Utilizing a nanobody recruitment approach for assessing serine palmitoyltransferase activity in ER sub-compartments of yeast

Abstract

Sphingolipids (SP) are one of the three major lipid classes in eukaryotic cells and serve as38 structural components of the plasma membrane. The rate-limiting step in SP biosynthesis is39 catalyzed by serine palmitoyltransferase (SPT). In yeast, SPT consists of two catalytic subunits40 (Lcb1 and Lcb2), a regulatory subunit (Tsc3), negative regulators (Orm1 and Orm2), and the41 phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1, collectively known as the SPOTS42 complex. Regulating SPT activity enables cells to adapt SP metabolism to changing43 environmental conditions. Therefore, the Orm proteins are phosphorylated by two signaling44 pathways originating from either the plasma membrane localized target of rapamycin (TOR)45 complex 2 or the lysosomal/vacuolar TOR complex 1. Moreover, uptake of exogenous serine46 is necessary for the regulation of SP biosynthesis, which suggests the existence of differentially47 regulated SPT pools based on their intracellular localization.