The First Transmembrane Domain of LCB1 is Essential for Regulation of Serine Palmitoyltransferase (SPT) by the ORMs

SPT catalyzes the first step of sphingolipid biosynthesis. Yeast SPT is comprised of three ER‐associated transmembrane proteins, Lcb1p, Lcb2p and Tsc3p. Basal activity of the Lcb1p‐Lcb2p heterodimer is stimulated several fold by the Tsc3p subunit, which also alters acyl‐CoA substrate preference. Although there are no homologs of Tsc3p in higher eukaryotes, we have recently discovered the ssSPT family of functional orthologs, conserved from S. pombe to humans. In experiments to elucidate how these small subunits interact with the heterodimer, we have observed an interaction with the first transmembrane domain (TMD1) of LCB1. However, this interaction is not critical for activation. Interestingly, the yeast ORMs, recently reported to bind and negatively regulate SPT, also bind to TMD1 of LCB1. This interaction is critical for ORM function as LCB1ΔTMD1 mutants accumulate high level of sphingolipids similar to those observed in the orm1 Δ orm2 Δ mutants, whether or not the ORMs are present. This raises the possibility that the ORMs regulate sphingolipid biosynthesis by modulating activation by the ssSPTs. Although the ORMs bind to the Lcb1p‐Lcb2p heterodimer in absence of the small subunit, it is unknown whether they inhibit basal activity of the heterodimer. We are therefore testing whether the ORMs directly inhibit the heterodimer or act only on the heterotrimer. These studies were supported by USPHS grant NS47717.