Phosphatidic acid (PA) is both a central phospholipid biosynthetic intermediate and a multifunctional lipid second messenger produced at several discrete subcellular locations. Organelle-specific PA pools are believed to play distinct physiological roles, but tools with high spatiotemporal control are lacking for unraveling these pleiotropic functions. Here, we present an approach to precisely generate PA on demand on specific organelle membranes. We exploited a microbial phospholipase D (PLD), which produces PA by phosphatidylcholine hydrolysis, and the CRY2–CIBN light-mediated heterodimerization system to create an optogenetic PLD (optoPLD). Directed evolution of PLD using yeast membrane display and IMPACT, a chemoenzymatic method for visualizing cellular PLD activity, yielded a panel of optoPLDs whose range of catalytic activities enables mimicry of endogenous, physiological PLD signaling. Finally, we applied optoPLD to elucidate that plasma membrane, but not intracellular, pools of PA can attenuate the oncogenic Hippo signaling pathway. OptoPLD represents a powerful and precise approach for revealing spatiotemporally defined physiological functions of PA.

中文翻译：

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利用光遗传学、工程化磷脂酶 D 对磷脂酸信号传导进行时空控制


磷脂酸 (PA) 既是中央磷脂生物合成中间体，又是在几个离散的亚细胞位置产生的多功能脂质第二信使。人们认为细胞器特异性的 PA 库发挥着独特的生理作用，但缺乏具有高度时空控制的工具来揭示这些多效性功能。在这里，我们提出了一种在特定细胞器膜上按需精确生成 PA 的方法。我们利用微生物磷脂酶 D (PLD)（通过磷脂酰胆碱水解产生 PA）和 CRY2-CIBN 光介导的异二聚化系统来创建光遗传学 PLD (optoPLD)。使用酵母膜展示和 IMPACT（一种用于可视化细胞 PLD 活性的化学酶方法）对 PLD 进行定向进化，产生了一组 optoPLD，其催化活性范围能够模拟内源性生理 PLD 信号传导。最后，我们应用 optoPLD 来阐明质膜而非细胞内的 PA 池可以减弱致癌的 Hippo 信号通路。 OptoPLD 代表了一种强大而精确的方法，用于揭示 PA 的时空定义的生理功能。