From the Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298.
the Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, and.
J Biol Chem. 2019 Mar 29;294(13):5146-5156. doi: 10.1074/jbc.RA118.007291. Epub 2019 Jan 30.
Sphingolipids compose a lipid family critical for membrane structure as well as intra- and intercellular signaling. sphingolipid biosynthesis is initiated by the enzyme serine palmitoyltransferase (SPT), which resides in the endoplasmic reticulum (ER) membrane. In both yeast and mammalian species, SPT activity is homeostatically regulated through small ER membrane proteins, the Orms in yeast and the ORMDLs in mammalian cells. These proteins form stable complexes with SPT. In yeast, the homeostatic regulation of SPT relies, at least in part, on phosphorylation of the Orms. However, this does not appear to be the case for the mammalian ORMDLs. Here, we accomplished a cell-free reconstitution of the sphingolipid regulation of the ORMDL-SPT complex to probe the underlying regulatory mechanism. Sphingolipid and ORMDL-dependent regulation of SPT was demonstrated in isolated membranes, essentially free of cytosol. This suggests that this regulation does not require soluble cytosolic proteins or small molecules such as ATP. We found that this system is particularly responsive to the pro-apoptotic sphingolipid ceramide and that this response is strictly stereospecific, indicating that ceramide regulates the ORMDL-SPT complex via a specific binding interaction. Yeast membranes harboring the Orm-SPT system also directly responded to sphingolipid, suggesting that yeast cells have, in addition to Orm phosphorylation, an additional Orm-dependent SPT regulatory mechanism. Our results indicate that ORMDL/Orm-mediated regulation of SPT involves a direct interaction of sphingolipid with the membrane-bound components of the SPT-regulatory apparatus.
鞘脂组成了一个脂质家族，对于膜结构以及细胞内和细胞间信号传递至关重要。鞘脂生物合成由位于内质网膜（ER）中的丝氨酸棕榈酰转移酶（SPT）酶起始。在酵母和哺乳动物物种中，SPT 活性通过 ER 膜上的小膜蛋白（酵母中的 Orm 和哺乳动物细胞中的 ORMDLs）进行同源调节。这些蛋白质与 SPT 形成稳定的复合物。在酵母中，SPT 的同源调节至少部分依赖于 Orm 的磷酸化。然而，对于哺乳动物 ORMDLs 来说，情况似乎并非如此。在这里，我们通过无细胞体系重建了 ORMDL-SPT 复合物的鞘脂调控，以探究潜在的调控机制。在分离的膜中，即基本不含胞质溶胶的情况下，证明了鞘脂和 ORMDL 对 SPT 的依赖性调控。这表明这种调控不需要可溶性胞质溶胶蛋白或小分子（如 ATP）。我们发现，该系统对促凋亡鞘脂神经酰胺特别敏感，而且这种反应具有严格的立体特异性，表明神经酰胺通过特定的结合相互作用来调节 ORMDL-SPT 复合物。含有 Orm-SPT 系统的酵母膜也直接对鞘脂产生反应，这表明除了 Orm 磷酸化之外，酵母细胞还具有另一种依赖 Orm 的 SPT 调节机制。我们的结果表明，ORMDL/Orm 介导的 SPT 调节涉及鞘脂与 SPT 调节装置的膜结合成分的直接相互作用。
