Sphingolipids (SP) are one of the three major lipid classes in eukaryotic cells and serve as structural components of the plasma membrane. The rate-limiting step in SP biosynthesis is catalyzed by the serine palmitoyltransferase (SPT). In yeast, SPT is negatively regulated by the two proteins, Orm1 and Orm2. Regulating SPT activity enables cells to adapt SP metabolism to changing environmental conditions. Therefore, the Orm proteins are phosphorylated by two signaling pathways originating from either the plasma membrane or the lysosome/vacuole. Moreover, uptake of exogenous serine is necessary for the regulation of SP biosynthesis, which suggests the existence of differentially regulated SPT pools based on their intracellular localization. However, measuring lipid metabolic enzyme activity in different cellular sub-compartments has been challenging. Combining a nanobody recruitment approach with sphingolipid flux analysis, we show that the nuclear ER localized SPT and the peripheral ER localized SPT pools are differentially active. Thus, our data add another layer to the complex network of SPT regulation. Moreover, combining lipid metabolic enzyme re-localization with flux analysis serves as versatile tool to measure lipid metabolism with sub-cellular resolution.

中文翻译：

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鉴定内质网亚区室中酵母丝氨酸棕榈酰转移酶的不同活性池。

鞘脂 (SP) 是真核细胞中三大脂质类别之一，是质膜的结构成分。SP 生物合成中的限速步骤由丝氨酸棕榈酰转移酶 (SPT) 催化。在酵母中，SPT 受到两种蛋白质 Orm1 和 Orm2 的负调节。调节 SPT 活性使细胞能够使 SP 代谢适应不断变化的环境条件。因此，Orm 蛋白被源自质膜或溶酶体/液泡的两条信号通路磷酸化。此外，外源丝氨酸的摄取对于 SP 生物合成的调节是必要的，这表明基于其细胞内定位存在差异调节的 SPT 库。然而，测量不同细胞亚区室中的脂质代谢酶活性一直具有挑战性。将纳米抗体招募方法与鞘脂通量分析相结合，我们发现核内质网局部 SPT 和外周内质网局部 SPT 池的活性存在差异。因此，我们的数据为复杂的 SPT 监管网络添加了另一层。此外，将脂质代谢酶重新定位与通量分析相结合，可作为以亚细胞分辨率测量脂质代谢的通用工具。