Copyright � 2011 by the Genetics Society of AmericaDOI: 10.1534/genetics.110.124347

Genetic Evidence That Polysumoylation Bypasses the Needfor a SUMO-Targeted Ub Ligase

Janet R. Mullen, Mukund Das and Steven J. Brill1

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854

Manuscript received July 19, 2010Accepted for publication November 1, 2010

ABSTRACT

Saccharomyces cerevisiae cells lacking the Slx5–Slx8 SUMO-targeted Ub ligase display increased levels ofsumoylated 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 insgs1D slx5D cells. To address this possibility, we isolated a second-site suppressor of sgs1D slx5D syntheticlethality and identified it as an allele of the ULP2 SUMO isopeptidase. The suppressor, ulp2–D623H,behaved like the ulp2D 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 sgs1D

slx5D synthetic lethality and the slx5D sporulation defect. Further, ulp2D’s growth sensitivities were found tobe suppressed in ulp2D slx5D double mutants. This mutual suppression indicates that SLX5–SLX8 and ULP2interact antagonistically. However, the suppressed strain sgs1D slx5D ulp2–D623H displayed even higherlevels of sumoylated proteins than the corresponding double mutants. Thus, sgs1D slx5D synthetic lethalitycannot be due simply to high levels of bulk sumoylated proteins. We speculate that the loss of ULP2suppresses the toxicity of the sumoylated proteins that accumulate in slx5D–slx8D cells by permitting theextension of poly-SUMO chains on specific target proteins. This additional modification might attenuatethe activity of the target proteins or channel them into alternative pathways for proteolytic degradation. Insupport of this latter possibility we find that the WSS1 isopeptidase is required for suppression by ulp2D.

UBIQUITIN (Ub) and the small ubiquitin-relatedmodifier (SUMO) are conjugated to target

proteins post-translationally where they perform func-tions that are essential for cell viability (Kerscher et al.2006). Chief among these functions is the role of Ub indirecting the proteasomal degradation of target pro-teins bearing a chain of K48-linked Ub moieties(Ciechanover and Schwartz 1998; Pickart andFushman 2004; Ravid and Hochstrasser 2008).Although SUMO regulates a wide variety of cellularprocesses, its functions are typically dependent on theligation of single SUMO moieties to target proteins(Johnson 2004). An additional distinction between Uband SUMO is that sumoylation is not known to directproteins to the proteasome. However, the recent iden-tification of a class of proteins termed SUMO-targetedUb ligases (STUbLs) has revealed that sumoylation canlead indirectly to the proteolysis of sumoylated proteins(Perry et al. 2008; Denuc and Marfany 2010). Theability of STUbLs to ubiquitinate sumoylated proteinsraises the question of specificity. That is, how doSTUbLs distinguish between hundreds of sumoylatedproteins and identify those destined for destruction?

One possibility is that specificity is conferred by dif-ferences in the SUMO modification itself.

Modification by SUMO, or Smt3 in Saccharomycescerevisiae, involves the formation of an isopeptide bondbetween the C terminus of a mature SUMO moiety andthe e-amino group of lysine side chains present intarget proteins ( Johnson 2004). This multistep pro-cess requires an ATP-dependent E1 activating enzyme(Aos1/Uba2), an E2 conjugating enzyme (Ubc9), andone of several SUMO E3 ligases. Sumoylation normallytakes place at lysine residues that fall within theconsensus sequence CKXE/D, where C is a hydropho-bic residue. Although single SUMO moieties arenormally conjugated to target proteins, poly-SUMOchains are observed in in vitro reactions and are knownto arise in vivo under certain circumstances (Tatham

et al. 2001; Bylebyl et al. 2003; Li et al. 2003; Fu et al.2005).

Equally important to the function of SUMO modifi-cation is the process of desumoylation. In budding yeastthis is carried out by the SUMO-specific proteases Ulp1and Ulp2/Smt4 (Li and Hochstrasser 1999, 2000;Strunnikov et al. 2001). Analogous activities are pro-vided by the sentrin-specific proteases (SENPs) 1–4 and6 and 7 in mammals (Mukhopadhyay and Dasso

2007). Ulp1 is essential for viability due to its uniquerole in processing Smt3(Y101) into its mature form

1Corresponding author: 679 Hoes Lane, CABM, Rutgers University,Piscataway, NJ 088554. E-mail: brill@cabm.rutgers.edu

Genetics 187: 73–87 ( January 2011)

Smt3(G98) (Li and Hochstrasser 1999, 2003). How-ever, Ulp1 must also play a role in desumolyatingsubstrate proteins, since ulp1D cells are sick even whenprovided with Smt3(G98) (Li and Hochstrasser 1999;Xie et al. 2007). Ulp1 is localized to the nuclear porecomplex although structure/function and cytoplasmictethering experiments suggest that it plays an importantrole in desumoylating cytoplasmic proteins (Li andHochstrasser 2003; Panse et al. 2003).

The Ulp2 isopeptidase is dispensable for viability andon the basis of its nucleoplasmic localization and itsmutant phenotypes, Ulp2 may act predominantly on nu-clear proteins (Li and Hochstrasser 2000; Strunnikov

et al. 2001). Cells lacking ULP2 display heat-sensitivegrowth, a nibbled colony phenotype due to a 2-mm circleoverreplication, a severe sporulation defect, and sensi-tivity to DNA damage resulting from treatment withmethyl methanesulfonate (MMS) or hydroxyurea (HU)(Li and Hochstrasser 2000; Schwienhorst et al.2000; Strunnikov et al. 2001; Bachant et al. 2002;Bylebyl et al. 2003; Chen et al. 2005; Dobson et al. 2005;Xiong et al. 2009). Characterization of this DNAdamage sensitivity revealed a unique role for Ulp2 inresuming growth following checkpoint arrest at mitosis(Schwartz et al. 2007). Ulp2 is a member of the‘‘editing’’ class of SUMO isopeptidases that is character-ized by a preference for cleaving poly-SUMO chains. Invitro assays demonstrate that Ulp2 and its closest humanhomolog SENP6 are more active on poly-SUMO chainsthan monosumoylated substrates (Li and Hochstrasser

2000; Bylebyl et al. 2003; Mukhopadhyay et al. 2006;Lima and Reverter 2008). Further, as expected for anenzyme that reduces the lengths of poly-SUMO chains,ulp2D cells accumulate poly-SUMO conjugates. SUMOcontains multiple lysine residues that can potentiallyserve to interlink SUMO moieties; however, poly-SUMOchains form primarily through the K11 residue ofmammalian SUMO-2/3 and the three N-terminal lysineresidues (K11, K15, and K19) of yeast Smt3 (Tatham

et al. 2001; Bylebyl et al. 2003). These polymers havebeen shown to be responsible for some of ulp2D’sphenotypes since replacement of Smt3’s 3 N-terminallysine residues with nonconjugable arginine residuessuppresses many of the above defects associated withulp2D cells (Bylebyl et al. 2003). These results supportthe idea that poly-SUMO chain formation has a bi-ological function that is carefully regulated. On theother hand, replacement of all nine of Smt3’s lysineresidues with arginine (smt3–allR) results in a slow-growth phenotype, not lethality. So although poly-SUMO chains may be imporant for robust growth, theyare not essential for viability in yeast. Finally, buddingyeast contains a third SUMO protease known as Wss1.Wss1 is a metalloprotease that deconjugates poly-SUMOchains but is also capable of deconjugating a Ub–SUMOisopeptide conjugate in vitro (Mullen et al. 2010).These and other results have led to the proposal that

Wss1 plays a specific role in removing SUMO andUb moieties from proteins undergoing proteasomaldegradation.

SLX5 and SLX8 encode subunits of a SUMO-targetedUb ligase, which is important for genome stability. Bothgenes are essential for viability in the absence of theSGS1 DNA helicase, and cells lacking SLX5 or SLX8display genomic instability in the form of elevatedrates of mitotic recombination and gross chromosomalrearrangements (Mullen et al. 2001; Zhang et al. 2006;Burgess et al. 2007). Interestingly, these mutants displaysome of the same phenotypes observed in ulp2D

mutants. These include a nibbled-colony morphology,which is dependent on both a 2-mm circle and theRAD51-independent recombination pathway, reducedsporulation frequency, and sensitivity to HU (Mullen

et al. 2001; Burgess et al. 2007). Unlike ulp2D, thesemutants are not sensitive to continuous exposure toMMS (Mullen et al. 2001). Importantly, slx5D and slx8D

cells, like their corresponding mutants in Schizosacchar-omyces pombe, display an increase in poly-SUMO chains,which is reminiscent of that observed in the absence ofULP2 (Wang et al. 2006; Ii et al. 2007; Kosoy et al. 2007;Prudden et al. 2007; Sun et al. 2007; Uzunova et al.2007). In this case, however, SUMO chains accumulatedue to a decrease in proteasomal-dependent degrada-tion as opposed to the loss of the SUMO editingfunction of Ulp2.

