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. 2011 Jan;187(1):73-87.
doi: 10.1534/genetics.110.124347. Epub 2010 Nov 8.

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

Janet R Mullen  1 , Mukund Das, Steven J Brill
Affiliations

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

Janet R Mullen et al. Genetics. 2011 Jan.

Abstract

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

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Figures

F<sc>igure</sc> 1.—
Figure 1.—
Mammalian RNF4 complements the sgs1Δ slx5/8Δ synthetic-lethal phenotype. (A) Strains NJY2462 (sgs1Δ slx5Δ, upper) and JMY2820 (sgs1Δ slx8Δ, lower), each carrying balancer plasmid pJM500 (SGS1/URA3/ADE3/CEN), were transformed with either pRS415 alone (vector) or pRS415 containing wild-type rat RNF4, RNF4–4CS, SLX5, or SLX8, as indicated, all under the control of the SLX5 promoter. The rat RNF4–4CS allele contains C-to-S mutations at the following ring finger positions: C136, C139, C177, and C180. After selecting for leucine prototrophy, the transformed strains were streaked in duplicate onto solid media containing 5-FOA to select against pJM500. Plates were photographed following 3 days growth at 30°. (B) Total denatured protein was isolated from an slx5Δ yeast strain carrying the indicated pRS415-based plasmids as described in A. Protein was resolved by 12.5% SDS–PAGE prior to immunoblotting with anti-Smt3 antibody. The filter was reprobed with anti-Rfa1 antibody as a control for protein loading.
F<sc>igure</sc> 2.—
Figure 2.—
The SSL5-1 mutation confers a nibbled-colony phenotype and is dominant for the suppression of sgs1Δslx5Δ synthetic lethality. (A) The FOA-resistant strain MDY2640 (sgs1Δ slx5Δ SSL5-1) was retransformed with pJM500 and then streaked onto a YPD plate. White sectors represent cells having lost plasmid pJM500, indicating that it is not required for cell viability. (B) Spore clones from five tetrads from an SSL5-1/SSL5 diploid (MDY2667/NJY2470) are shown; colonies with a nibbled morphology are circled. (C) Haploid or diploid strains with the indicated genotypes and carrying pJM500 were pregrown on YPD, streaked onto synthetic complete plates containing 5-FOA, and allowed to grow for 5 days at 30°. Left panel strains: NJY2808, JFY2481, and NJY2808/JFY2481. Right panel strains: JMY2778, JMY2819, and JMY2778/JMY2819.
F<sc>igure</sc> 3.—
Figure 3.—
The SSL5-1 mutation is recessive for heat, HU, and MMS sensitivity. (A) Ten-fold serial dilutions of haploid SSL5-1 and SSL5 strains were spotted onto YPD plates containing no drug, 0.1 m HU, or 0.03% MMS. (B) Serial dilutions of diploid cells with the indicated genotypes were treated as above. Unless otherwise indicated, the plates were incubated at 30° and photographed after 2 (YPD and HU) or 4 (MMS) days. Note that slx5Δ cells are slow growing compared to wt cells.
F<sc>igure</sc> 4.—
Figure 4.—
SSL5-1 is ulp2–D623H. (A) Strain MDY2817 (SSL5-1) was crossed to each of the 16 mapping strains that contain a centromere-linked GAL/URA3 construct. Diploids were selected, grown on YP galactose to destabilize a chromosome, and then streaked onto 5-FOA. The surviving hemizygous diploid strains were then serially diluted and spotted onto YPD plates containing either no drug, 0.1 m HU, or 0.03% MMS. Shown are the diploids, including those prior to 5-FOA/GAL selection, with marked chromosomes IX, X, XI, and XII. (B) Shown are schematic diagrams of the Ulp1 and Ulp2 proteins with the Ulp-specific region shaded, and a sequence comparison of the two Ulp-specific domains. The three conserved metal coordinating residues and the catalytic cysteine residues are highlighted. The arrow points to the ulp2–D623H mutation.
F<sc>igure</sc> 5.—
Figure 5.—
The phenotypes of ulp2–D623H are distinguishable from those of the ulp2Δ null allele. (A) Pairs of independent haploid yeast strains, whose relevant genotypes are indicated in the left-hand columns, were serially diluted, spotted onto various media, and treated as in Figure 3. (B) Haploid or diploid strains of the indicated genotype and containing pJM500 were streaked from a YPD plate onto minimal medium containing 5-FOA. Shown is a photograph of the plate following 6 days growth at 30°. Strains: JMY1922, NJY2462, and JMY1922/NJY2462.
F<sc>igure</sc> 6.—
Figure 6.—
