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.     

Yeast PIAS-type Ull1/Siz1 is composed of SUMO ligase and regulatory domains

SUMO (small ubiquitin-like modifier)/Smt3 (suppressor of mif two) is a member of the ubiquitin-related protein family and is known to conjugate with many proteins. In the sumoylation pathway, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme), and E3 (SUMO ligase) functions as an adaptor between E2 and each substrate. Yeast Ull1 (ubiquitin-like protein ligase 1)/Siz1, a PIAS (protein inhibitor of activated STAT)-type SUMO ligase, modifies both cytoplasmic and nuclear proteins. In this paper, we performed a domain analysis of Ull1/Siz1 by constructing various deletion mutants. A novel conserved N-terminal domain, called PINIT, as well as the RING-like domain (SP-RING) were required for the SUMO ligase activity in the in vitro conjugation system and for interaction with Smt3 in an in vitro binding assay. The most distal N-terminal region, which contains a putative DNA-binding SAF-A/B, Acinus, and PIAS (SAP) motif, was not required for the ligase activity but was involved in nuclear localization. A strong SUMO-binding motif was identified, which interacted with Smt3 in the two-hybrid system but was not necessary for the ligase activity. The most distal C-terminal domain was important for stable localization at the bud neck region and thereby for the substrate recognition of septins. Furthermore, the C-terminal half conferred protein instability on Ull1/Siz1. Taken together, we conclude that the SP-RING and PINIT of Ull1/Siz1 are core domains of the SUMO ligase, and the other domains are regulatory for protein stability and subcellular localization.     

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.     

SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by the sumoylation of topoisomerase II

The Smt3 (SUMO) protein is conjugated to substrate proteins through a cascade of E1, E2, and E3 enzymes. In budding yeast, the E3 step in sumoylation is largely controlled by Siz1p and Siz2p. Analysis of Siz- cells shows that SUMO E3 is required for minichromosome segregation and thus has a positive role in maintaining the fidelity of mitotic transmission of genetic information. Sumoylation of the carboxy-terminus of Top2p, a known SUMO target, is mediated by Siz1p and Siz2p both in vivo and in vitro. Sumoylation in vitro reveals that Top2p is an extremely potent substrate for Smt3p conjugation and that chromatin-bound Top2p can still be sumoylated, unlike many other SUMO substrates. By combining mutations in the TOP2 sumoylation sites and the SIZ1 and SIZ2 genes we demonstrate that the minichromosome segregation defect and dicentric minichromosome stabilization, both characteristic for Smt3p-E3-deficient cells, are mediated by the lack of Top2p sumoylation in these cells. A role for Smt3p-modification as a signal for Top2p targeting to pericentromeric regions was suggested by an analysis of Top2p-Smt3p fusion. We propose a model for the positive control of the centromeric pool of Top2p, required for high segregation fidelity, by Smt3p modification.     

The yeast homologue of the microtubule-associated protein Lis1 interacts with the sumoylation machinery and a SUMO-targeted ubiquitin ligase

Microtubules and microtubule-associated proteins are fundamental for multiple cellular processes, including mitosis and intracellular motility, but the factors that control microtubule-associated proteins (MAPs) are poorly understood. Here we show that two MAPs—the CLIP-170 homologue Bik1p and the Lis1 homologue Pac1p—interact with several proteins in the sumoylation pathway. Bik1p and Pac1p interact with Smt3p, the yeast SUMO; Ubc9p, an E2; and Nfi1p, an E3. Bik1p interacts directly with SUMO in vitro, and overexpression of Smt3p and Bik1p results in its in vivo sumoylation. Modified Pac1p is observed when the SUMO protease Ulp1p is inactivated. Both ubiquitin and Smt3p copurify with Pac1p. In contrast to ubiquitination, sumoylation does not directly tag the substrate for degradation. However, SUMO-targeted ubiquitin ligases (STUbLs) can recognize a sumoylated substrate and promote its degradation via ubiquitination and the proteasome. Both Pac1p and Bik1p interact with the STUbL Nis1p-Ris1p and the protease Wss1p. Strains deleted for RIS1 or WSS1 accumulate Pac1p conjugates. This suggests a novel model in which the abundance of these MAPs may be regulated via STUbLs. Pac1p modification is also altered by Kar9p and the dynein regulator She1p. This work has implications for the regulation of dynein''s interaction with various cargoes, including its off-loading to the cortex.     

