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.

通过 O-连接 β-N-乙酰氨基葡萄糖 （O-GlcNAc） 对细胞内蛋白进行翻译后修饰是多细胞生物中的一种保守调节机制。在 O-GlcNAc 转移酶 （OGT） 的催化下，这种动态修饰在信号转导、基因表达、细胞器功能和全身生理学中起着重要作用。在这里，我们介绍了 Opto-OGT，这是一种光遗传学探针，允许通过光刺激对 OGT 活性进行精确的时空控制。通过将光敏隐花色素蛋白与 OGT 融合，Opto-OGT 可以通过蓝光以高时间分辨率稳健且可逆地激活，并且在没有照明的情况下表现出最小的背景活性。Opto-OGT 的瞬时激活导致 mTORC 激活和 AMPK 抑制，这概括了营养感应信号传导。此外，Opto-OGT 可以定制以定位于特定的亚细胞位点。通过将 OGT 靶向质膜，我们证明了响应胰岛素刺激的位点特异性 AKT 磷酸化和信号输出的下调。因此，Opto-OGT 是定义 O-GlcNAcylation 在细胞信号传导和生理学中的作用的强大工具。