Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called 'checkpoints', mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. The hus5+ gene has been cloned and sequenced. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control, the phenotypes were characterized of the hus5 mutants and the hus5 gene disruption. While the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. It is concluded that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation (Al-Khodairy, 1995).
At least one essential function of Smt3p, a Saccharomyces cerevisiae ubiquitin-like protein similar to the mammalian protein SUMO-1 (see Drosophila SUMO), involves its posttranslational covalent attachment to other proteins. Using Smt3p affinity chromatography, the second enzyme of the Smt3p conjugation pathway has been isolated and it is identical to Ubc9p, a previously identified protein that has extensive sequence similarity to the ubiquitin-conjugating enzymes (E2s) and that is required for yeast to progress through mitosis. A hallmark of E2s is the ability to form a thioester bond-containing covalent intermediate with ubiquitin (Ub). While the formation of a Ub approximately Ubc9p thioester was not detected, Ubc9p was found to form a thioester with Smt3p, indicating that Ubc9p is the functional analog of E2s in the Smt3p pathway and that this step is distinct from the ubiquitin pathway. Ubc9p is required for attachment of Smt3p to other proteins in vitro, suggesting that it is the only such enzyme in S. cerevisiae. These results suggest that, like ubiquitination, Smt3p conjugation may be a critical modification in cell cycle regulation (Johnson, 1997).
Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. This study describes the identification of the SUMO-1 homologue of fission yeast that is required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of the pmt3+ gene, the Schizosaccharomyces pombe homologue of SMT3, is not lethal, but mutant cells carrying the disrupted gene grew more slowly. The pmt3Delta cells show various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, pmt3+ is required for telomere length maintenance. Loss of Pmt3p function causes a striking increase in telomere length. When Pmt3p synthesis is restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation (Tanaka, 1999).
SMT3 of Saccharomyces cerevisiae is an essential gene encoding a ubiquitin-like protein similar to mammalian SUMO-1. When a tagged Smt3 or human SUMO-1 was expressed from GAL1 promoter, either gene rescued the lethality of the smt3 disruptant. By indirect-immunofluorescent microscopy, the HA-tagged Smt3 was detected mostly in nuclei and also at the mother-bud neck just like septin fibers. Indeed immunoprecipitation experiments revealed that Cdc3, one of septin components, was modified with Smt3. Furthermore, the protein level of the Cdc3-Smt3 conjugate was reduced and the septin rings disappeared in a ubc9-1 mutant at a restrictive temperature, where the Smt3 conjugation system should be defective. Thus, it is concluded that Smt3 is conjugated to Cdc3 in septin rings localized at the mother-bud neck. Around the time of cytokinesis, the Cdc3-Smt3 conjugate disappeared. The biological significance of this Smt3 conjugation to a septin component is discussed (Takahashi, 1999).
SUMO1/Smt3, a ubiquitin-like protein modifier, is known to be conjugated to other proteins and modulate their functions in various important processes. Similar to the ubiquitin system, SUMO1/Smt3 is activated in an ATP-dependent reaction by thioester bond formation with E1 (activating enzyme), transferred to E2 (conjugating enzyme), and passed to a substrate lysine. It remained unknown, however, whether any SUMO1/Smt3 ligases (E3s) are involved in the final transfer of this modifier. This study reports a novel factor Siz1 (YDR409w) required for septin-sumoylation of budding yeast, possibly acting as E3. Siz1 is a member of a new family (Miz1, PIAS3, etc.) containing a conserved domain with a similarity to a zinc-binding RING-domain, often found in ubiquitin ligases. In the siz1 mutant, septin-sumoylation was completely abolished. A conserved cysteine residue in the domain was essential for this conjugation. Furthermore, Siz1 is localized at the mother-bud neck in the M-phase and physically binds to both E2 and the target proteins (Takahashi, 2001).
