Identification and characterization of a SUMO-targeted ubiquitin ligase (E3) complex in Saccharomyces cerevisiae

Ubiquitin and ubiquitin-like molecules (Ubls) can be covalently attached to a plethora of protein substrates. Such post-translational modification of proteins mediates diverse biological functions in the cell, including protein degradation, cell signaling, DNA repair and immune responses. Central to substrate conjugation is a mechanistically conserved enzymatic pathway containing E1 (activating), E2 (conjugating) and E3 (ligase) proteins. Among these enzyme families, E3 ligases function at the final step of ubiquitin/Ubl conjugation and dictate the substrate specificity. E3 proteins exist in great numbers and have adopted distinct methods to interact with diverse substrates. Therefore, the identification of E3 proteins and characterization of their mechanistic functions have been central to the study of ubiquitin system.;Hex3 (Slx5) and Slx8 are Saccharomyces cerevisiae proteins with important functions in DNA damage control and maintenance of genomic stability. Both proteins have RING domains common in ubiquitin and Ubl E3s, but little was known about the molecular functions of either protein. We identified HEX3 as a high-copy suppressor of a temperature-sensitive small ubiquitin-related modifier (SUMO) protease mutant, ulp1ts, suggesting that it may affect cellular SUMO dynamics. Hex3 forms a heterodimer with Slx8. I found that the Hex3·Slx8 complex has a robust substrate-specific E3 ubiquitin ligase activity. In this E3 complex, Slx8 appears to bear the core ligase function, with Hex3 strongly enhancing its activity. Notably, SUMO attachment to a substrate stimulates its Hex3·Slx8-dependent ubiquitination, primarily through direct noncovalent interactions between SUMO and Hex3. My data reveal a novel mechanism of substrate targeting in which sumoylation of a protein can help trigger its subsequent ubiquitination by recruiting a SUMO-binding ubiquitin ligase.;I also identified the Hex3·Slx8 complex in a genetic screen and found it is required for the turnover of the yeast transcription repressor MATalpha2. Hex3 Slx8 works with Ubc4 (E2) to ubiquitinate MATalpha2, which leads to proteasomal degradation of MATalpha2. However, this targeting mechanism is SUMO-independent insofar as neither SUMO conjugation deficiency nor a SUMO mutant defective for Hex3 interaction was able to inhibit MATalpha2 proteolysis. Instead, the physical association between Hex3 and MATalpha2 may account for the E3-substrate interaction, which nevertheless requires the domain of Hex3 critical for SUMO binding.;Overall, my thesis work has identified the yeast Hex3·Slx8 complex as a novel type of ubiquitin ligase. The two inter-related projects described herein have underscored the important functions of the Hex3·Slx8 E3 in the regulation of ubiquitin and SUMO dynamics in the cell. Homologs of Hex3·Slx8 have been identified by other groups, indicating the conservation of the SUMO-targeted ubiquitin ligase (STUbL) family throughout the eukaryotes. Notably, my discovery that Hex3·Slx8 directly targets MATalpha2 for degradation is the first report that an in vivo STUbL substrate can also be recognized without the SUMO signal. This indicates that Hex3·Slx8, as well as other STUbLs, may realize its full function in the cell by targeting a collection of substrates by either SUMO-dependent or -independent mechanisms.