Homeostatic regulation of sphingolipid synthesis by the ORMDL proteins: Molecular mechanisms
Authors: Wattenberg Binks , Siow Deanna , Sunkara Manjual , Morris Andrew 
Published: November 2023

Sphingolipids are a unique and essential family of lipids which are vital membrane components as well as powerful signaling molecules. Sphingolipids constitute a metabolically‐interrelated, structurally diverse family built on the sphingosine backbone. The overall levels of cellular sphingolipid are determined by the initiating and rate‐limiting enzyme in the pathway, serine‐palmitoyltransferase (SPT) and by the only enzyme which destroys the sphingosine backbone, sphingosine‐1‐phosphate lyase, while the levels of individual members of the family are determined by regulation of the synthetic and degradative enzymes that modify sphingosine and its downstream metabolites. It has been known for decades that serine palmitoyltransferase activity is homeostatically regulated in response to cellular levels of sphingolipids. This is hardly surprising given the essential roles that these lipids play balanced by the pathological consequences of overproduction of individual species. Work in yeast identified two membrane proteins of the endoplasmic reticulum (Orm1 and ‐2) as mediators of this homeostatic response. Subsequently we and others confirmed that the three mammalian Orm orthologues, ORMDL1‐3, also small membrane proteins, homeostatically regulate serine palmitoyltransferase activity in mammalian cells. Here we explore the mechanism by which the ORMDLs sense sphingolipid and regulate serine palmitoyltransferase activity. SPT consists of two major, membrane embedded subunits (SPTLC1 and ‐2) which comprise the active site of the enzyme, and a third, small subunit which enhances activity. We and others have found that the ORMDLs form a stable complex with SPT. We find that the stability of the complex is not altered by the changes in cellular sphingolipid levels. This indicates that sphingolipids trigger a change in conformation within a pre‐existing ORMDL/SPT complex rather than enhancing formation of the complex.An important advance in our studies is the establishment of a cell‐free system which reconstitutes ORMDL‐dependent regulation of SPT. Using this system we find that sphingolipid and ORMDL‐dependent regulation of SPT does not require cytosolic components or ATP, nor can we detect phosphorylation of ORMDL under any conditions. This stands in sharp contrast to regulation of the yeast Orms, which is mediated by phosphorylation. Our cell‐free system has allowed us to test the lipid species to which the ORMDLs respond and to determine the sphingolipid set‐point of this system. Our data indicate that the ORMDLs respond directly to membrane sphingolipid levels leading us to believe that there is a specific sphingolipid binding site within the ORMDLs which, when occupied, triggers a conformational change within the ORMDL/SPT complex, resulting in inhibition of SPT activity.Support or Funding InformationSupported by funds from the James Graham Brown Foundation and a Bridging Grant from the University of Louisville School of Medicine Office of the Vice Dean for Research and grants from the National Institutes of Health and Veterans Administration (HL120507 and RR024598), and work utilized resources from the Lexington Veterans Affairs Medical Center.