Several studies have suggested that SUMO may participate in the regulation of heterochromatin, but direct evidence is lacking. A direct link between sumoylation and heterochromatin stability is presented in this study. SUMO deletion impairs silencing at heterochromatic regions and induces histone H3 Lys4 methylation, a hallmark of active chromatin in fission yeast. The SUMO-conjugating enzyme Hus5/Ubc9 interacts 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. Swi6, Chp2, and Clr4 themselves can be sumoylated in vivo and defective sumoylation of Swi6 or Chp2 compromises 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 (Shin, 2005).
Covalent modification with SUMO alters protein function, intracellular localization, or protein-protein interactions. Target recognition is determined, in part, by the SUMO E2 enzyme, Ubc9, while Siz/Pias E3 ligases may facilitate select interactions by acting as substrate adaptors. A yeast conditional Ubc9P(123)L mutant is viable at 36°C yet exhibits enhanced sensitivity to DNA damage. To define functional domains in Ubc9 that dictate cellular responses to genotoxic stress versus those necessary for cell viability, a 1.75-A structure of yeast Ubc9 that demonstrates considerable conservation of backbone architecture with human Ubc9 was solved. Nevertheless, differences in side chain geometry/charge guides the design of human/yeast chimeras, where swapping domains implicated in (1) binding residues within substrates that flank canonical SUMOylation sites, (2) interactions with the RanBP2 E3 ligase, and (3) binding of the heterodimeric E1 and SUMO have distinct effects on cell growth and resistance to DNA-damaging agents. These findings establish a functional interaction between N-terminal and substrate-binding domains of Ubc9 and distinguish the activities of E3 ligases Siz1 and Siz2 in regulating cellular responses to genotoxic stress (van Waardenburg, 2006).
Activation of NF-kappaB is achieved by ubiquitination and proteasome-mediated degradation of IkappaBalpha. Modified IkappaBalpha has been detected, conjugated to the small ubiquitin-like protein SUMO-1, which is resistant to signal-induced degradation. In the presence of an E1 SUMO-1-activating enzyme, Ubch9 conjugates SUMO-1 to IkappaBalpha primarily on K21, which is also utilized for ubiquitin modification. Thus, SUMO-1-modified IkappaBalpha cannot be ubiquitinated and is resistant to proteasome-mediated degradation. As a result, overexpression of SUMO-1 inhibits signal-induced activation of NF-kappaB-dependent transcription. Unlike ubiquitin modification, which requires phosphorylation of S32 and S36, SUMO-1 modification of IkappaBalpha is inhibited by phosphorylation. Thus, while ubiquitination targets proteins for rapid degradation, SUMO-1 modification acts antagonistically to generate proteins resistant to degradation (Desterro, 1998).
Using a yeast two hybrid system and pull-down assays, mouse Dac (mDac) has been demonstrated to specifically bind mouse ubiquitin-conjugating enzyme mUbc9. In contrast to a direct interaction between Drosophila Dachshund and Eyes absent, mDac interaction with mEya2 could not be detected. mDac protein is found predominantly in the nucleus but translocates to the cytoplasm and condensates along the nuclear membrane in a cell-cycle dependent manner. Deletion analysis of mDac shows the intracellular localization and protein stability correlates with the binding to mUbc9. The C-terminal half of mDac, which associates with mUbc9, remains cytoplasmic and is degraded in proteasome whereas the non-interacting N-terminus is exclusively nuclear and more stable than the full-length mDac or its C-terminal portion. In situ hybridization on whole-mount embryos or tissue sections detects mUbc9 transcripts in complementary and overlapping areas with mDac expression, particularly in the proliferation zone of the limb buds, the spinal cord and forebrain. Mouse embryos stained with an anti-mDac antibody document that mDac is localized both in the nucleus and the cytoplasm with a cytoplasmic predominance in migrating neural crest cells. In the proliferation zone, visible nuclear envelopes are not formed and mDac is detected throughout the cells (Machon, 2000).
Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis. The yeast two-hybrid screen was used to identify proteins interacting with Vsx-1. Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1 was isolated. Despite its interaction with Ubc9, Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH(2) terminus of the homeodomain. In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker. However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation (Kurtzman, 2001).
