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

鞘磷脂是一类脂(脂)，在植物、酵母和哺乳动物的质膜外层中含量丰富。适当的鞘磷脂动态平衡是支持多种途径的必要条件，如蛋白质的分选和运输、内化、信号对刺激的反应以及对高温的抵抗。鞘脂合成的第一步是由一种名为丝氨酸棕榈酰基转移酶(SPT)的酶催化的。在酵母中，SPT复合体由三种单独的蛋白质组成，即Lcb1、Lcb2和Tsc3，它们位于内质网中。当细胞有足够的鞘磷脂时，SPT复合体被关闭。然而，SPT复合体并不直接感受鞘磷脂。相反，另外两种蛋白质Orm1和Orm2感知鞘脂并抑制SPT复合体。SPT复合体、脂质磷酸酶SAC1和Orm1/2一起组成了一个更大的复合体，称为斑点复合体。人们对斑点复合体的组织知之甚少。这一建议旨在提高我们对两个同源Orm蛋白的结构/功能以及SPT和SPOTS复合体的组织的理解。我们将结合酵母遗传学和细胞生物学以及蛋白质和脂肪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(一种神经鞘脂脂)结合，以检测其丰度。第二，ORM1/2在物理上与SPT酶相互作用并抑制其活性。斑点成分的缺失或部分功能丧失会导致多种生长缺陷。例如，ORM1和ORM2的丢失会导致细胞不能在低温下生长。利用这种表型，我们经过随机诱变筛选，分离到了几个突变体。下一步将是确定为什么这些突变体是无功能的，目的是确定蛋白质的关键功能部分。最终，我们希望更清楚地了解鞘脂水平是如何受到调节的。在植物、酵母和动物中，适当的鞘磷脂动态平衡对于支持细胞存活和适应高温或低温生长是必不可少的。

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.