Li Xiang-Ling, Tei Reika, Uematsu Masaaki, Baskin Jeremy M
Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, 14853, USA.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
bioRxiv. 2023 Aug 31:2023.08.31.555787. doi: 10.1101/2023.08.31.555787.
Phosphatidic acid (PA) is a multifunctional lipid with important metabolic and signaling functions, and efforts to dissect its pleiotropy demand strategies for perturbing its levels with spatiotemporal precision. Previous membrane editing approaches for generating local PA pools used light-mediated induced proximity to recruit a PA-synthesizing enzyme, phospholipase D (PLD), from the cytosol to the target organelle membrane. Whereas these optogenetic PLDs exhibited high activity, their residual activity in the dark led to undesired chronic lipid production. Here, we report ultralow background membrane editors for PA wherein light directly controls PLD catalytic activity, as opposed to localization and access to substrates, exploiting a LOV domain-based conformational photoswitch inserted into the PLD sequence and enabling their stable and non-perturbative targeting to multiple organelle membranes. By coupling organelle-targeted LOVPLD activation to lipidomics analysis, we discovered different rates of metabolism for PA and its downstream products depending on the subcellular location of PA production. We also elucidated signaling roles for PA pools on different membranes in conferring local activation of AMP-activated protein kinase signaling. This work illustrates how membrane editors featuring acute, optogenetic conformational switches can provide new insights into organelle-selective lipid metabolic and signaling pathways.
磷脂酸（PA）是一种具有重要代谢和信号传导功能的多功能脂质，剖析其多效性需要能够以时空精确性扰动其水平的策略。先前用于产生局部PA池的膜编辑方法利用光介导的诱导接近作用，将PA合成酶磷脂酶D（PLD）从细胞质募集到靶细胞器膜上。虽然这些光遗传学PLD表现出高活性，但它们在黑暗中的残余活性导致了不期望的慢性脂质产生。在这里，我们报道了用于PA的超低背景膜编辑器，其中光直接控制PLD的催化活性，而不是定位和对底物的获取，利用插入PLD序列中的基于LOV结构域的构象光开关，并使其能够稳定且无干扰地靶向多种细胞器膜。通过将细胞器靶向的LOVPLD激活与脂质组学分析相结合，我们发现PA及其下游产物的代谢速率因PA产生的亚细胞位置而异。我们还阐明了不同膜上的PA池在赋予AMP激活的蛋白激酶信号局部激活方面的信号传导作用。这项工作说明了具有急性光遗传学构象开关的膜编辑器如何能够为细胞器选择性脂质代谢和信号传导途径提供新的见解。