The class II deacetylase histone deacetylase 4 (HDAC4) negatively regulates the transcription factor MEF2. HDAC4 is believed to repress MEF2 transcriptional activity by binding to MEF2 and catalyzing local histone deacetylation. HDAC4 also controls MEF2 by a novel SUMO E3 ligase activity. HDAC4 interacts with the SUMO E2 conjugating enzyme Ubc9 and is itself sumoylated. The overexpression of HDAC4 leads to prominent MEF2 sumoylation in vivo, whereas recombinant HDAC4 stimulates MEF2 sumoylation in a reconstituted system in vitro. Importantly, HDAC4 promotes sumoylation on a lysine residue that is also subject to acetylation by a MEF2 coactivator, the acetyltransferase CBP, suggesting a possible interplay between acetylation and sumoylation in regulating MEF2 activity. Indeed, MEF2 acetylation is correlated with MEF2 activation and dynamically induced upon muscle cell differentiation, while sumoylation inhibits MEF2 transcriptional activity. Unexpectedly, it was found that HDAC4 does not function as a MEF2 deacetylase. Instead, the NAD+-dependent deacetylase SIRT1 can potently induce MEF2 deacetylation. These studies reveal a novel regulation of MEF2 transcriptional activity by two distinct classes of deacetylases that affect MEF2 sumoylation and acetylation (Zhao, 2005).
Sumoylation regulates the activities of several members of the ETS transcription factor family. To provide a molecular framework for understanding this regulation, the conjugation of Ets-1 with SUMO-1 was characterized. Ets-1 is modified in vivo predominantly at a consensus sumoylation motif containing Lys-15. This lysine is located within the unstructured N-terminal segment of Ets-1 preceding its PNT domain. Using NMR spectroscopy, it has been demonstrated that the Ets-1 sumoylation motif associates with the substrate binding site on the SUMO-conjugating enzyme UBC9 [K(d) approximately 400 microm] and that the PNT domain is not involved in this interaction. Ets-1 with Lys-15 mutated to an arginine still binds UBC9 with an affinity similar to the wild type protein, but is no longer sumoylated. NMR chemical shift and relaxation measurements reveal that the covalent attachment of mature SUMO-1, via its flexible C-terminal Gly-97, to Lys-15 of Ets-1 does not perturb the structure or dynamic properties of either protein. Therefore sumoylated Ets-1 behaves as 'beads-on-a-string' with the two proteins tethered by flexible polypeptide segments containing the isopeptide linkage. Accordingly, SUMO-1 may mediate interactions of Ets-1 with signaling or transcriptional regulatory macromolecules by acting as a structurally independent docking module, rather than through the induction of a conformational change in either protein upon their covalent linkage. It is also hypothesized that the flexibility of the linking polypeptide sequence may be a general feature contributing to the recognition of SUMO-modified proteins by their downstream effectors (Macauley, 2006).
Covalent modification of proteins by the small ubiquitin-related modifier SUMO regulates diverse biological functions. Sumoylation usually requires a consensus tetrapeptide, through which the binding of the SUMO-conjugating enzyme Ubc9 to the target protein is directed. However, additional specificity determinants are in many cases required. To gain insights into SUMO substrate selection, the differential sumoylation of highly similar loop structures within the DNA-binding domains of heat shock transcription factor 1 (HSF1) and HSF2 were used. Site-specific mutagenesis in combination with molecular modeling revealed that the sumoylation specificity is determined by several amino acids near the consensus site, which are likely to present the SUMO consensus motif to Ubc9. Importantly, sumoylation of the HSF2 loop impedes HSF2 DNA-binding activity, without affecting its oligomerization. Hence, SUMO modification of the HSF2 loop contributes to HSF-specific regulation of DNA binding and broadens the concept of sumoylation in the negative regulation of gene expression (Anckar, 2006)
Human p66alpha and p66beta are two potent transcriptional repressors that interact with the methyl-CpG-binding domain proteins MBD2 and MBD3. An analysis of the molecular mechanisms mediating repression resulted in the identification of two major repression domains in p66alpha and one in p66beta. Both p66alpha and p66beta are SUMO-modified in vivo: p66alpha at two sites (Lys-30 and Lys-487) and p66beta at one site (Lys-33). Expression of SUMO1 enhances the transcriptional repression activity of Gal-p66alpha and Gal-p66beta. Mutation of the SUMO modification sites or using a SUMO1 mutant or a dominant negative Ubc9 ligase results in a significant decrease of the transcriptional repression of p66alpha and p66beta. The Mi-2/NuRD components MBD3, RbAp46, RbAp48, and HDAC1 bind to both p66alpha and p66beta in vivo. Most of the interactions are not affected by the SUMO site mutations in p66alpha or p66beta, with two exceptions. HDAC1 binding to p66alpha is lost in the case of a p66alphaK30R mutant, and RbAp46 binding is reduced in the case of a p66betaK33R mutant. These results suggest that interactions within the Mi-2/NuRD complex as well as optimal repression are mediated by SUMOylation (Gong, 2006).
