Structure and analysis of a complex between SUMO and Ubc9 illustrates features of a conserved E2-Ubl interaction

The SUMO E2 Ubc9 serves as a lynchpin in the SUMO conjugation pathway, interacting with the SUMO E1 during activation, with thioester linked SUMO after E1 transfer and with the substrate and SUMO E3 ligases during conjugation. Here, we describe the structure determination of a non-covalent complex between human Ubc9 and SUMO-1 at 2.4 A resolution. Non-covalent interactions between Ubc9 and SUMO are conserved in human and yeast insomuch as human Ubc9 interacts with each of the human SUMO isoforms, and yeast Ubc9 interacts with Smt3, the yeast SUMO ortholog. Structural comparisons reveal similarities to several other non-covalent complexes in the ubiquitin pathway, suggesting that the non-covalent Ubc9-SUMO interface may be important for poly-SUMO chain formation, for E2 recruitment to SUMO conjugated substrates, or for mediating E2 interactions with either E1 or E3 ligases. Biochemical analysis suggests that this surface is less important for E1 activation or di-SUMO-2 formation, but more important for E3 interactions and for poly-SUMO chain formation when the chain exceeds more than two SUMO proteins.     

Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1

E2 enzymes catalyze attachment of ubiquitin and ubiquitin-like proteins to lysine residues directly or through E3-mediated reactions. The small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes and is essential for cell-cycle progression in yeast. In contrast to most ubiquitin conjugation, the SUMO E2 enzyme Ubc9 is sufficient for substrate recognition and lysine modification of known SUMO targets. Crystallographic analysis of a complex between mammalian Ubc9 and a C-terminal domain of RanGAP1 at 2.5 A reveals structural determinants for recognition of consensus SUMO modification sequences found within SUMO-conjugated proteins. Structure-based mutagenesis and biochemical analysis of Ubc9 and RanGAP1 reveal distinct motifs required for substrate binding and SUMO modification of p53, IkappaBalpha, and RanGAP1.     

Distinct functional domains of Ubc9 dictate cell survival and resistance to genotoxic stress

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 was viable at 36 degrees C yet exhibited 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 demonstrated considerable conservation of backbone architecture with human Ubc9 was solved. Nevertheless, differences in side chain geometry/charge guided the design of human/yeast chimeras, where swapping domains implicated in (i) binding residues within substrates that flank canonical SUMOylation sites, (ii) interactions with the RanBP2 E3 ligase, and (iii) binding of the heterodimeric E1 and SUMO had distinct effects on cell growth and resistance to DNA-damaging agents. Our 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.     

Role of an N-terminal site of Ubc9 in SUMO-1, -2, and -3 binding and conjugation

Covalent posttranslational modification of target proteins with ubiquitin and ubiquitin-like proteins regulates many important cellular processes. However, the molecular mechanisms by which these proteins are activated and conjugated to substrates has yet to be fully understood. NMR studies have shown that the ubiquitin-like proteins SUMO-1, -2, and -3 interact with the same N-terminal region of the E2 conjugating enzyme Ubc9 with similar affinities. This is correlated to their almost identical utilization by Ubc9 in the SUMO conjugation pathway. To investigate the functional significance of this interaction, site-directed mutagenesis was used to alter residues in the SUMO binding surface of Ubc9, and the effect of the amino acid substitutions on binding and conjugation to SUMO-1 and target protein RanGAP1 was investigated by isothermal titration calorimetry and biochemical analysis. R13A/K14A and R17A/K18A mutations in Ubc9 disrupted the interaction with SUMO-1 but did not completely abolish the interaction with E1. While these Ubc9 mutants displayed a significantly reduced efficiency in the transfer of SUMO-1 from E1 to E2, their ability to recognize substrate and transfer SUMO-1 from E2 to the target protein was unaffected. These results suggest that the noncovalent binding site of SUMO-1 on Ubc9, although distant from the active site, is important for the transfer of SUMO-1 from the E1 to the E2. The conservation of E2 enzymes across the ubiquitin and ubiquitin-like protein pathways indicates that analogous N-terminal sites of E2 enzymes are likely to have similar roles in general.     

Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection

The conjugation of small ubiquitin-like modifiers SUMO-1, SUMO-2 and SUMO-3 onto target proteins requires the concerted action of the specific E1-activating enzyme SAE1/SAE2, the E2-conjugating enzyme Ubc9, and an E3-like SUMO ligase. NMR chemical shift perturbation was used to identify the surface of Ubc9 that interacts with the SUMO ligase RanBP2. Unlike known ubiquitin E2-E3 interactions, RanBP2 binds to the beta-sheet of Ubc9. Mutational disruption of Ubc9-RanBP2 binding affected SUMO-2 but not SUMO-1 conjugation to Sp100 and to a newly identified RanBP2 substrate, PML. RanBP2 contains a binding site specific for SUMO-1 but not SUMO-2, indicating that a Ubc9-SUMO-1 thioester could be recruited to RanBP2 via SUMO-1 in the absence of strong binding between Ubc9 and RanBP2. Thus we show that E2-E3 interactions are not conserved across the ubiquitin-like protein superfamily and identify a RanBP2-dependent mechanism for SUMO paralog-specific conjugation.     

UBC9 autosumoylation negatively regulates sumoylation of septins in Saccharomyces cerevisiae

Sumoylation regulates a wide range of cellular processes. However, little is known about the regulation of the SUMO machinery. In this study, we demonstrate that two lysine residues (Lys-153 and Lys-157) in the C-terminal region of the yeast E2-conjugating enzyme Ubc9 are the major and minor autosumoylation sites, respectively. Surprisingly, mutation of Lys-157 (ubc9(K157R)) significantly stimulates the level of Ubc9 autosumoylation at Lys-153. The functional role of Ubc9 autosumoylation is exemplified in our findings that cell cycle-dependent sumoylation of cytoskeletal septin proteins is inversely correlated with the Ubc9 autosumoylation level and that mutation of the Ubc9 autosumoylation sites results in aberrant cell morphology. Our study elucidates a regulatory mechanism that utilizes automodification of the E2 enzyme of the sumoylation machinery to control substrate sumoylation.     

DNA repair and global sumoylation are regulated by distinct Ubc9 noncovalent complexes

Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that differential recruitment of Ubc9 by SUMO or Rad60 provides a novel means for such selectivity. Indeed, deconvoluting Ubc9 function by disrupting either the Ubc9:SLD2 or Ubc9:SUMO noncovalent complex reveals distinct roles in facilitating sumoylation. Ubc9:SLD2 acts in the Nse2 SUMO E3 ligase-dependent pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation and chain formation, via the Pli1 E3 SUMO ligase. Moreover, this Pli1-dependent SUMO chain formation causes the genome instability phenotypes of SUMO-targeted ubiquitin ligase (STUbL) mutants. Overall, we determine that, unexpectedly, Ubc9 noncovalent partner choice dictates the role of sumoylation in distinct cellular pathways.     

Ubiquitin-dependent proteolytic control of SUMO conjugates

Posttranslational protein modification with small ubiquitin-related modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.     

Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation

The ubiquitin-related modifier SUMO regulates a wide range of cellular processes by post-translational modification with one, or a chain of SUMO molecules. Sumoylation is achieved by the sequential action of several enzymes in which the E2, Ubc9, transfers SUMO from the E1 to the target mostly with the help of an E3 enzyme. In this process, Ubc9 not only forms a thioester bond with SUMO, but also interacts with SUMO noncovalently. Here, we show that this noncovalent interaction promotes the formation of short SUMO chains on targets such as Sp100 and HDAC4. We present a crystal structure of the noncovalent Ubc9-SUMO1 complex, showing that SUMO is located far from the E2 active site and resembles the noncovalent interaction site for ubiquitin on UbcH5c and Mms2. Structural comparison suggests a model for poly-sumoylation involving a mechanism analogous to Mms2-Ubc13-mediated ubiquitin chain formation.     

Enhanced SUMOylation of proteins containing a SUMO-interacting motif by SUMO-Ubc9 fusion

Identifying new targets for SUMO and understanding the function of protein SUMOylation are largely limited by low level of SUMOylation. It was found recently that Ubc9, the SUMO E2 conjugating enzyme, is covalently modified by SUMO at a lysine 14 in the N-terminal alpha helix, and that SUMO-modified Ubc9 has enhanced conjugation activity for certain target proteins containing a SUMO-interacting motif (SIM). Here, we show that, compared to intact Ubc9, the SUMO-Ubc9 fusion protein has higher conjugating activity for SIM-containing targets such as Sp100 and human cytomegalovirus IE2. Assays using an IE2 SIM mutant revealed the requirement of SIM for the enhanced IE2 SUMOylation by SUMO-Ubc9. In pull-down assays with cell extracts, the SUMO-Ubc9 fusion protein bound to more diverse cellular proteins and interacted with some SIM-containing proteins with higher affinities than Ubc9. Therefore, the devised SUMO-Ubc9 fusion will be useful for identifying SIM-containing SUMO targets and producing SUMO-modified proteins.     

