SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus inSaccharomyces cerevisiae

Abstract

In this report, we identify cellular targets of Ulp2, one of two Saccharomyces cerevisiae small ubiquitin-related modifier (SUMO) proteases, and investigate the function of SUMO modification of these proteins. PolySUMO conjugates from ulp2D and ulp2D slx5D cells were isolated using an engineered affinity reagent containing the four SUMO-interacting motifs (SIMs) of Slx5, a component of the Slx5/Slx8 SUMO-targeted ubiquitin ligase (STUbL). Two proteins identified, Net1 and Tof2, regulate ribosomal DNA (rDNA) silencing and were found to be hypersumoylated in ulp2D, slx5D, and ulp2D slx5D cells. The increase in sumoylation of Net1 and Tof2 in ulp2D, but not ulp1ts cells, indicates that these nucleolar proteins are specific substrates of Ulp2. Based on quantitative chromatin-immunoprecipitation assays, both Net1 and Tof2 lose binding to their rDNA sites in ulp2D cells and both factors largely regain this association in ulp2D slx5D. A parsimonious interpretation of these results is that hypersumoylation of these proteins causes them to be ubiquitylated by Slx5/ Slx8, impairing their association with rDNA. Fob1, a protein that anchors both Net1 and Tof2 to the replication-fork barrier (RFB) in the rDNA repeats, is sumoylated in wild-type cells, and its modification levels increase specifically in ulp2D cells. Fob1 experiences a 50% reduction in rDNA binding in ulp2D cells, which is also rescued by elimination of Slx5. Additionally, overexpression of Sir2, another RFBassociated factor, suppresses the growth defect of ulp2D cells. Our data suggest that regulation of rDNA regulatory proteins by Ulp2 and the Slx5/Slx8 STUbL may be the cause of multiple ulp2D cellular defects. KEYWORDS SUMO; Ulp2; rDNA; RENT complex; Fob1 P OST-TRANSLATIONAL protein modifications provide a common mechanism for regulating protein-protein interactions, protein localization, and protein degradation. The small ubiquitin-related modifier (SUMO) protein family modifies target proteins through covalent attachment to lysine side chains (Johnson 2004). The function of substrate "sumoylation" varies with the protein that is sumoylated, but a common role is to modulate the assembly of large protein complexes either by creating additional binding sites or by blocking existing sites (Kerscher 2007).Protein sumoylation occurs through an enzymatic cascade similar to ubiquitylation (Johnson 2004). In Saccharomyces cerevisiae, a single gene, SMT3, codes for SUMO, and as in most species, SUMO ligation is essential for viability. Removal of SUMO from target proteins is also an important regulatory step. S. cerevisiae has two SUMO proteases, Ulp1 and Ulp2 (Hickey et al. 2012). Most Ulp1 is bound to the nuclear pore complex (NPC), and the enzyme is essential for cell-cycle progression. NPC tethering regulates Ulp1 activity by restricting its access to sumoylated proteins (Li and Hochstrasser 2003;Panse et al. 2003). Ulp1 also processes the SUMO precursor by removing its last three residues, thereby exposing the C-terminal Gly-Gly motif required for SU...

Loss of the SUMO protease Ulp2 triggers a specific multichromosome aneuploidy

“…Taken together, our results reveal a previously unsuspected phenotype of ulp2Δ mutant cells. Because the Slx5-Slx8 STUbL is likely to process poly-SUMO-conjugated substrates of Ulp2 in ways that are toxic to cells (Gillies et al 2016), the new data imply that aneuploidy is also a response to the action of this STUbL on Ulp2 substrates.…”

“…The SUMO pathway is linked to the ubiquitin-proteasome system through the Slx5-Slx8 heterodimer (Uzunova et al 2007;Xie et al 2007). Slx5-Slx8 is a SUMO targeted ubiquitin ligase (STUbL), and deletion of SLX5 in a ulp2Δ background suppresses the temperature sensitivity of ulp2Δ cells (Gillies et al 2016). The mechanism behind this suppression is uncertain but could reflect restoration of proper transcriptional regulation in the ulp2Δ slx5Δ double mutant.…”

SUMO-Mediated Regulation of Nuclear Functions and Signaling Processes

“…SUMO removal from substrates by desumoylases provides another way of counteracting the effects of STUbLs. For example, the yeast Ulp2 desumoylase protects rDNA-bound sumoylated proteins from STUbL-mediated degradation, thus promoting ribosomal DNA stability (Gillies et al, 2016; Liang et al, 2017). …”

SUMOylation is required for fungal development and pathogenicity in the rice blast fungus Magnaporthe oryzae

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