Orm2 promotes nitrogen-induced sphingolipid production and endocytosis via Orm1 phosphorylation.

The Orm family of proteins inhibit serine palmitoyltransferase-the enzyme that catalyzes the first step in sphingolipid synthesis. In S. cerevisiae, the two Orm proteins are thought to function redundantly in suppressing sphingolipid production. Here, we show that Orm2, in contrast, promotes production of complex sphingolipids by regulating Ypk1-dependent phosphorylation of Orm1 through control of long-chain base (LCB) levels, the initial precursors in the sphingolipid biosynthesis pathway. Using targeted lipidomic analysis of orm1Δ versus orm2Δ strains, the results showed that Orm1 regulates complex sphingolipid levels, whereas Orm2 primarily modulates the production of LCBs. We then show that reduced Orm1 phosphorylation in orm2Δ cells was mediated by LCB-dependent inactivation of AGC family protein kinase Ypk1. We further demonstrate that this Orm2-LCBs-Ypk1-Orm1 regulatory module is responsive to nitrogen availability, promoting sphingolipid synthesis under nitrogen-rich conditions. Functionally, this pathway is required for nitrogen-induced endocytosis of the general amino acid permease Gap1. Together, our findings reveal that Orm2 governs sphingolipid production and downstream endocytic events via a nitrogen-responsive LCBs-Ypk1-Orm1 signaling pathway, linking nitrogen status to sphingolipid metabolism and membrane trafficking.