Studies of the effects of phosphorylation and protein interactions on ATP-binding casette transporter activity

磷酸化和蛋白质相互作用对 ATP 结合盒转运蛋白活性影响的研究

• 批准号: RGPIN-2015-05372
• 负责人: Kanelis, Voula
• 金额: $ 2.19万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613510
• 关键词: Studies; effects; phosphorylation; protein; interactions

The protein Ycf1p is a member of a large superfamily of membrane proteins found in all organisms. By utilizing the energy of ATP, known as the energy currency of the cell, these proteins actively transport solutes into and out of the cell, regulate the function of other proteins, or open pores or channels to allow charged species to pass through. These ATP-using membrane proteins have a conserved molecular architecture that contains two regions embedded in the cellular envelope (known as the membrane) and two regions located inside the cell (known as the cytoplasm). Proper regulation of this process is essential, as many genetic diseases, including cystic fibrosis and adrenoleukodystrophy, result from dysfunction of different proteins in this ubiquitous superfamily. Ycf1p transports toxins from one part of the cell (cytoplasm) to a membrane-bound compartment that is also found inside the cell. In this way, Ycf1p detoxifies the cell by sequestering poisonous molecules. Ycf1p is a prototype of a specific subclass of the ATP-using protein superfamily and shares similarily with the multidrug-resistance proteins that cause tumors to be resistant to chemotherapy drugs. Ycf1p and Ycf1p-like proteins posses a unique structural region in addition to the minimum architecture shared by the entire superfamily. Ycf1p action is regulated in a complex fashion. Biochemical modification of Ycf1p in the cell, which results in the addition of a charged group to specific sites, can activate or deactivate protein depending on the site modified. Ycf1p also binds a number of other proteins, which regulate Ycf1p transport function or ascribe other functions to Ycf1p. This proposal describes biochemical, biophysical, and cell biology-based experiments designed to gain insights into how Ycf1p activity is controlled, which will be applicable to other members of superfamily. To date, few studies have been done to address understand the mechanism of regulation in this important class of proteins. We will investigate interactions of individual Ycf1p protein segments that are involved in relaying information from the utilization of ATP to transport of toxins, and that bind accessory proteins. We will further address how biochemical modification changes the structure and interactions of Ycf1p. Finally, we will conduct experiments to obtain a 3D picture of Ycf1p, which will yield much sought-after information on the organization of the unique region in Ycf1p with the minimum architecture shared by the entire superfamily. The proposed research will elucidate some of the complex mechanisms by which Ycf1p activity is controlled. Because Ycf1p is part of a large superfamily of proteins that share common structural and mechanistic features, our Ycf1p studies will yield information about this class of proteins, which are conserved throughout biology.

Ycf1p蛋白是在所有生物体中发现的一个膜蛋白超家族的成员。通过利用被称为细胞能量货币的ATP的能量，这些蛋白质主动地将溶质运进和运出细胞，调节其他蛋白质的功能，或打开毛孔或通道，允许带电物质通过。这些使用atp的膜蛋白具有保守的分子结构，包含两个嵌入细胞包膜（称为膜）的区域和两个位于细胞内（称为细胞质）的区域。这一过程的适当调节是必不可少的，因为许多遗传疾病，包括囊性纤维化和肾上腺脑白质营养不良，都是由这个普遍存在的超家族中不同蛋白质的功能障碍引起的。