Drs2p Function in Clathrin-coated Vesicle Budding

描述（由申请人提供）：生物膜形成屏障，控制哪些物质被允许进出细胞，也定义了大多数细胞内细胞器的边界。构成细胞膜的磷脂分子可以自由地在双层结构的单叶内快速扩散，但从细胞膜的一侧到另一侧，却面临着转移或翻转的实质性障碍。然而，膜含有一种叫做翻转酶和翻转酶的酶，这种酶可以将磷脂转运到双分子层上，并似乎负责不同磷脂在小叶之间的不对称分布。不对称质膜的医学意义在血细胞中得到最好的理解，其中细胞外小叶中磷脂酰丝氨酸（PS）的调节暴露诱导血液凝固。垂死的细胞也在细胞外小叶中暴露PS，以促进它们被其他细胞识别和吞噬。此外，一些floppases能够将抗癌和抗菌药物从细胞中抽离出来。这些酶的过度表达导致多药耐药肿瘤和病原体，这是全世界的一个主要健康问题。催化flip-flop的酶的特性尚不清楚，最佳候选酶是用于flip-flop的Drs2/ATPase II亚家族中的p型atp酶和用于flip-flop的ABC转运蛋白。酵母Drs2/ATPase II亚家族成员（Drs2p, Neo1p, Dnf1p， Dnf2p和Dnf3p）的表征允许应用强大的遗传工具来解剖这些潜在翻转酶的生化和细胞生物学功能。令人惊讶的是，这些atp酶与分泌和内吞途径中囊泡介导的蛋白质运输密切相关。例如，Drs2p是晚期高尔基膜翻转酶活性和从该细胞器萌发的特定类型的胞外转运囊泡形成所必需的。这些囊泡的形成还需要一种叫做ARF的小的gtp结合蛋白和网格蛋白外壳蛋白。本研究的长期目标是确定翻转酶在囊泡介导的蛋白质转运中的分子机制。这些研究将进一步确定Drs2p的翻转酶活性及其在高尔基复合体形成网格蛋白包被囊泡中的作用。这些研究还将确定在晚期高尔基膜中发现的floppase活性，并确定floppase是否在囊泡形成中拮抗Drs2p功能。

DESCRIPTION (provided by applicant): Biological membranes form the barrier that controls which substances are allowed to pass in and out of the cell and also defines the boundary of most intracellular organelles. Phospholipid molecules composing membranes of cells are free to diffuse rapidly within one leaflet of the bilayer structure but face a substantial barrier to translocation, or flip-flop, from one side of the membrane to the other. However, membranes contain enzymes called flippases and floppases that translocate phospholipid across the bilayer and appear responsible for the asymmetric distribution of different phospholipids between the leaflets. The medical significance of an asymmetric plasma membrane is best understood in blood cells where regulated exposure of phosphatidylserine (PS) in the extracellular leaflet induces blood clotting. Dying cells also expose PS in the extracellular leaflet facilitating their recognition and phagocytosis by other cells. In addition, some floppases are capable of pumping anti-cancer and antimicrobial drugs out of cells. Overexpression of these enzymes lead to multi-drug resistant tumors and pathogens, a major health concern throughout the world. The identities of enzymes catalyzing flip-flop are poorly characterized with the best candidates being P-type ATPases in the Drs2/ATPase II subfamily for the flippases and ABC transporters for the floppases. Characterization of the yeast members of the Drs2/ATPase II subfamily (Drs2p, Neo1p, Dnf1p, Dnf2p and Dnf3p) allowed application of powerful genetic tools to dissect the biochemical and cell biological functions of these potential flippases. Surprisingly, these ATPases are intimately linked to vesicle-mediated protein transport in the secretory and endocytic pathways. For example, Drs2p is required for a flippase activity in late Golgi membranes and for the formation of a specific class of exocytic transport vesicles that bud from this organelle. Formation of these vesicles also requires a small GTP-binding protein called ARF and the clathrin coat protein. The long-term goal of this research is to define the molecular mechanism of flippase function in vesicle-mediated protein transport. The proposed studies will further define the flippase activity of Drs2p and its role in forming clathrin-coated vesicles from the Golgi complex. These studies will also define a floppase activity discovered in late Golgi membranes and determine if the floppase antagonizes Drs2p function in vesicle formation.