SUMO modification of septin-interacting proteins in Candida albicans

The initiation of bud and hyphal growth in the opportunistic fungal pathogen Candida albicans both involve polarized morphogenesis. However, there are many differences including the function of the septin proteins, a family of proteins involved in membrane organization in a wide range of organisms. Septins form a characteristic ring on the inner surface of the plasma membrane at the bud neck, whereas the septins are diffusely localized across emerging hyphal tips. In addition, septin rings are maintained at sites of septum formation in hyphae rather than being disassembled immediately after cytokinesis. The possibility that C. albicans septins are regulated by the small ubiquitin-like protein SUMO was examined in this study because the Saccharomyces cerevisiae septins were shown previously to be modified by SUMO (Smt3p). However, SUMO conjugation to septins was not detected during budding or hyphal morphogenesis in C. albicans. These results are supported by the lack of conserved SUMO consensus motifs between septins from the two organisms even after adjusting the predicted Cdc3p and Cdc12p septin sequences to account for mRNA splicing in C. albicans. Interestingly, a homolog of the Smt3p SUMO was identified in the C. albicans genome, and an epitope tagged version of Smt3p was conjugated to a variety of proteins. Immunofluorescence analysis showed prominent Smt3p SUMO localization at bud necks and sites of septum formation in hyphae similar to the septins. However, Smt3p was primarily detected on the mother cell side of the septin ring. A subset of these Smt3p-modified proteins co-immunoprecipitated with the septin Cdc11p. These results indicate that septin-associated proteins and not the septins themselves are the key target of SUMO modification at the bud neck in C. albicans.     

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.     

Stimulation of in vitro sumoylation by Slx5-Slx8: evidence for a functional interaction with the SUMO pathway

The yeast genes SLX5 and SLX8 were identified based on their requirement for viability in the absence of the Sgs1 DNA helicase. Loss of these genes results in genome instability, nibbled colonies, and other phenotypes associated with defects in sumoylation. The Slx5 and Slx8 proteins form a stable complex and each subunit contains a single RING-finger domain at its C-terminus. To determine the physiological function of the Slx5-8 complex, we explored its interaction with the SUMO pathway. Curing 2micro circle from the mutants, suppressed their nibbled colony phenotype and partially improved their growth rate, but did not affect their sensitivity to hydroxyurea. The increase in sumoylation observed in slx5Delta and slx8Delta mutants was found to be dependent on the Siz1 SUMO ligase. Physical interactions between the Slx5-8 complex and both Ubc9 and Smt3 were identified and characterized. Using in vitro reactions, we show that Slx5, Slx8, or the Slx5-8 complex stimulates the formation of SUMO chains and the sumoylation of a test substrate. Interestingly, a functional RING-finger domain is not required for this stimulation in vitro. These biochemical data demonstrate for the first time that the Slx5 and Slx8 complex is capable of interacting directly with the SUMO pathway.     

The spindle positioning protein Kar9p interacts with the sumoylation machinery in Saccharomyces cerevisiae

Accurate positioning of the mitotic spindle is important for the genetic material to be distributed evenly in dividing cells, but little is known about the mechanisms that regulate this process. Here we report that two microtubule-associated proteins important for spindle positioning interact with several proteins in the sumoylation pathway. By two-hybrid analysis, Kar9p and Bim1p interact with the yeast SUMO Smt3p, the E2 enzyme Ubc9p, an E3 Nfi1p, as well as Wss1p, a weak suppressor of a temperature-sensitive smt3 allele. The physical interaction between Kar9p and Ubc9p was confirmed by in vitro binding assays. A single-amino-acid substitution in Kar9p, L304P disrupted its two-hybrid interaction with proteins in the sumoylation pathway, but retained its interactions with the spindle positioning proteins Bim1p, Stu2p, Bik1p, and Myo2p. The kar9-L304P mutant showed defects in positioning the mitotic spindle, with the spindle located more distally than normal. Whereas wild-type Kar9p-3GFP normally localizes to only the bud-directed spindle pole body (SPB), Kar9p-L304P-3GFP was mislocalized to both SPBs. Using a reconstitution assay, Kar9p was sumoylated in vitro. We propose a model in which sumoylation regulates spindle positioning by restricting Kar9p to one SPB. These findings raise the possibility that sumoylation could regulate other microtubule-dependent processes.     

