A Genetic Screening Method for Mammalian SUMOylated Proteins Using Split Fluorescence Protein Reconstitution
Maki Komiyaa, Mizuki Endoa and Takeaki Ozawaa
from: SUMOylation and Ubiquitination: Current and Emerging Concepts (Edited by: Van G. Wilson). Caister Academic Press, U.K. (2019) Pages: 123-134.
Abstract
SUMOylation is an essential post-translational protein modification in various cellular functions. To clarify the role of SUMOylation, numerous screening approaches have been reported for the discovery of novel SUMOylated proteins. However, the reversibility of SUMOylation and the highly varied SUMOylation levels among targets have made it difficult to detect infrequently-SUMOylated proteins, especially in mammalian cells. Here, we describe a newly-developed screening system for mammalian SUMOylated proteins in living cells, which is based on split fluorescence protein reconstitution and fluorescence-based cell sorting technique. The experiments demonstrated that SUMOylation by SUMO2 was detectable as a fluorescence signal in living mammalian cells, which enabled exploration of SUMOylation candidates without cell destructive processes. The system successfully identified 36 unreported SUMO2-substrate candidates, of which Atac2 was shown as SUMOylated at a lysine 408. We summarized the applicability to other SUMO isoforms and various cell types, which will be able to contribute to broader exploration of the roles of SUMOylation in numerous biological phenomena read more ...
