RUI: Sphingolipid Metabolism in Plants

9723484 Lynch先前对植物中鞘脂代谢所涉及的酶促反应的表征为所提出的研究奠定了基础。将使用细胞分级分离和标记物测定法在豆类和玉米组织中确定鞘脂合成和周转中涉及的各个酶促步骤的细胞内位置。还将测定分离的膜组分中的特定鞘脂含量和组成。将通过耦合鞘脂的体内标记和分级分离程序来跟踪标记的鞘脂通过细胞的转移。使用这种方法，细胞内鞘脂转运的途径和动力学的信息将获得。将通过检查反应对蛋白水解酶的敏感性来研究完整膜囊泡中葡糖神经酰胺合成的拓扑结构。同时，将进一步研究甾醇葡萄糖苷作为该反应的葡萄糖供体的作用。最后，酶促反应催化合成的神经酰胺磷酸肌醇，肌醇磷酸神经酰胺的生物合成前体，将被证明和表征的微粒体制剂从豆类和玉米。随后，其亚细胞分布和拓扑结构将被表征。拟议的研究将提供植物鞘脂代谢、细胞内运输/区室化以及被认为参与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的拓扑结构的综合观点。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的一类复杂鞘脂。 鞘脂存在于除细菌外的所有生物体中，并且已知其作为细胞中的结构组分和作为各种生物过程的调节剂起作用。然而，对植物中鞘脂的功能和代谢知之甚少。拟议的研究是NSF支持的先前工作的延续，将提供鞘脂在细胞中产生、运输和降解的位置和方式的综合视图。它还将首次研究肌醇磷酸神经酰胺的合成，这是一种新颖而多样的复杂植物脂质。这些研究的结果将有助于了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.