Previous studies have indicated that STUbLs such asyeast Slx5–Slx8 and human RNF4 prefer to ubiquitinatetarget proteins carrying poly-SUMO chains (Uzunova

et al. 2007; Mullen and Brill 2008; Tatham et al. 2008).In the best-characterized case, RNF4 was found to berequired for the proteasomal destruction of polysumoy-lated PML protein (Lallemand-Breitenbach et al.2008; Tatham et al. 2008). RNF4 polyubiquitinated PMLprotein carrying polymers of SUMO-2/3 and the Ub wasfound to be conjugated directly to the PML protein aswell as to the poly-SUMO chain (Tatham et al. 2008). Invitro experiments in the yeast system revealed a strongpreference for Slx5–Slx8 to ubiquitinate the N terminusof a poly-SUMO chain (Mullen and Brill 2008).Under the conditions of this in vitro reaction only oneto a few Ubs were added to the chain and the biologicalsignificance of this ubiquitination is unknown. Thus,poly-SUMO chains may provide the specificity neededby these STUbLs to identify sumoylated proteins thatare destined for degradation. This specificity wouldalso explain the accumulation of poly-SUMO chainsin slx5D–slx8D mutants. However, the Slx5–Slx8 Ubligase has been implicated in the degradation ofmonosumoylated Mot1-301 mutant protein and inthe turnover of the Mata2 protein, which lackssumoylation altogether (Wang et al. 2006; Wang andPrelich 2009; Xie et al. 2010). Thus, the Slx5–Slx8 Ubligase may use multiple mechanisms to choose itstarget. Further, these results raise the possibility that

74 J. R. Mullen, M. Das and S. J. Brill

TABLE 1

Strains used in this study

Strain Genotype Reference/source

JMY1814 MATa ade2-1 ura3 his3 trp1-1 leu2-3,112 ulp2-1THIS3 lys2-801 can1-100 This studyJMY1885 MATa ade2-1 ura3 his3 leu2-3,112TLEU2Tubc9-1(ts) trp1-1 ulp2-1THIS3 ubc9DTTRP1 This studyJMY1859 MATa ade2-1 ura3 his3 trp1-1 leu2-3,112 ulp2-1THIS3 slx5-11THGR can1-100 This studyJMY1921 MATa ade2 ade3ThisG ura3 his3-11,15 trp1-1 leu2 lys2 slx5-10TTRP1 ulp2-1THIS3 can1-100 This studyJMY1922 MATa ade2 ade3ThisG ura3 his3-11,15 trp1-1 leu2 slx5-10TTRP1 ulp2-1THIS3 sgs1-20THGR

can1-100 1 pJM500 (SGS1/ADE3/URA3/CEN)This study

NJY2430 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 slx5DTNAT This studyNJY2444 MATa ade2-1 ade3ThisG his3-11,15 ura3-1 leu2-3,112 trp1-1 smt3DTKANTloxP sgs1-20THGR

plus pNJ7234 [SMT3(G98)/URA3/ADE3/CEN]This study

NJY2460 MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 slx5DTNAT This studyNJY2462 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 sgs1-20THGR slx5DTNAT plus

pJM500 (SGS1/ADE3/URA3)This study

JMY2470 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 This studyJMY2472 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 leu2-3,112 lys2 can1-100 This studyJFY2481 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 sgs1-20THGR slx5DTNAT

plus pJM500This study

JFY2488 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 smt3DTloxP plus pNJ7234[GPDpSMT3(G98)/URA3/ADE3/CEN]

This study

NJY2524 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 smt3DTKANTloxP slx5DTNATplus pNJ7234 [SMT3(G98)/URA3/ADE3/CEN]

This study

NJY2545 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 leu2-3,112 can1-100 slx5DTNAT This studyNJY2547 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 leu2-3,112 lys2 can1-100 slx5DTNAT This studyJMY2561 MATa ade2-1 ura3-1 his3-11,15 leu2-3,112 lys2 trp1-1 slx5-10TTRP1 can1-100 This studyNJY2568 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 sgs1-20THGR This studyNJY2569 MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 sgs1-20THGR This studyMJY2613 MATa ade2-1 ura3 his3 trp1-1 slx5-10TTRP1 ulp2-1THIS3 This studyJMY2622 MATa ade2-1 ade3ThisG ura3 his3 lys2-801 trp1-1 ulp2-1THIS3 can1-100 This studyMDY2640 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 sgs1-20THGR slx5DTNAT

SSL5-1(ulp2-D623H)This study

MDY2667 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 leu2-3,112 lys2 SSL5-1(ulp2-D623H) This studyJMY2778 MATa ade2 ade3ThisG ura3 his3-11,15 leu2 lys2 sgs1-20THGR slx8-10TKAN ulp2-D623H rad5-535 This studyNJY2808 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 lys2 sgs1-20THGR slx5DTNAT

SSL5-1(ulp2-D623H)This study

JMY2809 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 lys2 sgs1-20THGR slx5DTNATSSL5-1(ulp2-D623H)

This study

NJY2811 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 leu2-3,112 sgs1-20THGR ulp2-D623H This studyMDY2817 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 leu2-3,112 lys2 can1-100 ulp2-D623H This studyJMY2819 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 sgs1-20THGR slx8-10TKAN

can1-100 plus pJM500This study

JMY2820 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 sgs1-20THGR slx8-10TKANcan1-100 plus pJM500

This study

NJY2828 MATa ade2-1 ura3 his3 leu2-3,112 lys2 ulp2-1THIS3 can1-100 This studyNJY2832 MATa ade2-1 ura3 his3 trp1-1 leu2-3,112 lys2 slx5-10TTRP1 wss1-10TKANTloxP This studyNJY2839 MATa ade2-1 ura3-1 his3-11,15 leu2-3,112 lys2 ulp2-D623H This studyNJY2843 MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 ulp2-D623H This studyNJY2844 MATa ade2-1 ura3-1 his3-11,15 leu2-3,112 lys2 can1-100 ulp2-D623H This studyNJY2894 MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 ulp2-D623HTHIS3 This studyJMY2901 MATa ade2-1 ura3 his3 trp1-1 leu2-3,112 slx5-10TTRP1 ulp2-1THIS3 can1-100 This studyJMY2902 MATa ade2-1 ura3 his3 trp1-1 leu2-3,112 slx5-10TTRP1 ulp2THIS3 can1-100 This studyJMY2904 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 lys2 slx5DTNAT ulp2-D623H This studyJMY2905 MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 lys2 slx5DTNAT ulp2-D623H This studyJMY2906 MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 slx5DTNAT ulp2-D623H This studyJMY2920 MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 lys2 slx5DTNAT smt3DTloxP ulp2-1THIS3

plus p7234 [SMT3(G98)/URA3/ADE3/CEN]This study

JMY3029 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 trp1-1 leu2-3,112 uls1TKAN sgs1-20THGR slx5DTNATplus pJM500 (SGS1/ADE3/URA3/CEN)

This study

JMY3073 MATa ade2-1 ade3ThisG ura3 his3 trp1-1 leu2-3,112 sgs1-20THGR slx5DTNAT ulp2-1THIS3plus pJM500 (SGS1/ADE3/URA3/CEN)

This study

(continued )

Antagonism Between SLX5–SLX8 and ULP2 75

the role of Slx5–Slx8 in genome stability is independentof SUMO.

To address the role of the Slx5–Slx8 Ub ligase ingenome stability, we carried out a suppressor analysis ofsgs1D slx5D synthetic lethality. Two mechanisms wereidentified that support a role of SUMO in this pathway.Not only did expression of a mammalian STUbL sup-press sgs1D slx5D synthetic lethality, but the loss of ULP2isopeptidase did as well. This latter result was unex-pected, since the resulting triple mutant strain accumu-lated even larger amounts of hypersumoylated proteinsthan the corresponding double mutants. Further anal-ysis revealed that several phenotypes of ulp2D and slx5D

cells were reciprocally suppressed in the ulp2D slx5D

double mutant. These data indicate that the activities ofthe isopeptidase and the Ub ligase oppose one another.We propose that the suppression of synthetic lethality inthe sgs1D slx5D ulp2D triple mutant is due to increasedpolysumoylation, which shuttles sumoylated target pro-teins into a WSS1-dependent proteolysis pathway.

MATERIALS AND METHODS

Yeast strains and growth conditions: Yeast strains are listedin Table 1. Unless otherwise indicated, all strains are RAD5derivatives of W303 that were maintained at 30� on 1% yeastextract, 2% peptone, and 2% dextrose (YPD) medium.Genetic mapping strains have been described (Reid et al.2008) and were generously provided by Rodney Rothstein.Strain growth, transformation, and preparation of minimalmedia followed standard procedures (Adams et al. 1997).Where employed, PCR-mediated gene disruptions were de-signed to replace complete open reading frames (ORFs) with theindicated antibiotic resistance marker as described (Guldener

et al. 1996). MMS sensitivity was tested by adding methylmetha-nesulfonate to a final concentration of 0.03% in YPD agarbefore pouring into plates. The solidified plates were used 24 hrlater. To spot cell dilutions, cells were scraped from freshlygrowing plates, resuspended in water, and their optical density(OD) at 600 nm was determined. Cells were then transferred tomicrotiter plates at an initial OD¼ 3.0 and serially diluted 1:10in water. A pin-type replica plater was then used to transfer�5 mlfrom each well to MMS and YPD plates.

