Abstract
The relationship between serine palmitoyltransferase (SPT) activity and ORMDL regulation of sphingolipid biosynthesis was investigated in mammalian HEK293 cells. Each of the three human ORMDLs reduced the increase in long-chain base synthesis seen after overexpression of wild-type SPT or SPT containing the C133W mutation in hLCB1, which produces the non-catabolizable sphingoid base, 1-deoxySa. ORMDL-dependent repression of sphingoid base synthesis occurred whether SPT was expressed as individual subunits or as a heterotrimeric single-chain SPT fusion protein. Overexpression of the single-chain SPT fusion protein under the control of a tetracycline-inducible promoter in stably transfected cells resulted in increased endogenous ORMDL expression. This increase was not transcriptional; there was no significant increase in any of the ORMDL mRNAs. Increased ORMDL protein expression required SPT activity since overexpression of a catalytically inactive SPT with a mutation in hLCB2a had little effect. Significantly, increased ORMDL expression was also blocked by myriocin inhibition of SPT as well as fumonisin inhibition of the ceramide synthases, suggesting that increased expression is a response to a metabolic signal. Moreover, blocking ORMDL induction with fumonisin treatment resulted in significantly greater increases in in vivo SPT activity than was seen when ORMDLs were allowed to increase, demonstrating the physiological significance of this response.
Highlights
In contrast to their yeast orthologues, the mechanism by which mammalian ORMDLs regulate serine palmitoyltransferase is not understood
The ORMDLs Inhibit in Vivo serine palmitoyltransferase (SPT) Activity—We have previously shown that overexpression of hLCB1 and hLCB2a results in a modest increase in catalytic activity of the heterodimer in vitro, which can be increased nearly 100-fold by the coexpression of an ssSPT isoform [11]
Despite clear evidence that SPT activity is tightly regulated, there is no evidence that either of the catalytic subunits or the ssSPTs are themselves regulated in response to changes in cellular sphingolipid levels at the level of transcription or translation [15], leading to the conclusion that there must be other regulatory components of the SPT complex responsible for sphingolipid homeostasis
Summary
In contrast to their yeast orthologues, the mechanism by which mammalian ORMDLs regulate serine palmitoyltransferase is not understood. Results: Overexpression of serine palmitoyltransferase in HEK293 cells results in increased long-chain base synthesis and an activity-dependent increase in ORMDL expression. Overexpression of the singlechain SPT fusion protein under the control of a tetracyclineinducible promoter in stably transfected cells resulted in increased endogenous ORMDL expression. Roelants et al demonstrated that the yeast Ypk and Ypk kinases phosphorylate this domain and that their deletion results in loss of viability, presumably as a result of constitutive repression of long-chain base (LCB) synthesis. This hypothesis is supported by the fact that deletion of the Orms rescues the ypk1⌬ypk2⌬ kinase knock-out mutant [3]. While highly homologous to their yeast counterparts, they lack the major portion of the N-terminal domain of the yeast Orm proteins that is phosphorylated by Ypk, Ypk,
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