<scp>ORMDL</scp>  Proteins Turnover via Proteasome and Autophagy Is Cell‐Type Dependent and Tied to Ceramide Homeostasis

ORMDL proteins are essential negative regulators of the serine palmitoyltransferase (SPT) complex, thereby controlling the rate of de novo sphingolipid synthesis. Although mammalian ORMDLs undergo rapid turnover, the mechanisms regulating their stability remain unclear, with conflicting observations across studies. Here, we combined lipidomics, proteomics, and biochemical assays to investigate ORMDL regulation in HEK293, RPE-1, and primary mouse bone marrow-derived mast cells (BMMCs). Inhibition of SPT by myriocin or of ceramide synthases by fumonisin B₁ (FB₁) profoundly altered sphingolipid composition but induced minimal global proteomic changes while consistently reducing ORMDL protein levels. In contrast, overexpression of a single-chain SPT increased ORMDLs alongside elevated sphingolipids, an effect reversed by myriocin or FB₁. ORMDL loss closely correlated with ceramide depletion and, in HEK293 and RPE-1 cells, was prevented by proteasome inhibition, whereas autophagy inhibition had no effect. In BMMCs, both pathways contributed to ORMDL regulation, consistent with high basal autophagy reflected by elevated LC3-II. The p97/valosin-containing protein ATPase was involved in the regulation of ORMDL turnover in all tested cell lines. Mutation of conserved asparagines (N11/N13) in ORMDL3, which mediate ceramide binding and stabilization of the inhibitory conformation, disrupted association with SPTLC1 and SPTLC2, mimicking myriocin-induced complex dissociation, while FB₁ had a weaker effect. Together, these findings suggest that ceramide depletion is the primary trigger for ORMDL degradation in HEK293, RPE-1, and BMMCs and reveal a proteasome-dependent pathway that can be supplemented by autophagy in cells with high basal autophagic activity.