The SUMO pathway plays a role in RENT complex rDNA binding

The post-translational modification of eukaryotic proteins with other proteins, most notably ubiquitin and small ubiquitin-related modifier (SUMO), is a common mechanism of cellular regulation. Modification with ubiquitin typically leads to degradation of the modified protein by the proteasome, and the ubiquitin-proteasome system (UPS) regulates a myriad of cellular processes. SUMO does not directly target proteins for degradation, but rather modulates the activity or protein-protein interactions of the modified protein. While the SUMO system has been linked to multiple processes and many SUMO-modified proteins have been identified, unappreciated substrates and functions of the SUMO system are expected. This thesis describes novel insights into the role of the SUMO pathway in the regulation of rDNA, which encodes the RNA component of the ribosome, the universal machinery for biological protein synthesis.;The SUMO system is dynamic, with SUMO modification reversed by enzymes called SUMO proteases. The model yeast Saccharomyces cerevisiae expresses two SUMO proteases: UIp1 and UIp2. Genetic studies have connected Ulp1 and UIp2 with the heterodimeric SUMO-targeted ubiquitin ligase (STUbL) complex: Slx5-Slx8. STUbL enzymes enable the addition of ubiquitin to sumoylated proteins, and proteasome-mediated degradation of these co-modified proteins is thought to be an alternative mechanism to de-sumoylation for counteracting the effects of SUMO on a given process. To identify substrates of UIp2 and SIx5-SIx8, I developed a strategy for large-scale isolation of High-Molecular Weight (HMW)-SUMO conjugates from ulp2Delta and ulp2Delta slx5Delta cells and identified the modified proteins by mass spectrometry.;Two of the proteins identified as SUMO-modified proteins, Net1 and Tof2, are involved in the regulation of rDNA silencing. I show that both Net1 and Tof2 are targets of Ulp2 and that Net1 and Tof2 lose binding to rDNA in ulp2Delta and ulp1ts cells. I also show that Fob1, the replication fork barrier (RFB) binding protein that anchors both Net1 and Tof2 to the RFB, displays reduced rDNA binding in ulp2Delta cells, but retains normal binding to the rDNA in ulp1ts cells. Fob1 is poly-sumoylated in wild type cells and the sumoylation levels decrease in ulp1ts cells and increase in ulp2Delta cells. Overexpressing the regulator of nucleolar silencing and telophase exit (RENT) complex subunits Sir2 or Net1 suppresses some growth defects of ulp2Delta cells, suggesting a defect in RENT complex formation, dissolution, or activity in ulp2Delta cells. While the exact mechanism of SUMO regulation of the RENT complex remains obscure, the results of this thesis imply an important role for the SUMO proteases UIp1 and UIp2 and the STUbL Slx5/Slx8 in rDNA regulation by the RENT complex.