Role of SUMO/Ubc9 in DNA Damage Repair and Tumorigenesis

DNA damage repair is an important cell function for genome integrity and its deregulation can lead to genomic instability and development of malignancies. Sumoylation is an increasingly important ubiquitin-like modification of proteins affecting protein stability, enzymatic activity, nucleocytoplasmic trafficking, and protein-protein interactions. In particular, several important DNA repair enzymes are subject to sumoylation, which appears to play a role in copping with DNA damage insults. Recent reports indicate that Ubc9, the single SUMO E2 enzyme catalyzing the conjugation of SUMO to target proteins, is overexpressed in certain tumors, such as lung adenocarcinoma, ovarian carcinoma and melanoma, suggestive of its clinic significance. This review summarizes the most important DNA damage repair pathways which are potentially affected by Ubc9/SUMO and their role in regulating the function of several proteins involved in the DNA damage repair machinery.     

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.     

In Vitro Studies Reveal a Sequential Mode of Chain Processing by the Yeast SUMO (Small Ubiquitin-related Modifier)-specific Protease Ulp2

Sumoylation is a post-translational modification essential in most eukaryotes that regulates stability, localization, activity, or interaction of a multitude of proteins. It is a reversible process wherein counteracting ligases and proteases, respectively, mediate the conjugation and deconjugation of SUMO molecules to/from target proteins. Apart from attachment of single SUMO moieties to targets, formation of poly-SUMO chains occurs by the attachment of additional SUMO molecules to lysine residues in the N-terminal extensions of SUMO. In Saccharomyces cerevisiae there are apparently only two SUMO(Smt3)-specific proteases: Ulp1 and Ulp2. Ulp2 has been shown to be important for the control of poly-SUMO conjugates in cells and to dismantle SUMO chains in vitro, but the mechanism by which it acts remains to be elucidated. Applying an in vitro approach, we found that Ulp2 acts sequentially rather than stochastically, processing substrate-linked poly-SUMO chains from their distal ends down to two linked SUMO moieties. Furthermore, three linked SUMO units turned out to be the minimum length of a substrate-linked chain required for efficient binding to and processing by Ulp2. Our data suggest that Ulp2 disassembles SUMO chains by removing one SUMO moiety at a time from their ends (exo mechanism). Apparently, Ulp2 recognizes surfaces at or near the N terminus of the distal SUMO moiety, as attachments to this end significantly reduce cleavage efficiency. Our studies suggest that Ulp2 controls the dynamic range of SUMO chain lengths by trimming them from the distal ends.     

Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as an adaptor between conjugating enzyme and substrates

SUMO1/Smt3, a ubiquitin-like protein modifier, is known to conjugate to other proteins and modulate their functions in various important processes. Similar to the ubiquitin conjugation system, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme). In our previous report (Takahashi, Y., Toh-e, A., and Kikuchi, Y. (2001) Gene 275, 223-231), we showed that Siz1/Ull1 (YDR409w) of budding yeast, a member of the human PIAS family containing a RING-like domain, is a strong candidate for SUMO1/Smt3 ligase because the SUMO1/Smt3 modification of septin components was abolished in the ull1 mutant and Ull1 associated with E2 (Ubc9) and the substrates (septin components) in immunoprecipitation experiments. Here we have developed an in vitro Smt3 conjugation system for a septin component (Cdc3) using purified recombinant proteins. In this system, Ull1 is additionally required as well as E1 (Sua1.Uba2 complex), E2 (Ubc9), and ATP. A cysteine residue of the RING-like domain was essential for the conjugation both in vivo and in vitro. Furthermore, a region containing the RING-like domain directly interacted with Ubc9 and Cdc3. Thus, this SUMO/Smt3 ligase functions as an adaptor between E2 and the target proteins.     