Plasmid construction: Unless otherwise stated, genes werecloned by PCR amplification using specific primers and PhusionDNA polymerase followed by ligation into the pRS400 series ofyeast shuttle vectors (Sikorski and Hieter 1989). Detailsregarding plasmid construction are available on request.

Suppressor screen: One hundred milliliters of a saturatedculture of yeast strain NJY2462 was mutated using ethyl

methanesulfonate (EMS) as follows: Cells were grown over-night in YPD media, harvested by centrifugation, and resus-pended in 0.1 m NaPO4 (pH 7) to give the final volume of 1 mlat 2 3 109 cells/ml. The cells were treated with 50 ml of EMSat 30� with gentle shaking. A total of 0.1 ml of mixture wasremoved every 15, 30, 45, and 60 min and added to 4 ml of 5%sodium thiosulfate to neutralize the mutagen. These neutral-ized cultures were stored at 4� while a portion was dilutedappropriately and plated on YPD to determine cell viability.The sample treated with EMS for 45 min displayed 60%lethality and the 60-min treatment resulted in 90% lethality.About 105, 106, and 107 cells from the 45-min and 60-min EMS-treated cells were plated on YPD and replica plated onto 5-FOA plates containing 5 mg/ml adenine. From these plates atotal of 22 colonies were found to acquire 5-FOA resistance.One of these colonies was found to be SGS1 negative.

Preparation of yeast extracts for anti-Smt3 immunoblot-ting: Yeast extracts were prepared as follows. A total of 5 ml ofactively growing yeast at OD600¼ 1.0 were pelleted, washed with1 ml of water, transferred to microcentrifuge tubes, and pelletedagain. Pellets were resuspended in 0.5 ml ice-cold lysis buff-er (1.85 N NaOH, 1 m b-mercaptoethanol (bME), 5 mg/mlleupeptin, 10 mg/ml pepstatinA, 20 mm N-ethylmaleimide) andincubated 5 min on ice. Ice-cold 50% TCA (0.5 ml) was added,mixed quickly by vortexing, and incubated 5 min on ice. Totalprotein was pelleted in a microfuge for 10 min at 4�. Pellets werewashed with 1 ml 90% acetone, incubated 5 min on dry ice, thenpelleted in a microcentrifuge for 10 min at 4�. Dried pellets wereresuspended in 300 ml solution I (0.5 m Tris base, 6% SDS) bysonicating three times 8 sec each. An equal volume (300 ml) ofsolution II (25% glycerol, 1.1 m bME, bromophenol blue) wasadded to each tube, samples were mixed and heated to 95� for10 min, insoluble debris was pelleted in a microfuge at highspeed for 2 min, supernatants were transferred to fresh tubes,and 10 ml from each was loaded onto protein gels for im-munoblotting. Samples were stored at �80�.

RESULTS

Suppression of SGS1–SLX5/8 synthetic lethality by aheterologous SUMO-dependent Ub ligase: To test thehypothesis that SGS1–SLX5/8 synthetic lethality is dueto the accumulation of sumolyated proteins, we exam-ined whether the distantly related mammalian SUMO-targeted Ub ligase, rat RNF4, could functionally replaceSlx5–Slx8 in this assay. Human RNF4 has previouslybeen shown to complement some of the growth defectsof slx5D and slx8D mutants in S. cerevisiae (Uzunova et al.2007) and their homologs in S. pombe (Prudden et al.2007; Sun et al. 2007). As shown in Figure 1A, rat RNF4provided robust growth to both sgs1D slx5D and sgs1D

slx8D cells, and complementation was dependent on an

TABLE 1

(Continued)

Strain Genotype Reference/source

HKY579-10A MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 H. KLEIN

HKY580-10D MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 H. KLEIN

NJY3068 MATa ade2-1 ura3 his3 trp1-1 leu2-3,112 sgs1-20THGR slx8-10TKAN ulp2-1THIS3 pluspJM500(SGS1/ADE3/URA3)

This study

JMY3098 MATa ade2-1 ade3ThisG ura3-1 his3-11,15 leu2-3,112 lys2 can1-100 This study

76 J. R. Mullen, M. Das and S. J. Brill

intact ring finger domain. Although we cannot rule outthe possibility that these related Ub ligases have con-served their ability to ubiquitinate specific nonsumoy-lated proteins (Xie et al. 2010), a simpler explanationis that they are targeting a similar set of sumoylatedproteins. Consistent with this idea, immunoblotting ofslx5D cells carrying the RNF4 plasmid displayed reducedlevels of sumoylated proteins (Figure 1B). The level ofsumoylated proteins approximated that obtained withan SLX5 plasmid and the reduced levels were depen-dent on RNF4’s ring finger. These results are in line withthe idea that sgs1D slx5D synthetic lethality is due to theaccumulation of one or more sumoylated proteins, andthey suggest that one mechanism of suppressing thelethality is to lower the overall levels of sumoylation.

Isolation of an extragenic suppressor of sgs1D slx5Dsynthetic lethality: Mutations that suppress the syntheticlethality of sgs1D slx5D cells do not arise spontaneouslyin our hands. To isolate such mutations, we mutagen-ized strain NJY2462 with EMS. Strain NJY2462 (sgs1D

slx5D) requires plasmid pJM500 (SGS1/URA3/ADE3) forviability and forms red colonies on media with limitingadenine due to the plasmid-borne ADE3 gene. Follow-ing mutagenesis, the cells were spread onto YPD platesand allowed to grow for 24 hr. The surviving cells werethen replica plated onto 5-FOA to select against pJM500.Of the 22 FOA-resistant clones that were obtained, 9formed white colonies, suggesting that pJM500 hadbeen lost. These white strains were then screened byPCR to detect the helicase domain of SGS1. Only onestrain lacked the SGS1 PCR fragment and was namedMDY2640. When pJM500 was reintroduced into MDY2640it produced red colonies that readily sectored, indicat-ing that SGS1 was not needed for viability (Figure 2A).MDY2640 was slow growing, temperature sensitive (ts),and formed nibbled colonies as expected for an slx5D

strain. However, when MDY2640 was backcrossed to acompletely wild-type (wt) strain, spore clones wt forboth SGS1 and SLX5 were identified that displayed aweak nibbled-colony phenotype. This nibbled-colony phe-notype segregated 21:2� in five additional backcrossesallowing us to conclude that it was due to a single mutantgene (Figure 2B). Cosegregating with this phenotypewas heat-, MMS-, and HU-sensitive growth (see below).When such progeny were crossed to sgs1D slx5D strains(containing pJM500), about half of the sgs1D slx5D

progeny inheriting pJM500 were able to grow on 5-FOA,suggesting that this single gene was responsible forboth the suppression of synthetic lethality and theadditional growth defects. We referred to this mutationas SSL5-1 (Suppressor of SLX5) because diploids ofthe genotype sgs1D/sgs1D slx5D/slx5D SSL5-1/1 grewwell on 5-FOA (Figure 2C). Thus, SSL5-1 is dominantfor the suppression of synthetic lethality. As expected,SSL5-1 suppressed sgs1D slx8D synthetic lethality, al-though in this case suppression was recessive to wt SSL5(Figure 2C).

To further characterize the SSL5-1 mutation, progenyfrom wt backcrosses were tested for growth in thepresence of heat (37�), MMS, and HU. All of the SSL5-1 haploid progeny were sensitive to these treatments(Figure 3A). Interestingly, when SSL5-1/1 diploids wereassayed, we found that, unlike the suppression ofsynthetic lethality, the heat, MMS, and HU sensitivitiesof SSL5-1 were recessive (Figure 3B, top half). The loss

Figure 1.—Mammalian RNF4 complements the sgs1D slx5/8D synthetic-lethal phenotype. (A) Strains NJY2462 (sgs1Dslx5D, upper) and JMY2820 (sgs1D slx8D, lower), each carryingbalancer plasmid pJM500 (SGS1/URA3/ADE3/CEN), weretransformed with either pRS415 alone (vector) or pRS415containing wild-type rat RNF4, RNF4–4CS, SLX5, or SLX8, asindicated, all under the control of the SLX5 promoter. Therat RNF4–4CS allele contains C-to-S mutations at the followingring finger positions: C136, C139, C177, and C180. After se-lecting for leucine prototrophy, the transformed strains werestreaked in duplicate onto solid media containing 5-FOA toselect against pJM500. Plates were photographed following3 days growth at 30�. (B) Total denatured protein was isolatedfrom an slx5D yeast strain carrying the indicated pRS415-based plasmids as described in A. Protein was resolved by12.5% SDS–PAGE prior to immunoblotting with anti-Smt3 an-tibody. The filter was reprobed with anti-Rfa1 antibody as acontrol for protein loading.

