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

鞘脂是一类丰富存在于植物、酵母和哺乳动物质膜外层的脂类（脂肪）。正确的鞘脂稳态支持多种途径，如蛋白质的分类和传递、内化、对刺激的信号反应和对高温的抵抗。鞘脂合成的第一步是由一种叫做“丝氨酸棕榈酰转移酶”（SPT）的酶催化。在酵母中，SPT复合体由三种单独的蛋白Lcb1、Lcb2和Tsc3组成，它们存在于内质网中。当细胞有足够的鞘脂时，SPT复合物被关闭。然而，SPT复合物并不直接感知鞘脂。相反，另外两种蛋白，Orm1和Orm2，感知鞘脂并抑制SPT复合物。SPT复合体、脂质磷酸酶Sac1和Orm1/2一起构成一个更大的复合体，称为spot复合体。人们对斑点复合体的结构知之甚少。这一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spot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，以感知其丰富程度。第二ORM1/2与SPT酶物理相互作用并抑制SPT酶。斑点复合物成分的缺失或部分功能丧失导致多种生长缺陷。例如，ORM1和ORM2的缺失会导致细胞无法在低温下生长。利用这种表型，我们在随机诱变筛选后分离出几个突变体。接下来的步骤将是确定为什么这些突变体没有功能，目标是确定蛋白质的关键功能部分。最终，我们希望能更清楚地了解鞘脂水平是如何被调节的。在植物、酵母和动物中，适当的鞘脂稳态是支持细胞活力和适应高温或低温生长的必要条件。

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.