Drs2p Function in Clathrin-coated Vesicle Budding

描述(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一个小叶内迅速扩散，但面临着从膜的一侧到另一侧的移位或触发的实质障碍。然而，膜含有被称为翻转酶和软脂酶的酶，它们将磷脂转移到整个双层，并似乎对不同磷脂在小叶之间的不对称分布负责。不对称质膜的医学意义在血细胞中得到最好的理解，在血细胞中，细胞外小叶中磷脂酰丝氨酸(PS)的调节暴露可诱导血液凝结。垂死的细胞也暴露在细胞外小叶中的PS，促进它们被其他细胞识别和吞噬。此外，一些软脂酶能够将抗癌和抗微生物药物泵出细胞。这些酶的过度表达会导致多药耐药肿瘤和病原体，这是世界各地的一个主要健康问题。催化触发器的酶的同源性很差，最好的候选者是DRs2/ATPase II亚家族中的P型ATPase和Flopase的ABC转运蛋白。对酵母中的Drs2/ATPase II亚家族成员(Drs2p、Neo1p、Dnf1p、Dnf2p和Dnf3p)的鉴定使我们能够应用强大的遗传工具来剖析这些潜在的翻转酶的生化和细胞生物学功能。令人惊讶的是，这些ATPase与囊泡介导的蛋白质在分泌和内吞途径中的运输密切相关。例如，在高尔基体晚期膜上的翻转酶活性以及从该细胞器发芽的一类特定类型的胞外运输囊泡的形成都需要Drs2p。这些囊泡的形成还需要一种名为ARF的小GTP结合蛋白和笼状蛋白外壳蛋白。本研究的长期目标是明确Flppase在囊泡介导的蛋白质运输中的作用的分子机制。拟议的研究将进一步确定Drs2p的翻转酶活性及其在从高尔基复合体形成笼状蛋白包裹的囊泡中的作用。这些研究还将确定在高尔基体晚期膜中发现的一种软脂酶活性，并确定该软脂酶是否拮抗囊泡形成中的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.