Antagonism Between SLX5–SLX8 and ULP2 77

of SLX5 in these diploids had no synergistic effect on thegrowth of either SSL5-1/1 or SSL5-1/SSL5-1 in thepresence of MMS or HU. However, loss of SLX5 partiallysuppressed the temperature sensitivity of SSL5-1/SSL5-1yeast (Figure 3B, bottom half).

Identification of SSL5-1 as ulp2–D623H: The recessivephenotypes of SSL5-1 allowed us to map the mutation tochromosome IX using a set of yeast strains specificallydesigned for this purpose (Reid et al. 2008). Each of the16 strains in this set has a GAL1 promoter–URA3construct integrated adjacent to one of its centromeressuch that individual centromere function is inhibited bygrowth in galactose. The SSL5-1 strain MDY2817 wasmated to each of the 16 mapping strains and diploidswere selected. Following growth in galactose, the diploids

were transferred to 5-FOA medium to ensure loss of thedestabilized chromosome. The resulting loss of hetero-zygosity for individual chromosomes allows recessivephenotypes to appear, thus identifying the mutant chro-mosome. As shown in Figure 4A, loss of the GAL1–URA3-marked chromosome IX exposed MMS- and HU-sensitivegrowth phenotypes that resembled those of the SSL5-1haploid strain.

Earlier backcrosses of the SSL5-1 mutant producedfewer-than-expected tetratype tetrads with the centromere-linked TRP1 gene (data not shown). This suggested thatSSL5-1 was weakly centromere linked. ULP2 is locatedon chromosome IX, is slightly centromere linked, and isknown to confer phenotypes similar to SSL5-1 whendeleted. These phenotypes include heat-, MMS-, and

Figure 2.—The SSL5-1 mutation confers anibbled-colony phenotype and is dominant forthe suppression of sgs1D slx5D synthetic lethality.(A) The FOA-resistant strain MDY2640 (sgs1Dslx5D SSL5-1) was retransformed with pJM500and then streaked onto a YPD plate. Whitesectors represent cells having lost plasmidpJM500, indicating that it is not required for cellviability. (B) Spore clones from five tetrads froman SSL5-1/SSL5 diploid (MDY2667/NJY2470) areshown; colonies with a nibbled morphology arecircled. (C) Haploid or diploid strains with theindicated genotypes and carrying pJM500 werepregrown on YPD, streaked onto synthetic com-plete plates containing 5-FOA, and allowed togrow for 5 days at 30�. Left panel strains:NJY2808, JFY2481, and NJY2808/JFY2481. Rightpanel strains: JMY2778, JMY2819, and JMY2778/JMY2819.

Figure 3.—The SSL5-1 mutation isrecessive for heat, HU, and MMS sensi-tivity. (A) Ten-fold serial dilutions ofhaploid SSL5-1 and SSL5 strains werespotted onto YPD plates containingno drug, 0.1 m HU, or 0.03% MMS.(B) Serial dilutions of diploid cells withthe indicated genotypes were treated asabove. Unless otherwise indicated, theplates were incubated at 30� and photo-graphed after 2 (YPD and HU) or 4(MMS) days. Note that slx5D cells areslow growing compared to wt cells.

78 J. R. Mullen, M. Das and S. J. Brill

HU-sensitive growth, as well as a severe nibbled-colonymorphology (Li and Hochstrasser 2000). When theULP2 gene from the SSL5-1 strain was sequenced, weidentified a mutation (G1867C) that results in theamino acid change D623H. The D623 residue is locatedimmediately adjacent to the catalytic cysteine of Ulp2and is conserved in the Ulp-specific domain of Ulp1(Figure 4B). As expected, transformation of the SSL5-1strain with a centromeric plasmid carrying the wt ULP2gene complemented its heat-, MMS-, and HU-sensitivegrowth phenotypes (data not shown). We hereafterrefer to this mutation as ulp2–D623H.

ulp2–D623H is a novel allele: To characterize theulp2–D623H allele, we compared the phenotypes of theulp2–D623H and ulp2D haploid strains singly and incombination with slx5D. As expected, the ulp2D and theulp2–D623H alleles were heat, MMS, and HU sensitive(Figure 5A, rows 2 and 6). Removing SLX5 from theulp2D strain suppressed all three phenotypes, whileremoving SLX5 from the ulp2–D623H strain suppressedonly its heat sensitivity (Figure 5A). This difference insuppression indicates that ulp2–D623H is distinct fromthe null allele.

We next asked whether the ulp2D null mutation wascapable of suppressing sgs1D slx5D synthetic lethality.The haploid triple mutant strain JMY1922 (sgs1D slx5D

ulp2D) carrying pJM500 was pregrown on YPD to allowplasmid loss and then streaked onto 5-FOA mediumwhere it was able to form small slow-growing colonies

(Figure 5B). In contrast to ulp2–D623H, however, ulp2D

was recessive in that it could not suppress sgs1D slx5D

synthetic lethality in the presence of the wt ULP2 allele(Figure 5B). On the basis of differences in the pheno-types generated by ulp2–D623H and ulp2D, we concludethat ulp2–D623H is not null.

To further test this notion, we compared ulp2–D623H toulp2–C624S, which replaces the catalytic cysteine to pro-duce a nonfunctional protease. We first tested the abilityof these alleles to complement the severe growth defect ofulp2D cells. Because ulp2D cells are sick and difficult totransform, we first complemented strain NJY2828 withthe balancer plasmid pJM7384 (ULP2/URA3/CEN). Wethen introduced the above ULP2 alleles on a LEU2/CENplasmid and spotted dilutions of the cells on mediacontaining 5-FOA to select against pJM7384. Under theseconditions, ulp2–D623H conferred growth that was not asrobust as that obtained with wt ULP2. In contrast, neitherulp2–C624S nor the empty vector were able to promotesignificant growth in 3 days at 30� (Figure 6A).

We next tested ulp2–C624S for the ability to comple-ment sgs1D slx8D synthetic lethality using the triplemutant strain JMY3068 (sgs1D slx8D ulp2D) carryingpJM500 (SGS1/URA3/ADE3/CEN). We introduced theabove LEU2/CEN plasmids and streaked the cells ontosolid media lacking leucine but containing 5-FOA toselect against pJM500. Under these conditions, ulp2–D623H and the empty vector (i.e., ulp2D) were capableof promoting the formation of moderately sized colo-

Figure 4.—SSL5-1 is ulp2–D623H.(A) Strain MDY2817 (SSL5-1) wascrossed to each of the 16 mappingstrains that contain a centromere-linked GAL/URA3 construct. Diploidswere selected, grown on YP galactoseto destabilize a chromosome, and thenstreaked onto 5-FOA. The survivinghemizygous diploid strains were thenserially diluted and spotted onto YPDplates containing either no drug, 0.1 m

HU, or 0.03% MMS. Shown are the dip-loids, including those prior to 5-FOA/GAL selection, with marked chromo-somes IX, X, XI, and XII. (B) Shownare schematic diagrams of the Ulp1and Ulp2 proteins with the Ulp-specificregion shaded, and a sequence compar-ison of the two Ulp-specific domains.The three conserved metal coordinatingresidues and the catalytic cysteine resi-dues are highlighted. The arrow pointsto the ulp2–D623H mutation.

Antagonism Between SLX5–SLX8 and ULP2 79

nies after 5 days growth (Figure 6B). In contrast, thecatalytic null allele ulp2–C624S produced only a fewslow-growing colonies and, as expected, wt ULP2 waslethal in the sgs1D slx8D background. We conclude that,although ulp2–D623H resembles ulp2D in the sup-pression of synthetic lethality, it does not act as asimple catalytic null allele. Taken together, the aboveresults suggest that there are two methods of suppress-ing sgs1D slx5D and sgs1D slx8D synthetic lethality. Onemethod is to completely eliminate the Ulp2 protein.However if the Ulp2 protein is present, then it musthave an altered protease activity. It is possible thatthe failure of the catalytically null Ulp2–C624S pro-tein to efficiently suppress this phenotype is because itretains the ability to bind substrates, albeit nonpro-ductively, and sequester them from alternative pro-cessing pathways.

ULP2 is known to interact with UBC9, the SUMOconjugating enzyme. We therefore tested whether ulp2–D623H was capable of suppressing the temperature-sensitive growth of ubc9-1 cells, as shown previously forulp2D (Li and Hochstrasser 2000). A ubc9-1 ulp2D

strain was transformed with plasmids containing eitherULP2, ulp2–C624S, ulp2–D623H, or no insert. As shownin Figure 6C, ulp2–C624S and ulp2–D623H promotedthe growth of ubc9-1 cells at the nonpermissive tem-

perature like ulp2D. This result is consistent with theidea that ulp2–D623H is a hypomorphic allele, sincesurvival of ubc9-1 cells requires a low level of SUMO-deconjugating activity.

