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

Abstract

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.

ORMDL2 Deficiency Potentiates the ORMDL3-Dependent Changes in Mast Cell Signaling

“…The extent of identity between ORMDL2 and ORMDL3 at the protein level is 83% in mice and 80% in humans ( 7 ). The graphical comparison of the primary amino acid sequence of mouse ORMDL2 and ORMDL3, based on the recently determined topology of ORMDL1 by Davis et al ( 46 ), is shown in Figure 2A . The difference between proteins is subtle, and most of the dissimilar amino acids are in the membrane region.…”

“… The role of ORMDL2 and ORMDL3 and their redundancy in the metabolism of sphingolipids in BMMCs. (A) Left: predicted topology of murine ORMDL2 based on recently described ORMDL1 topology ( 46 ). Residues marked in the murine ORMDL2 by green color indicate conserved amino acids, in red are shown residues which are different in mORMDL3.…”

Evolution and insights into the structure and function of the DedA superfamily containing TMEM41B and VMP1

“…Once a protein is conjugated with PEG-maleimide, it becomes larger and can be separated from an unconjugated form by SDS-PAGE (Fig. 5B) (Davis et al, 2019). As PEG-maleimide is cell-impermeable, specific labeling is achieved only after membrane permeabilization with a detergent (Fig.…”

Sphingosine‐1‐phosphate controls endothelial sphingolipid homeostasis via ORMDL

“…Interestingly, S1P‐mediated PHD inhibition was, at least in part, mediated by AKT signaling, in agreement with previous studies. Considering the proposed structure of ORMDLs (Davis et al , 2019a), P137 has been predicted to be the penultimate amino acid in the second transmembrane domain, which does not necessarily preclude the accessibility of this amino acids by PHDs. For instance, enzymatic‐mediated palmitoylation of cysteines located inside or close to the transmembrane domain has been shown for several proteins (Charollais & Van Der Goot, 2009).…”