Investigating sphingolipid metabolism in budding yeast using stable isotope-labeled and caged vacuole-specific probes

Abstract

Sphingolipids constitute a class of lipid compounds which carry out important structural and signaling functions in the cells. However, the regulation of their biosynthesis is not completely understood. By combining genetics, lipidomics and metabolomics our study shows that localization of serine palmitoyltransferase in the nuclear ER increase ceramide biosynthesis rate. Our knowledge of the metabolism and transport of sphingolipids between intracellular organelles is limited. In this study we characterize caged vacuole-targeted sphingolipid probes which can be released by UV-irradiation. We established a method to investigate the transport of sphingolipids from the vacuole to the ER in vivo. We observed that the depletion of contact sites between the endoplasmic reticulum and the vacuole decreases metabolic conversion and transport of sphingolipids released from the vacuole. Overexpression of contact site protein Mdm1, which expands the contacts between the endoplasmic reticulum and the vacuoles, increases sphingolipid transport