Role of ULP2 activity in other slx5D–slx8D pheno-types: Diploids homozygous for either slx5D–slx8D orulp2D are unable to sporulate (Li and Hochstrasser

2000; Mullen et al. 2001). To identify additional in-teractions between SLX5–SLX8 and ULP2, we comparedthe sporulation efficiencies of diploid strains containingvarious SLX5 and ULP2 alleles. As expected, diploidsindividually homozygous for slx5D, ulp2–D623H, orulp2D failed to sporulate (Figure 7A). However, replac-ing a single copy of ULP2 with either ulp2–D623H orulp2D increased the sporulation efficiency of an slx5D/slx5D diploid from ,1% to about half the wt level. Thissuggests that the sporulation defect of slx5D/slx5D

strains is due, in part, to ‘‘excess’’ ULP2 activity, since adecrease in ULP2 activity partially restores the ability tosporulate. Interestingly, the sporulation efficiencies ofheterozygous ulp2–D623H/1 (38%) and ulp2D/1 (16%)strains were easily distinguishable in a wt SLX5/SLX5background (Figure 7A). This provides additional ev-idence that ulp2–D623H is not a null allele.

Because the loss of ULP2 suppresses certain slx5D

defects and the loss of SLX5 suppresses certain ulp2D

Figure 5.—The phenotypes of ulp2–D623Hare distinguishable from those of the ulp2D nullallele. (A) Pairs of independent haploid yeaststrains, whose relevant genotypes are indicatedin the left-hand columns, were serially diluted,spotted onto various media, and treated as in Fig-ure 3. (B) Haploid or diploid strains of the indi-cated genotype and containing pJM500 werestreaked from a YPD plate onto minimal mediumcontaining 5-FOA. Shown is a photograph of theplate following 6 days growth at 30�. Strains:JMY1922, NJY2462, and JMY1922/NJY2462.

80 J. R. Mullen, M. Das and S. J. Brill

defects, we conclude that the activities of ULP2 andSLX5–SLX8 need to be balanced in wt cells. To confirmthis, SLX5 and slx5D strains were sequentially trans-formed with a single-copy SLX5/URA3 plasmid and ahigh-copy ULP2/LEU2 plasmid. The double transform-ants were then streaked onto media containing 5-FOAbut lacking leucine to select for retention of the high-copy ULP2 plasmid and the loss of the SLX5 plasmid. Asshown in Figure 7B, the slx5D strain could not survive inthe presence of excess ULP2 activity.

sgs1D slx5D synthetic lethality cannot be due simplyto the accumulation of bulk poly-SUMO chains: One ofthe consequences of the loss of SLX5 is that sumoylatedand polysumoylated proteins accumulate in vivo (Wang

et al. 2006; Ii et al. 2007; Uzunova et al. 2007; Wang andPrelich 2009). Deletion of SGS1 has also been associ-ated with an increase in poly-SUMO levels (Mullen andBrill 2008). It is therefore reasonable to hypothesizethat the synthetic lethality of sgs1D slx5D cells is due tothe accumulation of one or more sumoylated or poly-sumoylated proteins. If this is true, then the isolation of asuppressor mutation in ULP2 is paradoxical, given thatthe primary effect of a ulp2D mutation is to increase, notdecrease, polysumoylation (Bylebyl et al. 2003). Thisraised the possibility that ulp2–D623H might haveunexpected effects on polysumoylation levels.

To test this idea, we examined the in vivo SUMO levelsof the relevant mutant strains by immunoblot. Proteinextracts were isolated from wild-type and mutant strainsunder denaturing conditions and resolved by SDS–PAGE prior to immunoblotting with anti-Smt3 anti-bodies (Figure 8). As shown previously, sumoylationlevels in the slx5D, sgs1D, and ulp2D single mutants wereall elevated relative to wt (Figure 8, lanes 4–9, 14, and15). On its own, the ulp2–D623H suppressor mutationproduced an increase in the level of polysumoylatedproteins albeit less than that obtained with the ulp2D

single mutant. Specifically, there was an increase in thepoly-SUMO signal that migrates in the stacking gelrelative to wt, as well as an increase in signal migrating inthe high molecular-weight region of the resolving gel.Sumoylation levels were further elevated in the slx5D

ulp2–D623H and the sgs1D ulp2–D623H double mutantsas the poly-SUMO signal in the stacking gel from thesestrains approached that of the ulp2D null (Figure 8,lanes 10–13). Unexpectedly, sumoylated and polysu-moylated levels were highest in the suppressed triplemutant sgs1D slx5D ulp2–D623H (Figure 8B, lanes 16and 17). Because suppression of sgs1D slx5D syntheticlethality is associated with an increase in the level ofbulk sumoylation in the cell, the lethality of sgs1D slx5D

cells cannot be due simply to high levels of sumoylated

Figure 6.—The phenotypes of ulp2–D623Hare distinguishable from those of the ulp2–C624S catalytic null allele. (A) Strain NJY2828(ulp2D) was first transformed with pJM7384(ULP2/URA3/CEN) and then transformed witha LEU2/CEN-based vector containing either ULP2(pJM7359), ulp2–C624S (pJM7360), ulp2–D623H(pJM7361), or no insert (pRS415). Cells werepregrown on media lacking leucine to allow lossof pJM7384, and serial dilutions were spotted onmedia lacking leucine with or without 5-FOA asindicated. Plates were photographed following3 days growth at 30�. (B) Strain NJY3068 (sgs1Dslx8D ulp2D) containing pJM500 (SGS1/URA3/ADE3) was transformed with the same ULP2 plas-mids as above. As indicated in the key at right,transformants were streaked in duplicate ontosolid media lacking leucine but containing5-FOA to select against pJM500. The plate wasphotographed following 5 days growth at 30�.(C) Strain JMY1885 (ubc9–1D ulp2D) was trans-formed with a URA3/CEN-based vector contain-ing either ULP2 (pJM7384), ulp2–C624S(pJM7396), ulp2–D623H (pJM7397), or no insert(pRS416). As indicated in the schematic in B,transformants were streaked in duplicate ontomedia lacking uracil and incubated at 25� (3days) or 33� (4 days).

Antagonism Between SLX5–SLX8 and ULP2 81

proteins. However, this does not rule out the possibilitythat the distribution of poly-SUMO chains on varioustarget proteins may have changed or that alternativeforms of sumoylation are taking place under theseconditions.

A role for polysumoylation in sgs1D and slx5Dmutants: The ability of a mutation in ULP2 to suppressslx5D phenotypes (Figures 2, 5, and 7) suggests thatpolysumoylation is important in the absence of theSlx5–Slx8 Ub ligase. This idea is consistent with thefinding that a form of SUMO that is unable to formconventional poly-SUMO chains, Smt3–3KR, is toxic inslx5D uls1D double mutants (Uzunova et al. 2007). Totest the requirement for polysumoylation in the slx5D

single mutant, we attempted to replace the wt SMT3gene, present on a URA3-based plasmid, with mutantversions in which individual or multiple lysine residuesof Smt3 were mutated to arginine. After streaking thecells onto 5-FOA media, to select against the wt SMT3plasmid, we observed that the loss of all three N-terminal lysine resides (K11, -15, and -19) encoded bysmt3–3KR was lethal in an slx5D mutant (Figure 9A).This result is consistent with idea that the mechanismof suppression of slx5D–slx8D phenotypes by ulp2D

involves the formation of conventional poly-SUMOchains. Further, the inviability of slx5D smt3–3KR cellscould not be rescued by ulp2D (Figure 9B).

Similar experiments were carried out in the sgs1D

mutant background. Although sgs1D cells tolerated smt3–

3KR as did wt cells (Figure 9C), they were especiallysensitive to the smt3–allR allele, which encodes Smt3 inwhich all nine lysine residues are changed to arginine.This implies that sgs1D cells may also be dependent onsome form of polysumoylation. To test this idea, wefound that restoring any single lysine reside, other thanat position 40, partially suppressed this synthetic growthdefect (Figure 9D). Restoring a lysine residue at po-sition 11, 15, or 19 was especially effective at suppressingthe growth defect. Thus, both slx5D and sgs1D cellsappear to require conjugable forms of Smt3. But,whereas slx5D cells are critically dependent on the threeN-terminal lysine residues of Smt3, sgs1D mutantsappear to be able to use the other six lysine residuesin their absence.