The RanBP2/RanGAP1*SUMO1/Ubc9 complex is a multisubunit SUMO E3 ligase

RanBP2/Nup358 is an essential protein with roles in nuclear transport and mitosis, and is one of the few known SUMO E3 ligases. However, why RanBP2 functions in vivo has been unclear: throughout the cell cycle it stably interacts with RanGAP1*SUMO1 and Ubc9, whose binding sites overlap with the E3 ligase region. Here we show that cellular RanBP2 is quantitatively associated with RanGAP1, indicating that complexed rather than free RanBP2 is the relevant E3 ligase. Biochemical reconstitution of the RanBP2/RanGAP1*SUMO1/Ubc9 complex enabled us to characterize its activity on the endogenous substrate Borealin. We find that the complex is a composite E3 ligase rather than an E2-E3 complex, and demonstrate that complex formation induces activation of a catalytic site that shows no activity in free RanBP2. Our findings provide insights into the mechanism of an important E3 ligase, and extend the concept of multisubunit E3 ligases from ubiquitin to the SUMO field.     

A SUMO-targeted ubiquitin ligase is involved in the degradation of the nuclear pool of the SUMO E3 ligase Siz1

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.     

Identification of a multifunctional binding site on Ubc9p required for Smt3p conjugation

Ubiquitin-like proteins (ub-lps) are conjugated by a conserved enzymatic pathway, involving ATP-dependent activation at the C terminus by an activating enzyme (E1) and formation of a thiolester intermediate with a conjugating enzyme (E2) prior to ligation to the target. Ubc9, the E2 for SUMO, synthesizes polymeric chains in the presence of its E1 and MgATP. To better understand conjugation of ub-lps, we have performed mutational analysis of Saccharomyces cerevisiae Ubc9p, which conjugates the SUMO family member Smt3p. We have identified Ubc9p surfaces involved in thiolester bond and Smt3p-Smt3p chain formation. The residues involved in thiolester bond formation map to a surface we show is the E1 binding site, and E2s for other ub-lps are likely to bind to their E1s at a homologous site. We also find that this same surface binds Smt3p. A mutation that impairs binding to E1 but not Smt3p impairs thiolester bond formation, suggesting that it is the E1 interaction at this site that is crucial. Interestingly, other E2s and their relatives also use this same surface for binding to ubiquitin, E3s, and other proteins, revealing this to be a multipurpose binding site and suggesting that the entire E1-E2-E3 pathway has coevolved for a given ub-lp.     

Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component of septin rings at the mother-bud neck in budding yeast.

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, we conclude that Smt3 was conjugated to Cdc3 in septin rings localized at the mother-bud neck. Around the time of cytokinesis the Cdc3-Smt3 conjugate disappeared. We discuss the biological significance of this Smt3 conjugation to a septin component.     

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.     

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.     

A global S. cerevisiae small ubiquitin‐related modifier (SUMO) system interactome

The small ubiquitin‐related modifier (SUMO) system has been implicated in a number of biological functions, yet the individual components of the SUMO machinery involved in each of these activities were largely unknown. Here we report the first global SUMO system interactome. Using affinity purification coupled with mass spectrometry, we identify >450 protein–protein interactions surrounding the SUMO E2, Siz type E3s and SUMO‐specific proteases in budding yeast. Exploiting this information‐rich resource, we validate several Siz1‐ and Siz2‐specific substrates, identify a nucleoporin required for proper Ulp1 localization, and uncover important new roles for Ubc9 and Ulp2 in the maintenance of ribosomal DNA.     

The role of karyopherins in the regulated sumoylation of septins

In the yeast Saccharomyces cerevisiae, several components of the septin ring are sumoylated during anaphase and then abruptly desumoylated at cytokinesis. We show that septin sumoylation is controlled by the interactions of two enzymes of the sumoylation pathway, Siz1p and Ulp1p, with the nuclear transport machinery. The E3 ligase Siz1p is imported into the nucleus by the karyopherin Kap95p during interphase. In M phase, Siz1p is exported from the nucleus by the karyopherin Kap142p/Msn5p and subsequently targeted to the septin ring, where it participates in septin sumoylation. We also show that the accumulation of sumoylated septins during mitosis is dependent on the interactions of the SUMO isopeptidase Ulp1p with Kap121p and Kap95p-Kap60p and the nuclear pore complex (NPC). In addition to sequestering Ulp1 at the NPC, Kap121p is required for targeting Ulp1p to the septin ring during mitosis. We present a model in which Ulp1p is maintained at the NPC during interphase and transiently interacts with the septin ring during mitosis.