Spatiotemporal control of subcellular O-GlcNAc signaling using Opto-OGT

The post-translational modification of intracellular proteins through O-linked β- N -acetylglucosamine ( O -GlcNAc) is a conserved regulatory mechanism in multicellular organisms. Catalyzed by O -GlcNAc transferase (OGT), this dynamic modification has an essential role in signal transduction, gene expression, organelle function and systemic physiology. Here, we present Opto-OGT, an optogenetic probe that allows for precise spatiotemporal control of OGT activity through light stimulation. By fusing a photosensitive cryptochrome protein to OGT, Opto-OGT can be robustly and reversibly activated with high temporal resolution by blue light and exhibits minimal background activity without illumination. Transient activation of Opto-OGT results in mTORC activation and AMPK suppression, which recapitulate nutrient-sensing signaling. Furthermore, Opto-OGT can be customized to localize to specific subcellular sites. By targeting OGT to the plasma membrane, we demonstrate the downregulation of site-specific AKT phosphorylation and signaling outputs in response to insulin stimulation. Thus, Opto-OGT is a powerful tool for defining the role of O -GlcNAcylation in cell signaling and physiology. An optogenetic tool called Opto-OGT has been developed that enables researchers to probe pathways involving modifications with O-linked β- N -acetylglucosamine ( O -GlcNAc) such as OGT, mTOR and AMPK signaling with high spatiotemporal precision. The method is based on fusing a photosensitive cryptochrome protein to an O -GlcNAc transferase and enables OGT to be reversibly activated with blue light.