Finally, we tested the roles of WSS1 and ULS1 in thesuppression of sgs1D slx5D lethality by ulp2D. WSS1 andULS1 have been implicated in the proteolytic destruc-tion of sumoylated proteins and represent potentialpathways for eliminating polysumoylated substrates thatform due to the loss of ULP2. As shown in Figure 10,ULS1 was not required for suppression as the quadruplemutant sgs1D slx5D ulp2D uls1D remained viable. How-ever, the quadruple mutant sgs1D slx5D ulp2D wss1D

failed to survive in the absence of the balancer plasmidbearing SGS1. As controls, the corresponding ULP2triple mutants were inviable, suggesting that neitherwss1D nor uls1D is capable of suppressing sgs1D slx5D

in the presence of ULP2. The simplest interpretation

Figure 7.—Opposing activities of ULP2 andSLX5–SLX8. (A) Diploid strains, whose relevantgenotypes are indicated on the x-axis, were pre-grown on YPD for 24 hr, patched onto sporula-tion plates, and incubated at room temperaturefor 7 days. The fraction of sporulated cells wasdetermined microscopically by counting thenumber of four-spored asci among the totalnumber of diploid cells. The values representan average of at least two trials counting 350–650 cells per trial. Where possible, asci were sub-jected to tetrad dissection and spore viability wasdetermined. Genotypes: 1, wt; �, ulp2–D623H;and D, null. (B) SLX5 (NJY2472) and slx5D(NJY2460) strains carrying pNJ6502 (SLX5/URA3) were transformed with multicopy plasmidpRS425 containing either no insert (vector) orULP2 (pJM7371) by selecting on media lackingleucine. Cells were then streaked onto solid me-dia containing 5-FOA but lacking leucine andphotographed following 3 days growth at 30�.OE, overexpression.

82 J. R. Mullen, M. Das and S. J. Brill

of this result is that ulp2D suppresses sgs1D slx5D

lethality by creating substrates for the Wss1 SUMOisopeptidase.

DISCUSSION

Antagonism between SLX5–SLX8 and ULP2: Thisstudy has identified a novel allele of ULP2 as a sup-pressor of sgs1D slx5D synthetic lethality. The allele,ULP2–D623H, displays many of the same recessive phe-notypes as ulp2D, but it is dominant for the suppressionof synthetic lethality. However, since ulp2D also suppres-sed lethality, we must conclude that a simple reductionin Ulp2 isopeptidase activity is sufficient to restoreviability to sgs1D slx5D cells. The simplest explanationfor the mechanism of suppression is that the reductionin Ulp2 activity compensates for the loss of the Slx5–Slx8Ub ligase. This is consistent with the findings thatoverexpression of ULP2 is detrimental to the growthof slx5D cells and that, in diploid cells, the loss of onecopy of ULP2 partially suppressed the slx5D/slx5D

sporulation defect. Interestingly, SLX5–SLX8 was alsorequired for multiple ulp2D phenotypes. Here, the lossof SLX5 supressed the heat, HU, and MMS sensitivitiesof ulp2D cells. Thus, ULP2 and SLX5–SLX8 appear toencode antagonistic activities.

At first glance, this conclusion is difficult to reconcilewith either the enzymatic activities of Ulp2 and Slx5–Slx8 or their mutant phenotypes. Both ulp2D and slx5D/slx8D mutants accumulate high levels of sumoylated

proteins and poly-SUMO chains (Li and Hochstrasser

2000; Bylebyl et al. 2003; Wang et al. 2006; Ii et al. 2007;Uzunova et al. 2007) through mechanisms that arethought to be well understood. Ulp2 is best known forreducing or eliminating poly-SUMO chains, as opposedto removing mono-SUMO moieties from target proteins(Mukhopadhyay et al. 2006; Mukhopadhyay andDasso 2007; Lima and Reverter 2008). Support forthis role comes from the fact that some ulp2D pheno-types are suppressed by replacing wt SUMO in ulp2D

cells with a version (Smt3–allR) that is unable to formpoly-SUMO chains (Bylebyl et al. 2003). Slx5–Slx8, aSUMO-dependent Ub ligase, appears to have a relatedfunction in eliminating sumoylated and polysumoylatedproteins, although in this case the Ub ligase targetsthem for proteasomal degradation. Slx5–Slx8 appearsto be conserved in many species and has functionalhomologs in S. pombe (Rfp1/2/Slx8) and humans(RNF4) (Prudden et al. 2007; Sun et al. 2007; Uzunova

et al. 2007; Xie et al. 2007; Lallemand-Breitenbach

et al. 2008; Mullen and Brill 2008; Tatham et al. 2008;Geoffroy and Hay 2009). The increase in poly-SUMOchains in these two different mutants would seem tosuggest that SLX5/SLX8 and ULP2 have redundantfunctions. Instead, SLX5/SLX8 appears to be redundantonly with ULP1. This conclusion is based on theisolation of SLX5 as a high-copy suppressor of ulp1tsand ulp1D (Xie et al. 2007) and the fact that a ulp1tsslx5D double mutant is inviable at the permissivetemperature (Ii et al. 2007; Xie et al. 2007). The simplest

Figure 8.—Suppression of sgs1D slx5D syn-thetic lethality is associated with an increase inbulk sumoylated proteins. Total denatured pro-tein was isolated from two independent isolatesof yeast strains with the indicated genotypesand resolved by 12.5% SDS–PAGE prior to anal-ysis for Smt3-protein conjugates by immunoblot-ting with anti-Smt3 antibody. The high molecularweight Smt3-protein conjugates that just enterthe stacking gel are considered to be poly-SUMOconjugates and are indicated by the arrow. A Pon-ceau stain of the filter immediately following theblotting step is presented as a control for proteinloading.

Antagonism Between SLX5–SLX8 and ULP2 83

interpretation of this data is that Slx5–Slx8 effectivelyeliminates sumoylated proteins in the absence of Ulp1,but antagonizes the activity of Ulp2. Another importantobservation is that ULP1 and ULP2 display an antago-nistic relationship to each other. For example, the heatsensitivities of both ulp1ts and ulp2D are suppressed inthe ulp1ts ulp2 double mutant (Li and Hochstrasser

2000). Thus, loss of either ULP1 or SLX5–SLX8 results inULP2-dependent phenotypes.

What then are the primary targets of the Slx5–Slx8 Ubligase, and are those targets also substrates for the Ulp1isopeptidase? Although satisfactory answers to thesequestions are lacking, we presume that one set of sub-strates includes monosumoylated proteins, such asMot1-301 (Wang and Prelich 2009), that are markedfor degradation by monosumoylation and subsequentubiquitination by Slx5–Slx8. We speculate that a secondset of substrates includes monosumoylated proteinswhose processing could be shared by Ulp1 and Slx5–Slx8. For example, some monosumoylated substratesmay be either desumoylated by Ulp1 directly, or shuntedinto the SLX5–SLX8 pathway for proteolytic destruction.Shunting could occur by simply extending the mono-SUMO moiety into a poly-SUMO chain, which is apreferred substrate of Slx5–Slx8 and RNF4 (Mullen

and Brill 2008; Tatham et al. 2008). If this is the case,

then editing of these poly-SUMO chains by Ulp2 wouldbe expected to have a negative effect by preventing thetarget proteins from entering the SLX5–SLX8 pathway.The increased half-life of these (mono)-sumoylatedtarget proteins would presumably have a toxic effect in

Figure 9.—Requirement forSmt3’s lysine residues in slx5Dand sgs1D single mutants. (A)Strain NJY2524 (slx5D smt3D) car-rying balancer plasmid pNJ7234[SMT3(G98)/URA3/ADE3] wastransformed with a pRS415-basedplasmid bearing an SMT3 allelethat encodes either wt Smt3 orthe indicated K-to-R mutation.Following growth on media lack-ing leucine, the transformantswere streaked onto solid mediacontaining 5-FOA to selectagainst pNJ7234. The plate wasphotographed following growthat 30�. (B) The slx5D smt3Dstrains JMY2524 (ULP2) andJMY2920 (ulp2D) carrying plas-mid pNJ7234 were transformedwith vector pRS415 containingSMT3(G98) (wt, pNJ723), smt3(G98)–3KR (3KR, pNJ7235), or no insert(vector). Transformants were trea-ted as in A. (C) Strains JFY2488(SGS1 smt3D, left) and NJY2444(sgs1D smt3D, right), each carry-ing pNJ7234, were transformedwith a pRS415-based plasmidbearing the indicated SMT3 al-

lele. Transformants were treated as in A. (D) Strain NJY2444 (sgs1D smt3D) carrying pNJ7234 was transformed with a pRS415-basedplasmid bearing either an SMT3 allele in which all lysine residues were mutated to arginine (smt3–allR) or an allele retaining thesingle lysine residue indicated on the left. Cells were resuspended at OD ¼ 3, serially diluted in 1/5 steps, and spotted on YPDplates or synthetic complete media containing 5-FOA to select against pNJ7234. The plates were then photographed followinggrowth at 30�.