SOX4 is a member of SOX transcriptional factor family that is crucial for many cellular processes. In this study, a yeast two-hybrid screening of human mammary cDNA library identified human ubiquitin-conjugating enzyme 9 (hUbc9) interaction with SOX4. This interaction was confirmed by GST pull-down in vitro and co-immunoprecipitation assays in vivo. Deletion mapping demonstrated that HMG-box domain of SOX4 is required to mediate the interaction with Ubc9 in yeast. Furthermore, confocal microscopy showed that Ubc9 co-localizes with SOX4 in the nucleus. Luciferase assays found that Ubc9 specifically represses SOX4 transcriptional activity in 293T cells. Ubc9 can functionally repress the transcriptional activity of endogenous SOX4 induced by progesterone in T47D cells. The C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, does not abolish the ability to repress SOX4 activity. It shows that Ubc9 interacts with SOX4 and represses its transcriptional activity independent of its SUMO-1-conjugating activity (Pan, 2006).
KLF8 (Kruppel-like factor 8) is a member of the Kruppel transcription factor family that binds CACCC elements in DNA and activates or represses their target genes in a context-dependent manner. Sumoylation is a novel mechanism that regulates KLF8 post-translationally. KLF8 can be covalently modified by small ubiqitin-like modifier (SUMO)-1, SUMO-2, and SUMO-3 in vivo. KLF8 interacts with the PIAS family of SUMO E3 ligases PIAS1, PIASy, and PIASxalpha but not with E2 SUMO-conjugating enzyme Ubc9. Furthermore, E2 and E3 ligases enhance the sumoylation of KLF8. In addition, site-directed mutagenesis identified lysine 67 as the major sumoylation site on KLF8. Lysine 67 to arginine mutation strongly enhances activity of KLF8 as a repressor or activator to its physiological target promoters and as an inducer of the G(1) cell cycle progression. Taken together, these results demonstrate that sumoylation of KLF8 negatively regulates its transcriptional activity and cellular functions (Wei, 2006)
Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis (Kovalenko, 1996).
Ubiquitin conjugating enzymes (UBCs) comprise a family of proteins directly involved in ubiquitination of proteins. Ubiquitination is known to be involved in control of a variety of cellular processes, including cell proliferation, through the targeting of key regulatory proteins for degradation. The ubc9 gene of the yeast Saccharomyces cerevisiae (Scubc9) is an essential gene which is required for cell cycle progression and is involved in the degradation of S phase and M phase cyclins. A human homolog of Scubc9 (termed hubc9) has been identified using the two hybrid screen for proteins that interact with the human papillomavirus type 16 E1 replication protein. The hubc9 encoded protein shares a very high degree of amino acid sequence similarity with ScUBC9 and with the homologous hus5+ gene product of Schizosaccharomyces pombe. Genetic complementation experiments in a S. cerevisiae ubc9ts mutant reveal that hUBC9 can substitute for the function of ScUBC9 required for cell cycle progression (Yasugi, 1996).
The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins. p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2). Ran-binding protein 2 [p340(RanBP2)] is a nuclear pore protein, and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier, and that SUMO-1 conjugation promotes RanGAP1's interaction with RanBP2. This study shows that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts (Saitoh, 1998).
