SPHINGOLIPID BIOSYNTHESIS IN YEAST

鞘磷脂在所有真核细胞中都是必不可少的，而 鞘磷脂代谢发生在许多疾病过程中。尽管一个 鞘脂及其代谢物的各种细胞功能有 已经提出，但对它们的生物合成和生理作用知之甚少。 角色。在真核生物模型中合成鞘磷脂，酿酒酵母将 采用遗传/生化相结合的方法进行研究。一个 神经鞘脂脂缺陷突变株(csg2、scs1-scs7)的收集 新陈代谢已经被分离出来，并将用于分离野生型 神经鞘脂合成所需的基因。SCS突变体将会是 转化基因组文库，克隆野生型基因 SCS表型的互补。每个SCS基因的功能将 通过确定破坏的生物化学后果来进行探索 通过将SCS基因产物的氨基酸序列与 蛋白质数据库，通过研究上位性关系 在SCS、CSG、SEC和PMR突变体之间，并通过鉴定细胞 基因产物的位置。上位性的确定 Csg2、scs、sec和pmr突变体之间的关系将提供 对生物合成途径的洞察(包括生物合成的作用顺序 生物合成酶及其细胞位置)。抑制者分析 SCS基因将用于识别其他基因和蛋白质 鞘磷脂合成在CSG和SCS集合中未发现。因为我们的 SCS突变体集合由每个SCS基因的几个等位基因组成， 改变相互作用蛋白质活性的突变以及 完全绕过SCS基因功能的基因将被识别出来。这个 这些实验的结果应该会提供关于 催化和调节鞘脂合成的基因和蛋白质 以及鞘脂在所有真核细胞中的作用。

Sphingolipids are essential in all eukaryotic cells and alterations in sphingolipid metabolism occur in many disease processes. Although a variety of cellular functions for sphingolipids and their metabolites have been proposed, little is known about their biosynthesis and physiological roles. Sphingolipid synthesis in the model eukaryote, S. cerevisiae will be investigated using a combined genetic/biochemical approach. A collection of mutants (csg2, scs1-scs7) with defects in sphingolipid metabolism has been isolated, and will be used to isolate the wild type genes required for sphingolipid synthesis. The scs mutants will be transformed with a genomic library and the wild type genes cloned by complementation of the scs phenotype. The function of each SCS gene will be probed by determining the biochemical consequence of disrupting the gene, by comparing the amino acid sequence of the SCS gene products with the protein data base, by investigating the epistatic relationships between scs, csg, sec and pmr mutants, and by identifying the cellular location of the gene products. Determination of the epistatic relationships between the csg2, scs, sec, and pmr mutants will provide insight into the biosynthetic pathway (both the order of action of the biosynthetic enzymes and their cellular location). Suppressor analysis of scs genes will be used to identify other genes and proteins in sphingolipid synthesis not found in the CSG and SCS collections. Since our scs mutant collection consists of several alleles of each scs gene, mutations that alter the activity in interacting proteins as well as those that bypass the scs gene function altogether will be identified. The results of these experiments should provide valuable information about the genes and proteins that catalyze and regulate sphingolipid synthesis as well as the roles of sphingolipids in all eukaryotic cells.