Distinct SUMO ligases cooperate with Esc2 and Slx5 to suppress duplication-mediated genome rearrangements

Abstract

Suppression of duplication-mediated gross chromosomal rearrangements (GCRs) is essential to maintain genome integrity in eukaryotes. Here we report that SUMO ligase Mms21 has a strong role in suppressing GCRs in Saccharomyces cerevisiae, while Siz1 and Siz2 have weaker and partially redundant roles. Understanding the functions of these enzymes has been hampered by a paucity of knowledge of their substrate specificity in vivo. Using a new quantitative SUMO-proteomics technology, we found that Siz1 and Siz2 redundantly control the abundances of most sumoylated substrates, while Mms21 more specifically regulates sumoylation of RNA polymerase-I and the SMC-family proteins. Interestingly, Esc2, a SUMO-like domain-containing protein, specifically promotes the accumulation of sumoylated Mms21-specific substrates and functions with Mms21 to suppress GCRs. On the other hand, the Slx5-Slx8 complex, a SUMO-targeted ubiquitin ligase, suppresses the accumulation of sumoylated Mms21-specific substrates. Thus, distinct SUMO ligases work in concert with Esc2 and Slx5-Slx8 to control substrate specificity and sumoylation homeostasis to prevent GCRs.

Figure 1. A new quantitative proteomics technology to characterize the SUMO-proteome.

A) Method to identify and quantify sumoylated proteins using SILAC and MS. Untagged cells and HF-SUMO cells were combined for purification of sumoylated proteins. Ulp1 was used to elute sumoylated proteins for MS analysis. B) Scatter plot of the identified proteins based on two replicate experiments. The majority of the proteins show a large abundance ratio between HF-SUMO purification versus mock purification, indicating a highly purified sample. Candidate SUMO targets are identified based on at least 10-fold abundance ratio between HF-SUMO and mock sample in both replicate experiments. C) Western blot analysis of several SUMO targets confirmed them being sumoylated. Ulp1 treatment was used to treat half of the anti-HA immunoprecipitated sample.

Figure 2. A summary of the effect of SUMO ligase-null mutations on the abundance of SUMO targets as measured by quantitative MS.

Data are shown in the same order of siz1Δ, siz2Δ, siz1Δ siz2Δ and mms21-11 mutants from left to right for each SUMO target. Log2 scale was used to calculate the relative abundance ratios. Occasionally, some SUMO targets were not identified and are indicated as not available (N/A). Additional information can be found in Tables S3, S4, S5, S6.

Figure 3. Identification of SUMO targets that are more specific to Mms21 than Siz1 and Siz2.

A) Grey bar shows the calculated ratio between Mms21-dependent ratio and Siz1/Siz2 dependent ratio, using the data shown in Figure 2. Black bar shows a direct comparison between mms21-11 and siz1Δ siz2Δ mutants using quantitative MS. B) Effects of mm21-11 and siz1Δ siz2Δ on the sumoylation of selected SUMO targets. Each SUMO target was purified by anti-HA antibody and probed for its sumoylation using anti-FLAG Western blot.

Figure 4. Effect of esc2Δ on the sumoylation of SUMO targets.

A) Bar graph shows the relative abundance of SUMO targets between esc2Δ and wild-type cells (black bars). As a comparison, the results of mms21-11 versus wild-type cells are shown in grey bars. B) Quantification of the relative abundance of SUMO targets between esc2Δ and mms21-11 mutants. C) Western blot analysis of the effect of esc2Δ on the sumoylation of SMC proteins and Rpa135. Each SUMO target was purified by anti-HA antibody and probed for its sumoylation by anti-FLAG Western blot.

Figure 5. Effect of slx5Δ on the sumoylation of SUMO targets.

A) Bar graph shows the relative abundance of SUMO targets between slx5Δ and wild-type cells (black bars). Results of mms21-11 versus wild-type are included for comparison (grey bars). B) Bar graph shows the relative abundance of SUMO targets between slx5Δ siz2Δ and wild-type cells (black bars). Results of siz2Δ versus wild-type are included for comparison (grey bars). C) A proposed model of the regulation of sumoylation homeostasis in cells. Mms21-specific sumoylation is positively regulated by Esc2 and negatively regulated by Slx5 to achieve a balance of sumoylation in cells, which is critical to suppress GCRs.