SLX8 and ULP2 interact antagonistically. However, the suppressed strain sgs1Δ slx5Δ ulp2-D623H displayed even higher levels of sumoylated proteins than the corresponding double mutants. Thus, sgs1Δ slx5Δ synthetic lethality cannot be due simply to high levels of bulk sumoylated proteins. We speculate that the loss of ULP2 suppresses the toxicity of the sumoylated proteins that accumulate in slx5Δ-slx8Δ cells by permitting the extension of poly-SUMO chains on specific target proteins. This additional modification might attenuate the activity of the target proteins or channel them into alternative pathways for proteolytic degradation. In support of this latter possibility we find that the WSS1 isopeptidase is required for suppression by ulp2Δ.     

Ubc9 is required for damage-tolerance and damage-induced interchromosomal homologous recombination in S. cerevisiae

Ubc9 is an enzyme involved in the conjugation of small ubiquitin related modifier (SUMO) to target proteins. A Saccharomyces cerevisiae ubc9 temperature sensitive (ts) mutant showed higher sensitivity to various DNA damaging agents such as methylmethanesulfonate (MMS) and UV at a semi-permissive temperature than wild-type cells. The sensitivity of ubc9ts cells was not suppressed by the introduction of a mutated UBC9 gene, UBC9-C93S, whose product is unable to covalently bind to SUMO and consequently fails to conjugate SUMO to target proteins. Diploid ubc9ts cells were more sensitive to various DNA damaging agents than haploid ubc9ts cells suggesting the involvement of homologous recombination in the sensitivity of ubc9ts cells. The frequency of interchromosomal recombination between heteroalleles, his1-1/his1-7 loci, in wild-type cells was remarkably increased upon exposure to MMS or UV. Although the frequency of spontaneous interchromosomal recombination between the heteroalleles in ubc9ts cells was almost the same as that of wild-type cells, no induction of interchromosomal recombination was observed in ubc9ts cells upon exposure to MMS or UV.     

Characterization of a novel posttranslational modification in polypyrimidine tract-binding proteins by SUMO1

Polypyrimidine tract-binding protein 1 (PTBP1) and its brainspecific homologue, PTBP2, are associated with pre-mRNAs and influence pre-mRNA processing, as well as mRNA metabolism and transport. They play important roles in neural differentiation and glioma development. In our study, we detected the expression of the two proteins in glioma cells and predicted that they may be sumoylated using SUMOplot analyses. We confirmed that PTBP1 and PTBP2 can be modified by SUMO1 with co-immunoprecipitation experiments using 293ET cells transiently co-expressing SUMO1 and either PTBP1 or PTBP2. We also found that SUMO1 modification of PTBP2 was enhanced by Ubc9 (E2). The mutation of the sumoylation site (Lys137) of PTBP2 markedly inhibited its modification by SUMO1. Interestingly, in T98G glioma cells, the level of sumoylated PTBP2 was reduced compared to that of normal brain cells. Overall, this study shows that PTBP2 is posttranslationally modified by SUMO1. [BMB Reports 2014; 47(4): 233-238]     

Smt3/SUMO and Ubc9 are required for efficient APC/C-mediated proteolysis in budding yeast

Ubiquitin-mediated proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for sister chromatid separation and the mitotic exit. Like ubiquitylation, protein modification with the small ubiquitin-related modifier SUMO appears to be important during mitosis, because yeast cells impaired in the SUMO-conjugating enzyme Ubc9 were found to be blocked in mitosis and defective in cyclin degradation. Here, we analysed the role of SUMOylation in the metaphase/anaphase transition and in APC/C-mediated proteolysis in Saccharomyces cerevisiae. We show that cells depleted of Ubc9 or Smt3, the yeast SUMO protein, mostly arrested with undivided nuclei and with high levels of securin Pds1. This metaphase block was partially relieved by a deletion of PDS1. The absence of Ubc9 or Smt3 also resulted in defects in chromosome segregation. Temperature-sensitive ubc9-2 mutants were delayed in proteolysis of Pds1 and of cyclin Clb2 during mitosis. The requirement of SUMOylation for APC/C-mediated degradation was tested more directly in G1-arrested cells. Both ubc9-2 and smt3-331 mutants were defective in efficient degradation of Pds1 and mitotic cyclins, whereas proteolysis of unstable proteins that are not APC/C substrates was unaffected. We conclude that SUMOylation is needed for efficient proteolysis mediated by APC/C in budding yeast.     

