Developing an in-vivo screening genetic approach for identifying the SUMO interactome in yeast and discovering new SUMOylation mediator proteins

SUMOylation, a covalent attachment of the small ubiquitin-related modifier (SUMO) to the target protein, has been implicated in a wide range of essential cellular functions through diverse mechanisms that remain to be fully understood. Using the yeast Saccharomyces cerevisiae, a model organism with a single essential SUMO gene (SMT3), we developed a plate- based screening assay in which the protein complementation assay (PCA), to systematically identify the global SUMO interactome.

In this system a strain carrying SMT3 fused to the N-terminal end of a reporter protein, is systematically crossed to an ordered array of a genomic library fused to the C-terminal end of this reporter. Upon interaction of Smt3 with one of the yeast proteins, the reporter protein fragments are brought into proximity, thus reconstituting the activity of the reporter, such that it provides a detectable signal.

We demonstrate the feasibility of this approach by identifying previously identified and novel proteins that interacts with SUMO. Selected interactions are now selected for validation and will be then subjected to the reverse PCA screening system, that was previously developed in our lab. This system enables the identification of genes whose products are required for modulating the physical interaction between SUMO and specific target protein. We believe that the combination of these approaches will reveal new components responsible for SUMO conjugation and deconjugation events which currently are largely unknown.