The phenotypes of ulp2–D623H are distinguishable from those of the ulp2–C624S catalytic null allele. (A) Strain NJY2828 (ulp2Δ) was first transformed with pJM7384 (ULP2/URA3/CEN) and then transformed with a LEU2/CEN-based vector containing either ULP2 (pJM7359), ulp2–C624S (pJM7360), ulp2–D623H (pJM7361), or no insert (pRS415). Cells were pregrown on media lacking leucine to allow loss of pJM7384, and serial dilutions were spotted on media lacking leucine with or without 5-FOA as indicated. Plates were photographed following 3 days growth at 30°. (B) Strain NJY3068 (sgs1Δ slx8Δ ulp2Δ) containing pJM500 (SGS1/URA3/ADE3) was transformed with the same ULP2 plasmids as above. As indicated in the key at right, transformants were streaked in duplicate onto solid media lacking leucine but containing 5-FOA to select against pJM500. The plate was photographed following 5 days growth at 30°. (C) Strain JMY1885 (ubc9–1Δ ulp2Δ) was transformed with a URA3/CEN-based vector containing 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 onto media lacking uracil and incubated at 25° (3 days) or 33° (4 days).
F<sc>igure</sc> 7.—
Figure 7.—
Opposing activities of ULP2 and SLX5–SLX8. (A) Diploid strains, whose relevant genotypes are indicated on the x-axis, were pregrown on YPD for 24 hr, patched onto sporulation plates, and incubated at room temperature for 7 days. The fraction of sporulated cells was determined microscopically by counting the number of four-spored asci among the total number of diploid cells. The values represent an average of at least two trials counting 350–650 cells per trial. Where possible, asci were subjected to tetrad dissection and spore viability was determined. Genotypes: +, wt; −, ulp2–D623H; and Δ, null. (B) SLX5 (NJY2472) and slx5Δ (NJY2460) strains carrying pNJ6502 (SLX5/URA3) were transformed with multicopy plasmid pRS425 containing either no insert (vector) or ULP2 (pJM7371) by selecting on media lacking leucine. Cells were then streaked onto solid media containing 5-FOA but lacking leucine and photographed following 3 days growth at 30°. OE, overexpression.
F<sc>igure</sc> 8.—
Figure 8.—
Suppression of sgs1Δ slx5Δ synthetic lethality is associated with an increase in bulk sumoylated proteins. Total denatured protein was isolated from two independent isolates of yeast strains with the indicated genotypes and resolved by 12.5% SDS–PAGE prior to analysis for Smt3-protein conjugates by immunoblotting with anti-Smt3 antibody. The high molecular weight Smt3-protein conjugates that just enter the stacking gel are considered to be poly-SUMO conjugates and are indicated by the arrow. A Ponceau stain of the filter immediately following the blotting step is presented as a control for protein loading.
F<sc>igure</sc> 9.—
Figure 9.—
Requirement for Smt3's lysine residues in slx5Δ and sgs1Δ single mutants. (A) Strain NJY2524 (slx5Δ smt3Δ) carrying balancer plasmid pNJ7234 [SMT3(G98)/URA3/ADE3] was transformed with a pRS415-based plasmid bearing an SMT3 allele that encodes either wt Smt3 or the indicated K-to-R mutation. Following growth on media lacking leucine, the transformants were streaked onto solid media containing 5-FOA to select against pNJ7234. The plate was photographed following growth at 30°. (B) The slx5Δ smt3Δ strains JMY2524 (ULP2) and JMY2920 (ulp2Δ) carrying plasmid pNJ7234 were transformed with vector pRS415 containing SMT3(G98) (wt, pNJ723), smt3(G98)–3KR (3KR, pNJ7235), or no insert (vector). Transformants were treated as in A. (C) Strains JFY2488 (SGS1 smt3Δ, left) and NJY2444 (sgs1Δ smt3Δ, right), each carrying pNJ7234, were transformed with a pRS415-based plasmid bearing the indicated SMT3 allele. Transformants were treated as in A. (D) Strain NJY2444 (sgs1Δ smt3Δ) carrying pNJ7234 was transformed with a pRS415-based plasmid bearing either an SMT3 allele in which all lysine residues were mutated to arginine (smt3–allR) or an allele retaining the single lysine residue indicated on the left. Cells were resuspended at OD = 3, serially diluted in 1/5 steps, and spotted on YPD plates or synthetic complete media containing 5-FOA to select against pNJ7234. The plates were then photographed following growth at 30°.
F<sc>igure</sc> 10.—
Figure 10.—
Suppression of slx5Δ sgs1Δ synthetic lethality by ulp2Δ is dependent on WSS1. Strains of the indicated genotypes, which also contained pJM500 as a balancer plasmid, were isolated following tetrad dissection of diploids JMY3029/JMY2613 or JMY3073/NJY2832. The strains were streaked onto media containing 5-FOA to select against pJM500 and the plate was photographed following 9 days growth at 30°. Where indicated, independent spore clones were streaked in duplicate.
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