Figure 10.—Suppression of slx5D sgs1D synthetic lethalityby ulp2D is dependent on WSS1. Strains of the indicated gen-otypes, which also contained pJM500 as a balancer plasmid,were isolated following tetrad dissection of diploidsJMY3029/JMY2613 or JMY3073/NJY2832. The strains werestreaked onto media containing 5-FOA to select againstpJM500 and the plate was photographed following 9 daysgrowth at 30�. Where indicated, independent spore cloneswere streaked in duplicate.

84 J. R. Mullen, M. Das and S. J. Brill

ulp1ts cells. This may be one reason why ULP2 expres-sion is harmful to ulp1ts cells.

The above reasoning can also be used to explain whythe Slx5–Slx8 Ub ligase is harmful to ulp2D cells. In theabsence of Ulp2, certain monosumoylated substratesare expected to become polysumoylated. If these poly-sumoylated substrates are ubiquitinated by Slx5–Slx8,they could be degraded prematurely. While the currentstudy has identified an antagonistic relationship be-tween Ulp2 and Slx5–Slx8, additional experimentsare needed to test these models, including the role ofUlp1.

Is ulp2–D623H a gain-of-function allele? A number ofobservations support the idea that ulp2–D623H is notnull. Not only does the ulp2–D623H single mutant growbetter than the null, but its level of poly-SUMO chains,its sporulation defect, and its nibbled-colony morphol-ogy are less severe than that of ulp2D cells. This leads usto ask whether ulp2–D623H is simply a hypomorphicallele or whether it is a neomorph that has acquired anew function. Several results are consistent with the ideathat ulp2–D623H is simply a hypomorph. This includesthe above phenotypes as well as the fact that ulp2–D623H cells share many ulp2D phenoptypes includingsensitivity to DNA damaging agents, and the ability ofulp2D to suppress sgs1D slx5D synthetic lethality. How-ever, if ulp2–D623H is a hypomorph, then the level ofactivity that suppresses synthetic lethality must fallwithin an extremely narrow range, given that hypo-morphs and nullomorphs of ULP2 have yet to be iso-lated spontaneously in the sgs1D slx5D background.Moreover, it seems unlikely that this level of activity iscoincidentally an amount that is dominant in slx5D/slx5D diploids. Indeed, the dominance of ulp2–D623His the strongest evidence that it is a gain-of-functionallele. Although it is possible that the dominant sup-pression of synthetic lethality by ulp2–D623H is due to adominant-negative effect on the wt Ulp2 protein,another possibility is that it has acquired an alteredfunction. What altered functions might the protein haveacquired? One possibility is that the relative activity ofUlp2–D623H protein on monomeric and polymericSUMO may be changed. For example, given that itsprimary role is in editing SUMO chains, the Ulp2–D623H protein may be relatively more active onsubstrates bearing monomeric SUMO. Alternatively,the protein may display enhanced activity on poly-SUMO chains that are assembled with alternative link-ages. Although there is currently no evidence to supportthe idea that poly-SUMO chains assemble in any formother than the canonical K11, -15, or -19–linked chains,this question has not been exhaustively addressed.Indeed, the idea that poly-SUMO chains, like poly-Ubchains, are distinguished by their linkages might beanticipated, given the increasing number of similaritiesbetween Ub and SUMO. Either of these two alternatives,reduced activity or altered cleavage specificity, is consis-

tent with the location of the Ulp2–D623H mutationimmediately adjacent to the active site cysteine. Addi-tional experiments should be able to answer thesequestions definitively. For example, downregulation ofUlp2 expression can test whether reduced expressionalone is capable of suppression, and biochemicalexperiments can determine whether Ulp2–D623H dis-plays altered cleavage activities.

The finding that Ulp2–D623H is dominant only inthe sgs1D slx5D background may be related to the factthat Slx8 is active as a Ub ligase on its own (Xie et al.2007). Specifically, the residual Ub ligase activity of Slx8may contribute to the ability of Ulp2–D623H to over-come the toxic effect of wt Ulp2 in sgs1D slx5D cells. Onespeculation is that the unregulated Ub ligase activity ofSlx8 ubiquitinates poly-SUMO chains inappropriately.This inappropriate ubiquitination may inhibit theactivity of wt Ulp2 or assist in the destruction of thelong polysumoylated targets via the WSS1 pathway.

sgs1D slx5D synthetic lethality and the mechanism ofulp2D suppression: The identification of ulp2D as asuppressor has provided limited insight into the cause ofsgs1D slx5D synthetic lethality. Initially, it seemed likelythat sgs1D slx5D lethality was simply due to the increasedlevels of hypersumoylated proteins in the doublemutant. Indeed, the ability of mammalian RNF4 to sup-press lethality suggests that sumoylated proteins are re-sponsible for the lethal phenotype. However, becausesuppression by ulp2–D623H leads to a further increasein the level of bulk sumoylated proteins in the triplemutant, we can rule out the possibility that hyper-sumoylation per se is responsible for the lethality. Thisresult makes it more likely that one or more specificsumoylated proteins are responsible for sgs1D slx5D

lethality. But if this is the case, how can an increase insumoylation lead to suppression? Borrowing from thearguments presented above, it may be that the loss ofULP2 leads to poly-SUMO chains that are sufficientlylong that they now trigger alternative or bypass pathwaysof degradation. Our data have eliminated the SUMO-targeted Ub ligase Uls1 as a candidate for this bypasspathway. This is consistent with the fact that uls1D sgs1D

cells display no obvious phenotype (J. R. Mullen,unpublished result). In contrast, WSS1 is required forthe viability of the sgs1D slx5D ulp2D triple mutant. Thisresult is compatible with the synthetic fitness defect ofwss1D sgs1D cells and with the fact that overexpression ofthe Wss1 SUMO protease suppresses sgs1D slx5D lethal-ity (Mullen et al. 2010). Therefore, one possiblemechanism to explain our results is that the Wss1protease is activated by the longer poly-SUMO chainspresent in sgs1D slx5D ulp2D cells. Under these con-ditions, Wss1 may promote the desumoylation and/orproteasome-mediated degradation of target proteinsthat would otherwise be toxic. The critical role of ulp2D-induced polysumoylation in slx5D–slx8D cells is consis-tent with the fact that smt3–allR is lethal in slx5D/slx8D

Antagonism Between SLX5–SLX8 and ULP2 85

cells (Figure 9) (Uzunova et al. 2007). That is, slx5D/slx8D cells may require polysumoylation as a way ofdirecting substrates into the WSS1 pathway.

Lastly, we find the synthetic lethality of sgs1D smt3–allR double mutants intriguing as it suggests thatalternative poly-SUMO linkages may have a biologicalrole. As mentioned above, there is no evidence thatbudding yeast uses any inter-SUMO linkages other thanthe three N-terminal lysine residues. If this is true, thensgs1D smt3–allR synthetic lethality may simply be due tothe fact that wt Smt3 structure is critically important inthe absence of SGS1. However the ease with which sgs1D

smt3–allR cells are rescued by a single lysine in any ofseveral positions in Smt3 suggests that what is importantin the absence of SGS1 is the ability of Smt3 topolymerize even if it is not a sanctioned linkage. Furtherexperimentation is clearly needed to identify a biolog-ical role for alternative SUMO linkages. Our currentresults suggest that mutants defective in genome stabil-ity may provide the appropriate genetic background tosearch for such a role.

The authors thank Rodney Rothstein, Alex Strunnikov, and JormaPalvimo for strains and plasmids. This work was supported by NationalInstitutes of Health grant GM071268 and a grant from the BuschBiomedical Foundation.

LITERATURE CITED

Adams, A., D. E. Gottschling, C. A. Kaiser and T. Stearns,1997 Methods in Yeast Genetics. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY.

Bachant, J., A. Alcasabas, Y. Blat, N. Kleckner and S. J. Elledge,2002 The SUMO-1 isopeptidase Smt4 is linked to centromericcohesion through SUMO-1 modification of DNA topoisomeraseII. Mol. Cell 9: 1169–1182.

Burgess, R. C., S. Rahman, M. Lisby, R. Rothstein and X. Zhao,2007 The Slx5-Slx8 complex affects sumoylation of DNA repairproteins and negatively regulates recombination. Mol. Cell. Biol.27: 6153–6162.

Bylebyl, G. R., I. Belichenko and E. S. Johnson, 2003 The SUMOisopeptidase Ulp2 prevents accumulation of SUMO chains inyeast. J. Biol. Chem. 278: 44113–44120.

Chen, X. L., A. Reindle and E. S. Johnson, 2005 Misregulation of 2micron circle copy number in a SUMO pathway mutant. Mol.Cell. Biol. 25: 4311–4320.

Ciechanover, A., and A. L. Schwartz, 1998 The ubiquitin-proteasome pathway: the complexity and myriad functions ofproteins death. Proc. Natl. Acad. Sci. USA 95: 2727–2730.

Denuc, A., and G. Marfany, 2010 SUMO and ubiquitin paths con-verge. Biochem. Soc. Trans. 38: 34–39.

