Eukaryotic cells utilize the dynamic addition and removal of post-translational modifications to modulate protein function. Two such modifiers are ubiquitin and SUMO (Small Ubiquitin-like MOdifier), which traditionally regulate their substrates in opposite ways. The discovery of SUMO-targeted ubiquitin ligases (STUbLs), E3 ligases that ubiquitylate sumoylated targets, offers an opportunity for cross-talk between the SUMO and ubiquitin pathways. STUbLs are crucial for the response to DNA damage and maintenance of genomic integrity, but currently only a few STUbL substrates are known. Recently, we observed a novel interaction between the yeast STUbL subunit Slx5 and the SUMO ligase Siz1 both in a yeast two-hybrid system and in vitro. The goals of this study were to develop protein extraction and purification protocols for the purpose of determining if Slx5 and Siz1 also interact in vivo. This study additionally seeks to determine if Siz1 is a target for ubiquitylation by Slx5 in vivo. Here we report our finding that Slx5 and Siz1Δ440 co-affinity purify in in vivo pulldown experiments, and that Siz1Δ440 is ubiquitylated in vivo in an Slx5-dependent manner. We also describe the intrinsic binding ability of the RING domain present in E3 ligases for metal affinity purification
