Structure/Function and Regulation of the Serine-Palmitoyltransferase Complex.

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Sphingolipids are a class of lipid (fat) that are abundant in the outer layer of the plasma membrane in plants, yeast and mammals. Proper sphingolipid homeostasis is required to support numerous pathways such as protein sorting and delivery, internalization, signaling in response to stimuli and resistance to high temperature. The first step in the synthesis of sphingolipids is catalyzed by an enzyme called "serine palmitoyltransferase" (SPT). In yeast, the SPT complex is comprised of three individual proteins, Lcb1, Lcb2 and Tsc3 that reside in the endoplasmic reticulum. When the cells have sufficient sphingolipids the SPT complex is turned off. However, the SPT complex does not directly sense sphingolipids. Instead, two additional proteins, Orm1 and Orm2, sense sphingolipids and inhibit the SPT complex. Together the SPT complex, a lipid phosphatase Sac1 and Orm1/2 comprise a larger complex termed the SPOTS complex. Little is known about the organization of the SPOTS complex. This proposal seeks to improve our understanding of the structure/function of the two homologous Orm proteins and the organization of the SPT and SPOTS complexes. We will use a combination of yeast genetics and cell biology together with protein and lipid biochemistry to address these questions. We hypothesize that Orm proteins possess two critical features. First, the protein can bind to ceramide (a sphingolipid) to sense its abundance. Second ORM1/2 physically interacts with and inhibits the SPT enzyme. Deletion or partial loss-of-function of components of the SPOTS complex results in with a variety of growth defects. For instance, loss of both ORM1 and ORM2 results in an inability of cells to grow at low temperature. Using this phenotype we have isolated a several mutants following a random mutagenesis screen. The next steps will be to determine why these mutants are non-functional with the goal of identifying the key functional portions of the protein. Ultimately, we hope to obtain a clearer picture of how sphingolipid levels are regulated. Proper sphingolipid homeostasis is an essential to support cell viability and for adaptation to growth at high or low temperatures in plants, yeast and animals.