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

Deanna L Davis,Kenneth Gable,John Suemitsu,Teresa M Dunn,Binks W Wattenberg

doi:10.1074/jbc.ra118.007291

Abstract

Sphingolipids 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.

Highlights

Sphingolipids compose a lipid family critical for membrane structure as well as intra- and intercellular signaling
Incubation with C8 ceramide strongly inhibited de novo sphingolipid biosynthesis. This inhibition was eliminated if the cells were depleted of all three ORMDL isoforms by siRNA transfection, similar to our previous findings [8], confirming that the ORMDLs are required for homeostatic regulation of serine palmitoyltransferase (SPT)
As before we found that in permeabilized cells C8 ceramide inhibits SPT activity, but that the response is eliminated in cells depleted of the ORMDLs

Summary

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Insight into the mechanism underlying homeostatic control of SPT has been advanced by the discovery of small membrane-bound proteins of the endoplasmic reticulum, the Orms in yeast and ORMDLs in mammals, which are required for this regulation [6, 8, 11, 12]. In yeast Orms are phosphorylated at N-terminal serine residues by the Ypk kinase, downstream of TORC2, in response to reduced cellular sphingolipid levels [6, 12, 16]. The mammalian ORMDLs, otherwise homologous to the yeast proteins, lack the N-terminal peptide region that is phosphorylated in yeast (reviewed in Ref. 17) This suggests that the mammalian ORMDL–SPT complex utilizes a distinct mechanism to both sense cellular sphingolipid. We utilize a cell-free reconstitution of sphingolipid regulation of the ORMDL–SPT complex to characterize the underlying mechanism

Results

Discussion

Experimental procedures