SUMO modification is involved in the maintenance of heterochromatin stability in fission yeast

Several studies have suggested that SUMO may participate in the regulation of heterochromatin, but direct evidence is lacking. Here, we present a direct link between sumoylation and heterochromatin stability. SUMO deletion impaired silencing at heterochromatic regions and induced histone H3 Lys4 methylation, a hallmark of active chromatin in fission yeast. Our findings showed that the SUMO-conjugating enzyme Hus5/Ubc9 interacted with the conserved heterochromatin proteins Swi6, Chp2 (a paralog of Swi6), and Clr4 (H3 Lys9 methyltransferase). Moreover, chromatin immunoprecipitation (ChIP) revealed that Hus5 was highly enriched in heterochromatic regions in a heterochromatin-dependent manner, suggesting a direct role of Hus5 in heterochromatin formation. We also found that Swi6, Chp2, and Clr4 themselves can be sumoylated in vivo and defective sumoylation of Swi6 or Chp2 compromised silencing. These results indicate that Hus5 associates with heterochromatin through interactions with heterochromatin proteins and modifies substrates whose sumoylations are required for heterochromatin stability, including heterochromatin proteins themselves.     

The CacyBP/SIP Protein is Sumoylated in Neuroblastoma NB2a Cells

The Calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein is highly expressed in mammalian brain as well as in neuroblastoma NB2a cells and pheochromocytoma PC12 cells. This protein interacts with several targets such as cytoskeletal proteins or ERK1/2 kinase and seems to be involved in many cellular processes. In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function. Since theoretical analysis of the amino acid sequence of CacyBP/SIP indicated several lysine residues which could potentially be sumoylated we checked experimentally whether this protein might be modified by SUMO attachment. We have shown that indeed CacyBP/SIP bound the E2 SUMO ligase, Ubc9, in neuroblastoma NB2a cell extract and was sumoylated in these cells. By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction. We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.     

Sumoylation of amyloid precursor protein negatively regulates Abeta aggregate levels

The proteolytic processing of amyloid precursor protein (APP) to produce Aβ peptides is thought to play an important role in the mechanism of Alzheimer’s disease. Here, we show that lysines 587 and 595 of APP, which are immediately adjacent to the site of β-secretase cleavage, are covalently modified by SUMO proteins in vivo. Sumoylation of these lysine residues is associated with decreased levels of Aβ aggregates. Further, overexpression of the SUMO E2 enzyme ubc9 along with SUMO-1 results in decreased levels of Aβ aggregates in cells transfected with the familial Alzheimer’s disease-associated V642F mutant APP, indicating the potential of up-regulating activity of the cellular sumoylation machinery as an approach against Alzheimer’s disease. The results also provide the first demonstration that the SUMO E2 enzyme (ubc9) is present within the endoplasmic reticulum, indicating how APP, and perhaps other proteins that enter this compartment, can be sumoylated.     

The Yeast Slx5-Slx8 DNA Integrity Complex Displays Ubiquitin Ligase Activity

Genetic studies in budding yeast have previously implicated SLX5 and SLX8 in the control of genome stability and sumoylation. These genes encode RING-finger domain proteins that form a complex of unknown function. Because RING-finger proteins comprise a large class of ubiquitin (Ub) ligases, Slx5 and Slx8 were tested for this activity. Here we show that the Slx5-Slx8 complex, but not its individual subunits, stimulates several human and yeast Ub conjugating enzymes, including Ubc1, 4, 5, and Ubc13-Mms2. The RING-finger domains of both subunits are genetically required for suppression of slx sgs1? synthetic-lethality, and point mutations that abolish Ub ligase activity in vitro also eliminate in vivo complementation. Targets of the in vitro ubiquitination reaction include the Slx5 and Slx8 subunits themselves, and the homologous recombination proteins Rad52 and Rad57. We propose that the Slx5-Slx8 complex functions as a two-component Ub ligase in vivo and that it controls genome stability and sumoylation via ubiquitination.     

The S. pombe Translation Initiation Factor eIF4G Is Sumoylated and Associates with the SUMO Protease Ulp2

SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering protein-protein interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination, resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated; in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and Mnk-binding sites respectively.     

An unusual transmembrane helix in the endoplasmic reticulum ubiquitin ligase Doa10 modulates degradation of its cognate E2 enzyme

In the endoplasmic reticulum (ER), nascent membrane and secreted proteins that are misfolded are retrotranslocated into the cytosol and degraded by the proteasome. For most ER-associated degradation (ERAD) substrates, ubiquitylation is essential for both their retrotranslocation and degradation. Yeast Doa10 is a polytopic membrane ubiquitin ligase (E3) that along with its cognate ubiquitin-conjugating enzymes (E2s), Ubc7 and the C-terminally membrane-anchored Ubc6, makes a major contribution to ER-associated degradation. Ubc6 is also a substrate of Doa10. One highly conserved Doa10 element, the uncharacterized ~130-residue TEB4-Doa10 domain, includes three transmembrane helices (TMs). We find that the first of these, TM5, includes an absolutely conserved ΦPΦXXG motif that is required for Doa10 function, as well as highly conserved negatively charged glutamate and aspartate residues. The conservative exchange of the TM5 glutamate to aspartate (doa10-E633D) results in complete stabilization of Ubc6 but has little if any effect on other substrates. Unexpectedly, mutating the glutamate to glutamine (doa10-E633Q) specifically accelerates Ubc6 degradation by ~5-fold. Other substrates are weakly stabilized in doa10-E633Q cells, consistent with reduced Ubc6 levels. Notably, catalytically inactive ubc6-C87A is degraded in doa10-E633Q but not wild-type cells, but an active version of Ubc6 is required in trans. Fusion of the Ubc6 TM to a soluble protein yields a protein that is degraded in a doa10-E633Q-dependent manner, whereas fusion of the C-terminal TM from an unrelated protein does not. These results suggest that the TEB4-Doa10 domain regulates Doa10 association with the Ubc6 membrane anchor, thereby controlling the degradation rate of the E2.     