Dobson, M. J., A. J. Pickett, S. Velmurugan, J. B. Pinder, L. A.Barrett et al., 2005 The 2 micron plasmid causes cell death inSaccharomyces cerevisiae with a mutation in Ulp1 protease. Mol.Cell. Biol. 25: 4299–4310.

Fu, C., K. Ahmed, H. Ding, X. Ding, J. Lan et al., 2005 Stabilizationof PML nuclear localization by conjugation and oligomerizationof SUMO-3. Oncogene 24: 5401–5413.

Geoffroy, M. C., and R. T. Hay, 2009 An additional role for SUMOin ubiquitin-mediated proteolysis. Nat. Rev. Mol. Cell Biol. 10:564–568.

Guldener, U., S. Heck, T. Fielder, J. Beinhauer and J. H. Hegemann,1996 A new efficient gene disruption cassette for repeated usein budding yeast. Nucleic Acids Res. 24: 2519–2524.

Ii, T., J. R. Mullen, C. E. Slagle and S. J. Brill, 2007 Stimulation ofin vitro sumoylation by Slx5-Slx8: evidence for a functional inter-action with the SUMO pathway. DNA Repair (Amst.) 6: 1679–1691.

Johnson, E. S., 2004 Protein modification by SUMO. Annu. Rev. Bi-ochem. 73: 355–382.

Kerscher, O., R. Felberbaum and M. Hochstrasser,2006 Modification of proteins by ubiquitin and ubiquitin-likeproteins. Annu. Rev. Cell Dev. Biol. 22: 159–180.

Kosoy, A., T. M. Calonge, E. A. Outwin and M. J. O’Connell,2007 Fission yeast Rnf4 homologs are required for DNA repair.J. Biol. Chem. 282: 20388–20394.

Lallemand-Breitenbach, V., M. Jeanne, S. Benhenda, R. Nasr,M. Lei et al., 2008 Arsenic degrades PML or PML-RARalphathrough a SUMO-triggered RNF4/ubiquitin-mediated pathway.Nat. Cell Biol. 10: 547–555.

Li, S. J., and M. Hochstrasser, 1999 A new protease required forcell-cycle progression in yeast. Nature 398: 246–251.

Li, S. J., and M. Hochstrasser, 2000 The yeast ULP2 (SMT4) geneencodes a novel protease specific for the ubiquitin-like Smt3 pro-tein. Mol. Cell. Biol. 20: 2367–2377.

Li, S. J., and M. Hochstrasser, 2003 The Ulp1 SUMO isopepti-dase: distinct domains required for viability, nuclear envelopelocalization, and substrate specificity. J. Cell Biol. 160: 1069–1081.

Li, Y., H. Wang, S. Wang, D. Quon, Y. W. Liu et al., 2003 Positiveand negative regulation of APP amyloidogenesis by sumoylation.Proc. Natl. Acad. Sci. USA 100: 259–264.

Lima, C. D., and D. Reverter, 2008 Structure of the human SENP7catalytic domain and poly-SUMO deconjugation activities forSENP6 and SENP7. J. Biol. Chem. 283: 32045–32055.

Mukhopadhyay, D., and M. Dasso, 2007 Modification in reverse:the SUMO proteases. Trends Biochem. Sci. 32: 286–295.

Mukhopadhyay, D., F. Ayaydin, N. Kolli, S. H. Tan, T. Anan et al.,2006 SUSP1 antagonizes formation of highly SUMO2/3-conjugatedspecies. J. Cell Biol. 174: 939–949.

Mullen, J. R., and S. J. Brill, 2008 Activation of the Slx5-Slx8 ubiq-uitin ligase by poly-SUMO conjugates. J. Biol. Chem. 283: 19912–19921.

Mullen, J. R., V. Kaliraman, S. S. Ibrahim and S. J. Brill,2001 Requirement for three novel protein complexes in theabsence of the Sgs1 DNA helicase in Saccharomyces cerevisiae.Genetics 157: 103–118.

Mullen, J. R., C. F. Chen and S. J. Brill, 2010 Wss1 is a SUMO-dependent isopeptidase that interacts genetically with theSlx5-Slx8 SUMO-targeted Ub ligase. Mol. Cell. Biol. 30: 3737–3748.

Panse, V. G., B. Kuster, T. Gerstberger and E. Hurt,2003 Unconventional tethering of Ulp1 to the transport chan-nel of the nuclear pore complex by karyopherins. Nat. Cell Biol.5: 21–27.

Perry, J. J., J. A. Tainer and M. N. Boddy, 2008 A SIM-ultaneousrole for SUMO and ubiquitin. Trends Biochem. Sci. 33: 201–208.

Pickart, C. M., and D. Fushman, 2004 Polyubiquitin chains: poly-meric protein signals. Curr. Opin. Chem. Biol. 8: 610–616.

Prudden, J., S. Pebernard, G. Raffa, D. A. Slavin, J. J. Perry et al.,2007 SUMO-targeted ubiquitin ligases in genome stability. EMBOJ. 18: 4089–4101.

Ravid, T., and M. Hochstrasser, 2008 Diversity of degradation sig-nals in the ubiquitin-proteasome system. Nat. Rev. Mol. Cell Biol.9: 679–690.

Reid, R. J., I. Sunjevaric, W. P. Voth, S. Ciccone, W. Du et al.,2008 Chromosome-scale genetic mapping using a set of 16 con-ditionally stable Saccharomyces cerevisiae chromosomes. Genetics180: 1799–1808.

Schwartz, D. C., R. Felberbaum and M. Hochstrasser, 2007 TheUlp2 SUMO protease is required for cell division following ter-mination of the DNA damage checkpoint. Mol. Cell. Biol. 27:6948–6961.

Schwienhorst, I., E. S. Johnson and R. J. Dohmen, 2000 SUMOconjugation and deconjugation. Mol. Gen. Genet. 263: 771–786.

Sikorski, R. S., and P. Hieter, 1989 A system of shuttle vectors andyeast host strains designed for efficient manipulation of DNA inSaccharomyces cerevisiae. Genetics 12: 19–27.

Strunnikov, A. V., L. Aravind and E. V. Koonin, 2001 Saccharo-myces cerevisiae SMT4 encodes an evolutionarily conserved proteasewith a role in chromosome condensation regulation. Genetics 158:95–107.

86 J. R. Mullen, M. Das and S. J. Brill

Sun, H., J. D. Leverson and T. Hunter, 2007 Conserved functionof RNF4 family proteins in eukaryotes: targeting a ubiquitinligase to SUMOylated proteins. EMBO J. 18: 4102–4112.

Tatham, M. H., E. Jaffray, O. A. Vaughan, J. M. Desterro, C. H.Botting et al., 2001 Polymeric chains of SUMO-2 andSUMO-3 are conjugated to protein substrates by SAE1/SAE2and Ubc9. J. Biol. Chem. 276: 35368–35374.

Tatham, M. H., M. C. Geoffroy, L. Shen, A. Plechanovova,N. Hattersley et al., 2008 RNF4 is a poly-SUMO-specific E3ubiquitin ligase required for arsenic-induced PML degradation.Nat. Cell Biol. 10: 538–546.

Uzunova, K., K. Gottsche, M. Miteva, S. R. Weisshaar, C. Glanemann

et al., 2007 Ubiquitin-dependent proteolytic control of SUMOconjugates. J. Biol. Chem. 282: 34167–34175.

Wang, Z., and G. Prelich, 2009 Quality control of a transcriptionalregulator by SUMO-targeted degradation. Mol. Cell. Biol. 29:1694–1706.

Wang, Z., G. M. Jones and G. Prelich, 2006 Genetic analysis con-nects SLX5 and SLX8 to the SUMO pathway in Saccharomycescerevisiae. Genetics 172: 1499–1509.

Xie, Y., O. Kerscher, M. B. Kroetz, H. F. McConchie, P. Sung et al.,2007 The yeast HEX3–SLX8 heterodimer is a ubiquitin ligasestimulated by substrate sumoylation. J. Biol. Chem. 282: 34176–34184.

Xie, Y., E. M. Rubenstein, T. Matt and M. Hochstrasser,2010 SUMO-independent in vivo activity of a SUMO-targetedubiquitin ligase toward a short-lived transcription factor. GenesDev. 24: 893–903.

Xiong, L., X. L. Chen, H. R. Silver, N. T. Ahmed and E. S. Johnson,2009 Deficient SUMO attachment to Flp recombinase leadsto homologous recombination-dependent hyperamplificationof the yeast 2 micron circle plasmid. Mol. Biol. Cell 20: 1241–1251.

Zhang, C., T. M. Roberts, J. Yang, R. Desai and G. W. Brown,2006 Suppression of genomic instability by SLX5 and SLX8 inSaccharomyces cerevisiae. DNA Repair (Amst.) 5: 336–346.

Communicating editor: E. Alani

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