Transmembrane topology of mammalian ORMDL proteins in the endoplasmic reticulum as revealed by the substituted cysteine accessibility method (SCAM™).

Sphingolipids are diverse lipids with essential, and occasionally opposing, functions in the cell and therefore tight control over biosynthesis is vital. Mechanisms governing this regulation are not understood. Initial steps in sphingolipid biosynthesis take place on the cytosolic face of the endoplasmic reticulum (ER). Serine palmitoyltransferase (SPT) is an ER-resident enzyme catalyzing the first-committed step in sphingolipid biosynthesis. Not surprisingly, SPT activity is tightly regulated. ORMDLs are ER-resident proteins recently identified as regulators of SPT activity. ORMDL proteins interact directly with SPT but the nature of this interaction is unknown. ORMDL protein sequences contain hydrophobic regions, yet algorithm-based predictions of transmembrane segments are highly ambiguous, making topology of this key regulator unclear. Here we report use of substituted cysteine accessibility to analyze topology of mammalian ORMDLs. We constructed multiple mutant ORMDLs, each containing a single cysteine strategically placed along the protein length. Combined use of selective membrane permeabilization with an impermeant cysteine modification reagent allowed us to assign transmembrane and cytosolic segments of ORMDL. We confirmed that mammalian ORMDL proteins transit the membrane four times, with amino- and carboxy termini facing the cytosol along with a large cytosolic loop. This model will allow us to determine details of the ORMDL-SPT interaction and identify regions acting as the "lipid sensor" to detect changes in cellular sphingolipid levels. We also observe that SPT and ORMDL are substantially resistant to extraction from membranes with non-ionic detergent, indirectly suggesting that both proteins reside in a specialized subdomain of the ER.