The ORMDL/Orm–serine palmitoyltransferase (SPT) complex is directly regulated by ceramide: Reconstitution of SPT regulation in isolated membranes

Abstract

Edited by George M. CarmanSphingolipids compose a lipid family critical for membrane structure as well as intra-and intercellular signaling. De novo 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. . 2 The abbreviations used are: LCB, long chain bases; SPT, serine palmitoyltransferase; scSPT, single chain SPT construct (SPTLC2-ssSPTa-SPTLC1 as a fusion protein in a single polypeptide); C(X), (phyto)ceramide-ceramide or phytoceramide, where X ϭ acyl chain length; TBS, Tris-buffered saline; PVDF, polyvinylidene difluoride.

Chlamydia trachomatis‐infected human cells convert ceramide to sphingomyelin without sphingomyelin synthases 1 and 2

“…1B). A recent paper demonstrated that ceramides directly inhibited serine palmitoyltransferase in an ORMDL-dependent manner [47]. We also previously observed that disruption of CERT caused some suppression of de novo sphingolipid biosynthesis including glycolipids in CHO cells [12].…”

Dynamics of sphingolipids and the serine palmitoyltransferase complex in rat oligodendrocytes during myelination

“…The ORMDLs are homeostatic regulators of SPT, inhibiting activity of this enzyme as cellular sphingolipids rise (reviewed in (17)). The ORMDL/SPT complex responds directly to levels of ceramide (65). There are three ORMDL isoforms.…”

Decreased sphingolipid synthesis in children with 17q21 asthma–risk genotypes

“…ORMDL3 inhibits serine-palmitoyltransferase (SPT), the rate-limiting enzyme complex of de novo sphingolipid synthesis (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI130860DS1) (8)(9)(10). Sphingolipids are essential building blocks of cellular membranes and constitute key cellular signaling mediators (11).…”

“…Sphinganine can be phosphorylated to sphinganine-1-phosphate or acylated to dihydroceramides. These analytes characterize the de novo sphingolipid synthesis pathway and are not generated through other means (8)(9)(10). Dihydroceramides are metabolized to ceramides that, together with sphingomyelins and complex glycosphingolipids, constitute the bulk of mammalian sphingolipids.…”