Thimet oligopeptidase cleaves the full-length Alzheimer amyloid precursor protein at a beta-secretase cleavage site in COS cells.

We developed an assay method using a novel quenched fluorescent substrate (QFS) flanking the beta-cleavage site of amyloid precursor protein (APP), and purified a candidate beta-secretase from bovine brain. N-terminal amino acid analysis showed the candidate to be thimet oligopeptidase (TOP). The cDNA for human TOP was cloned from a human brain cDNA library and expressed in COS cells. The enzyme was further purified on a Ni2+-agarose column. TOP cleaved the Swedish Alzheimer's substrate (SEVNLDAEFR) as well as the normal substrate (SEVKMDAEFR). We then coexpressed TOP with APP695 in COS cells, collected transfected cells and conditioned media, and analyzed them by immunoblotting. The antibody against the specific secreted APP cleaved by beta-secretase (sAPPbeta) detected the secretion of sAPPbeta only from APP/hTOP-overexpressing cells, and not from cells overexpressing of antisense hTOP cDNA. Finally, we analyzed the immunolocalization of overexpressed hTOP in COS cells. Most hTOP was localized in the nuclei, but a small amount was localized in the Golgi or other organelles around the nuclei. These results suggest that TOP has a beta-secretase-like activity responsible for the processing of APP.     

Topoisomerase I-dependent viability loss in saccharomyces cerevisiae mutants defective in both SUMO conjugation and DNA repair

Siz1 and Siz2/Nfi1 are the two Siz/PIAS SUMO E3 ligases in Saccharomyces cerevisiae. Here we show that siz1Delta siz2Delta mutants fail to grow in the absence of the homologous recombination pathway or the Fen1 ortholog RAD27. Remarkably, the growth defects of mutants such as siz1Delta siz2Delta rad52Delta are suppressed by mutations in TOP1, suggesting that these growth defects are caused by topoisomerase I activity. Other mutants that affect SUMO conjugation, including a ulp1 mutant and the nuclear pore mutants nup60Delta and nup133Delta, show similar top1-suppressible synthetic defects with DNA repair mutants, suggesting that these phenotypes also result from reduced SUMO conjugation. siz1Delta siz2Delta mutants also display TOP1-independent genome instability phenotypes, including increased mitotic recombination and elongated telomeres. We also show that SUMO conjugation, TOP1, and RAD27 have overlapping roles in telomere maintenance. Top1 is sumoylated, but Top1 does not appear to be the SUMO substrate involved in the synthetic growth defects. However, sumoylation of certain substrates, including Top1 itself and Tri1 (YMR233W), is enhanced in the absence of Top1 activity. Sumoylation is also required for growth of top1Delta cells. These results suggest that the SUMO pathway has a complex effect on genome stability that involves several mechanistically distinct processes.     

Perturbation of SUMOlation enzyme Ubc9 by distinct domain within nucleoporin RanBP2/Nup358.

Ubc9, a conjugation enzyme for the ubiquitin-related modifier SUMO, is present predominantly in the nucleus and at the nuclear pore complex. The functional significance of its subcellular compartmentalization, however, remains to be elucidated. Here, we define a Pro-Glu-Asp-Ser-Thr-rich element containing 129 amino acid residues, designated IR1+2, on the human nucleoporin RanBP2/Nup358, which binds directly to Ubc9 with high affinity both in vitro and in vivo. When IR1+2 tagged with green fluorescence protein at its amino terminus (GFP-IR1+2) was transfected into COS-7 cells, we found that approximately 90% of the nuclear Ubc9 was sequestered in the cytoplasm. We also observed that both SUMO-1 and SUMO-2/3 were mislocalized, and promyelocytic leukemia protein PML formed an enlarged aggregate in the nucleus. Moreover, the homologous recombination protein Rad51 mislocalized to the cytoplasm, and Rad51 foci, a hallmark of functional association of Rad51 with damaged DNA, did not form efficiently even in the presence of a DNA strand breaker. These findings emphasize that the IR1+2 domain is a useful tool for manipulating the nuclear localization of Ubc9 and perturbing the subcellular localization of SUMOs and/or SUMOlated proteins, and they emphasize the important role of nuclear Ubc9 in the Rad51-mediated homologous recombination pathway, possibly by modulating intracellular trafficking of Rad51.