Physical and Genetic Interactions Between Ris1 and the Slx5-Slx8 SUMO-Targeted Ubiquitin Ligase
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The Slx5-Slx8 complex is a ubiquitin ligase that preferentially ubiquitylates SUMOylated substrates, targeting them for proteolysis. Mutations in SLX5, SLX8, and other SUMO pathway genes were previously identified in our lab as genomic suppressors of a point mutation ( motl-301) in the transcriptional regulator MOT1. To further understand the links between the SUMO and ubiquitin pathways, a screen was performed for high copy suppressors of motl-301, yielding three genes (MOT3, MIT1 and RIS1). RIS1 is believed to encode another SUMO-targeted ubiquitin ligase (STUbL) that functionally overlaps with Slx5-Slx8 while MOT3 and MIT1 have characteristics of prions. Investigation of RIS1 suggests that the relationship between RIS1 and SLX5 is more complex than expected: Ris1 interacted with Slx5 physically by yeast two-hybrid and co-immunoprecipitation assays, a ris1 mutation that blocked the interaction between RIS1 and SLX5 interfered with RIS1 function, and genetic analyses indicated an antagonistic relationship between RIS1 and SLX5. Combined, our results challenge the assumption that Ris1 and Slx5 are simply partially overlapping STUbLs and begin to illuminate a regulatory relationship between these two proteins. Moreover, further studies reveal that the 2mu MOT3 and MIT1 phenotypes are dependent on Hsp104, a protein required for prion formation. Even though subsequent experiments suggest that 2mu MIT1 suppresses motl-301 through a mechanism other than prion formation, I discovered that Mit1 was SUMOylated. Therefore, these data together indicate a potential association between the SUMO pathway and prions.