PML is a nuclear phosphoprotein that was first identified as part of a translocated chromosomal fusion product associated with acute promyelocytic leukaemia (APL). PML localises to distinct nuclear multi-protein complexes termed ND10, Kr bodies, PML nuclear bodies and PML oncogenic domains (PODs); these complexes are disrupted in APL and are the targets for immediate early viral proteins, although little is known about their function. In a yeast two-hybrid screen, a ubiquitin-like protein named PIC1 (now known as SUMO-1) was identified, that interacts and co-localises with PML in vivo. More recent studies have now shown that SUMO-1 covalently modifies a number of target proteins including PML, RanGAP1 and IkappaBalpha and is proposed to play a role in either targeting modified proteins and/or inhibiting their degradation. The precise molecular role for the SUMO-1 modification of PML is unclear, and the specific lysine residues within PML that are targeted for modification and the PML sub-domains necessary for mediating the modification in vivo are unknown. This study shows that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger domain. Using site-specific mutagenesis, the primary PML-SUMO-1 modification site was identified as being part of the nuclear localisation signal (Lys487 or Lys490). However SUMO-1 modification is not essential for PML nuclear localisation since only nuclear PML is modified. The sequence of the modification site fits into a consensus sequence for SUMO-1 modification and several other nuclear proteins have been identified that could also be targets for SUMO-1. SUMO-1 modification appears to be dependant on the correct subcellular compartmentalisation of target proteins. The APL-associated fusion protein PML-RARA is efficiently modified in vitro, resulting in a specific and SUMO-1-dependent degradation of PML-RARA. These results provide significant insight into the role of SUMO-1 modification of PML in both normal cells and the APL disease state (Duprez, 1999).
In the absence of ligands the corepressor N-CoR mediates transcriptional repression by some nuclear hormone receptors. Several protein-protein interactions of N-CoR are known; of these, mainly complex formation with histone deacetylases (HDACs) leads to the repression of target genes. In contrast, the role of posttranslational modifications in corepressor function is not well established. This study shows that N-CoR is modified by Sumo-1. SUMO-E2-conjugating enzyme Ubc9 and SUMO-E3 ligase Pias1 are novel N-CoR interaction partners. The SANT1 domain of N-CoR mediates this interaction. K152, K1117, and K1330 of N-CoR can be conjugated to SUMO and mutation of all sites is necessary to fully block SUMOylation in vitro. Because these lysine residues are located within repression domains I and III, respectively, a possible correlation between the functions of the repression domains and SUMOylation was investigated. Coexpression of Ubc9 protein results in enhanced N-CoR-dependent transcriptional repression. Studies using SUMOylation-deficient N-CoR RDI mutants suggest that SUMO modification contributes to repression by N-CoR. Mutation of K152 to R in RD1, for example, not only significantly reduce repression of a reporter gene, but also abolish the effect of Ubc9 on transcriptional repression (Tiefenbach, 2006).
Human DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, it was shown that a poly-SUMO1 chain is assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate accumulates to a high level when the sumoylation assay is performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation (Yang, 2006).
Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, a conditional UBC9 mutant of chicken DT40 cells was constructed containing the UBC9 transgene under control of a tetracycline-repressible promoter and their loss of function phenotypes were characterized. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, no accumulation was observed of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9, but an increase was observed in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment (Hayashi, 2002).
Covalent modification by SUMO regulates a wide range of cellular processes, including transcription, cell cycle, and chromatin dynamics. To address the biological function of the SUMO pathway in mammals, mice were generated deficient for the SUMO E2-conjugating enzyme Ubc9. Ubc9-deficient embryos die at the early postimplantation stage. In culture, Ubc9 mutant blastocysts are viable, but fail to expand after 2 days and show apoptosis of the inner cell mass. Loss of Ubc9 leads to major chromosome condensation and segregation defects. Ubc9-deficient cells also show severe defects in nuclear organization, including nuclear envelope dysmorphy and disruption of nucleoli and PML nuclear bodies. Moreover, RanGAP1 fails to accumulate at the nuclear pore complex in mutant cells that show a collapse in Ran distribution. Together, these findings reveal a major role for Ubc9, and, by implication, for the SUMO pathway, in nuclear architecture and function, chromosome segregation, and embryonic viability in mammals (Nacerddine, 2005).