Saccharomyces cerevisiae Ub-conjugating enzyme Ubc4 binds the proteasome in the presence of translationally damaged proteins

Surveillance mechanisms that monitor protein synthesis can promote rapid elimination of misfolded nascent proteins. We showed that the translation elongation factor eEF1A and the proteasome subunit Rpt1 play a central role in the translocation of nascent-damaged proteins to the proteasome. We show here that multiubiquitinated proteins, and the ubiquitin-conjugating (E2) enzyme Ubc4, are rapidly detected in the proteasome following translational damage. However, Ubc4 levels in the proteasome were reduced significantly in a strain that expressed a mutant Rpt1 subunit. Ubc4 and Ubc5 are functionally redundant E2 enzymes that represent ideal candidates for ubiquitinating damaged nascent proteins because they lack significant substrate specificity, are required for the degradation of bulk, damaged proteins, and contribute to cellular stress-tolerance mechanisms. In agreement with this hypothesis, we determined that ubc4Delta ubc5Delta is exceedingly sensitive to protein translation inhibitors. Collectively, these studies suggest a specific role for Ubc4 and Ubc5 in the degradation of cotranslationally damaged proteins that are targeted to the proteasome.     

The essential Ubc4/Ubc5 function in yeast is HECT E3-dependent, and RING E3-dependent pathways require only monoubiquitin transfer by Ubc4

The ubiquitin (Ub)-conjugating enzymes Ubc4 and Ubc5 are involved in a variety of ubiquitination pathways in yeast, including Rsp5- and anaphase-promoting complex (APC)-mediated pathways. We have found the double deletion of UBC4 and UBC5 genes in yeast to be lethal. To investigate the essential pathway disrupted by the ubc4/ubc5 deletion, several point mutations were inserted in Ubc4. The Ubc4 active site mutation C86A and the E3-binding mutations A97D and F63A were both unable to rescue the lethal phenotype, indicating that an active E3/E2～Ub complex is required for the essential function of Ubc4/Ubc5. A mutation that specifically eliminates RING E3-catalyzed isopeptide formation but not HECT E3 transthiolation (N78S-Ubc4) rescued the lethal phenotype. Thus, the essential redundant function performed by Ubc4 and Ubc5 in yeast is with a HECT-type E3, likely the only essential HECT in yeast, Rsp5. Our results also suggest that Ubc1 can weakly replace Ubc4 to transfer mono-Ub with APC, but Ubc4 cannot replace Ubc1 for poly-Ub chain extension on APC substrates. Finally, the backside Ub-binding mutant S23R-Ubc4 has no observable effect in yeast. Together, our results are consistent with a model in which Ubc4 and Ubc5 are 1) the primary E2s for Rsp5 in yeast and 2) act as monoubiquitinating E2s in RING E3-catalyzed pathways, in contrast to the processive human ortholog UbcH5.     

Zebrafish Ubc13 is required for Lys63-linked polyubiquitination and DNA damage tolerance

Ubiquitination is an important post-translational protein modification that functions in diverse cellular processes of all eukaryotic organisms. Conventional Lys48-linked poly-ubiquitination leads to the degradation of specific proteins through 26S proteasomes, while Lys63-linked polyubiquitination appears to regulate protein activities in a non-proteolytic manner. To date, Ubc13 is the only known ubiquitin-conjugating enzyme capable of poly-ubiquitinating target proteins via Lys63-linked chains, and this activity absolutely requires a Ubc variant (Uev or Mms2) as a co-factor. However, Lys63-linked poly-ubiquitination and error-free DNA damage tolerance in zebrafish are yet to be defined. Here, we report molecular cloning and functional characterization of two zebrafish ubc13 genes, ubc13a and ubc13b. Analysis of their genomic structure, nucleotide and protein sequence indicates that the two genes are highly conserved during evolution and derived from whole genome duplication. Zebrafish Ubc13 proteins are able to physically interact with yeast or human Mms2 and both zebrafish ubc13 genes are able to functionally complement the yeast ubc13 null mutant for spontaneous mutagenesis and sensitivity to DNA damaging agents. In addition, upon DNA damage, the expression of zebrafish ubc13a and ubc13b is induced during embryogenesis and zebrafish Ubc13 is associated with nuclear chromatin. These results suggest the involvement of Lys63-linked poly-ubiquitylation in DNA damage response in zebrafish.