RUI: Sphingolipid Metabolism in Plants

9723484林奇之前对参与植物鞘脂新陈代谢的酶反应的描述是拟议研究的基础。在大豆和玉米组织中，涉及鞘磷脂合成和周转的各个酶步骤的细胞内位置将通过细胞分级和标记分析来确定。还将测定分离的膜组分中特定的鞘磷脂含量和组成。标记的鞘脂在细胞内的转移将通过体内鞘脂标记和分级过程相结合的方法来跟踪。使用这种方法，将获得有关神经鞘脂胞内转运的路线和动力学的信息。将通过检测反应对蛋白水解酶的敏感性来研究完整膜泡中葡萄糖神经酰胺合成的拓扑学。此外，还将进一步研究甾基糖苷作为该反应的葡萄糖供体的作用。最后，将在大豆和玉米的微粒体制剂中展示催化合成肌醇磷酸肌醇的酶反应，神经酰胺是肌醇磷酸神经酰胺的生物合成前体。随后，将对其亚细胞分布和拓扑结构进行描述。这项拟议的研究将提供关于植物鞘脂代谢、细胞内运输/区划以及关键酶的拓扑结构的综合观点，这些酶被认为是建立葡萄糖神经酰胺在质膜中不对称跨双层分布的关键酶。它还将提供有关肌醇磷酸神经酰胺合成的第一步的新信息，肌醇磷酸神经酰胺是植物中一种复杂的鞘糖脂。鞘磷脂存在于除细菌以外的所有生物体中，并被认为是细胞中的结构成分和各种生物过程的调节器。然而，人们对鞘脂在植物中的功能和代谢知之甚少。这项拟议的研究是NSF支持的先前工作的继续，将提供关于神经鞘脂脂在细胞中生产、运输和降解的位置和方式的综合观点。它还将首次研究肌醇磷酸神经酰胺的合成，这是一种新的和多样化的复杂植物类脂。这些研究的结果将有助于理解这些不寻常的脂质在植物生长和发育中的作用。

9723484 Lynch The previous characterization of enzymatic reactions involved in sphingolipid metabolism in plants serves as a foundation for the proposed research. The intracellular locations of the individual enzymatic steps involved in sphingolipid synthesis and turnover will be determined in bean and corn tissues using cell fractionation and marker assays. The specific sphingolipid content and composition in the isolated membrane fractions will also be determined. The transfer of labeled sphingolipids through the cell will be tracked by coupling in vivo labeling of sphingolipids and fractionation procedures. Using this approach, information on the route and kinetics of intracellular sphingolipid transport will be obtained. The topology of glucosylceramide synthesis in intact membrane vesicles will be investigated by examining the sensitivity of the reaction to proteolytic enzymes. As well, the role of steryl glucoside as the glucose donor for this reaction will be further investigated. Finally, the enzymatic reaction catalyzing the synthesis of ceramide phosphorylinositol, the biosynthetic precursor for inositol phosphorylceramides, will be demonstrated and characterized in microsomal preparations from bean and corn. Subsequently, its subcellular distribution and topology will be characterized. The proposed research will provide an integrated view of plant sphingolipid metabolism, intracellular transport/compartmentation, and the topology of key enzymes thought to be involved in establishing the asymmetric transbilayer distribution of glucosylceramide in the plasma membrane. It will also provide new information about the first step in the synthesis of inositol phosphorylceramides, a diverse class of complex sphingolipids in plants. Sphingolipids are present in all organisms except bacteria and are known to function as structural components in cells and as regulators of various biological processes. Little is known, however, about the function and metabolism of sphingolipids in plants. The proposed research, a continuation of previous work supported by the NSF, will provide an integrated view of where and how sphingolipids are produced, transported, and degraded in the cell. It will also investigate, for the first time, the synthesis of inositol phosphorylceramides, a novel and diverse class of complex plant lipids. The results from these studies will contribute to understanding the role of these unusual lipids in plant growth and development.