In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. The two-hybrid system in the yeast Saccharomyces cerevisiae was used to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II (Tarsounas, 1997).
During meiotic prophase, homologous chromosomes engage in a complex series of interactions that ensure their proper segregation at meiosis I. A central player in these interactions is the synaptonemal complex (SC), a proteinaceous structure elaborated along the lengths of paired homologs. In mutants that fail to make SC, crossing over is decreased, and chromosomes frequently fail to recombine; consequently, many meiotic products are inviable because of aneuploidy. This study investigated the role of the small ubiquitin-like protein modifier (SUMO) in SC formation during meiosis in budding yeast. SUMO localizes specifically to synapsed regions of meiotic chromosomes and this localization depends on Zip1, a major building block of the SC. A non-null allele of the UBC9 gene, which encodes the SUMO-conjugating enzyme, impairs Zip1 polymerization along chromosomes. The Ubc9 protein localizes to meiotic chromosomes, coincident with SUMO staining. In the zip1 mutant, SUMO localizes to discrete foci on chromosomes. These foci coincide with axial associations, where proteins involved in synapsis initiation are located. These data suggest a model in which SUMO modification of chromosomal proteins promotes polymerization of Zip1 along chromosomes. The ubc9 mutant phenotype provides the first evidence for a cause-and-effect relationship between sumoylation and synapsis (Hooker, 2006).
Posttranslational modification with small ubiquitin-related modifier (SUMO) has emerged as a central regulatory mechanism of protein function. However, little is known about the regulation of sumoylation itself. It has been reported that it is increased after exposure to various stresses including strong oxidative stress. Conversely, ROS (reactive oxygen species), at low concentrations, result in the rapid disappearance of most SUMO conjugates, including those of key transcription factors. This is due to direct and reversible inhibition of SUMO conjugating enzymes through the formation of (a) disulfide bond(s) involving the catalytic cysteines of the SUMO E1 subunit Uba2 and the E2-conjugating enzyme Ubc9. The same phenomenon is also observed in a physiological scenario of endogenous ROS production, the respiratory burst in macrophages. Thus, these findings add SUMO conjugating enzymes to the small list of specific direct effectors of H2O2 and implicate ROS as key regulators of the sumoylation-desumoylation equilibrium (Bossis, 2006).
Posttranslational modifications mediated by ubiquitin-like proteins have been implicated in regulating a variety of cellular pathways. Although small ubiquitin-like modifier (SUMO) is a new member of this family, it has caught a great deal of attention recently because of its novel and distinguished functions. Sumoylation is a multiple-step process, involving maturation, activation, conjugation and ligation. Ubc9 is an E2 conjugating enzyme essential for sumoylation. Suppression of sumoylation by a dominant negative Ubc9 mutant (Ubc9-DN) in the estrogen receptor (ER) positive MCF-7 cells is associated with alterations of tumor cell's response to anticancer drugs as well as tumor growth in a xenograft mouse carcinoma model. To dissect the underlying mechanism of Ubc9-associated alterations of drug responsiveness and tumor growth, gene expression for the cells expressing wild type Ubc9 (Ubc9-WT) and Ubc9-DN was profiled. Several tumorigenesis-related genes are downregulated in the Ubc9-DN cells. Within this group, over 10 genes are known to be regulated by ER. Experiments using the estrogen response element fused to the luciferase reporter showed that the basal level of luciferase activity is significantly reduced in the Ubc9-DN cells when compared to the vector alone or the Ubc9-WT cells. Furthermore, both the stability and the subcellular localization of steroid hormone receptor coactivator-1 (SRC-1) are altered in the Ubc9-DN cells. Together, these results suggest that Ubc9 might regulate bcl-2 expression through the ER signaling pathway, which ultimately contributes to the alterations of drug responsiveness and tumor growth (Lu, 2006)
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