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

鞘脂是一类富含于植物、酵母和哺乳动物质膜外层的脂质（脂肪）。需要适当的鞘脂稳态来支持许多途径，例如蛋白质分选和递送、内化、响应刺激的信号传导和对高温的抗性。鞘脂合成的第一步是由一种称为“丝氨酸棕榈酰转移酶”（SPT）的酶催化的。在酵母中，SPT复合物由三种单独的蛋白质组成，Lcb 1，Lcb 2和Tsc 3，它们位于内质网中。当细胞具有足够的鞘脂时，SPT复合物就会关闭。然而，SPT复合物不直接感测鞘脂。相反，两个额外的蛋白质，Orm 1和Orm 2，感觉鞘脂和抑制SPT复合物。SPT复合物、脂质磷酸酶Sac 1和Orm 1/2一起构成称为SPOTS复合物的较大复合物。人们对SPOTS综合体的组织结构知之甚少。该建议旨在提高我们对两种同源Orm蛋白的结构/功能以及SPT和SPOTS复合物的组织的理解。 我们将结合酵母遗传学和细胞生物学以及蛋白质和脂质生物化学来解决这些问题。我们假设Orm蛋白具有两个关键特征。首先，这种蛋白质可以与神经酰胺（一种鞘脂）结合，以感知其丰度。第二种ORM 1/2与SPT酶物理相互作用并抑制SPT酶。SPOTS复合物的组分的缺失或部分功能丧失导致各种生长缺陷。例如，ORM 1和ORM 2的缺失导致细胞在低温下无法生长。使用这种表型，我们已经分离出几个突变体后，随机诱变筛选。下一步将是确定为什么这些突变体是非功能性的，目的是确定蛋白质的关键功能部分。最终，我们希望能够更清楚地了解鞘脂水平是如何调节的。适当的鞘脂稳态对于支持细胞活力和适应植物、酵母和动物在高温或低温下的生长至关重要。

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.