Studies of how disordered regions, post-translational processing, and protein interactions affect the structure, dynamics, and activity of ABC transporters

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。由于许多遗传疾病，包括囊性纤维化和肾上腺脑白质营养不良，都是由于这个普遍存在的超家族中不同蛋白质的功能障碍造成的，因此对这些使用atp的蛋白质进行适当的调节是必不可少的。Ycf1p将毒素从细胞的一部分（细胞质）运输到细胞内的膜结合隔室。通过这种方式，Ycf1p通过隔离有毒分子为细胞解毒。Ycf1p是使用atp的膜蛋白超家族的一个特定亚类的原型，与导致肿瘤对化疗药物耐药的多药耐药蛋白有相似之处。Ycf1p和Ycf1p样蛋白除了具有整个超家族共有的最小结构外，还具有一个独特的结构区域。Ycf1p的作用以一种复杂的方式调节。细胞中Ycf1p的生化修饰导致在特定位点添加带电基团，可以根据修饰的位点激活或失活蛋白质。Ycf1p中特定化学键的断裂会影响输送的毒素类型。Ycf1p还结合许多其他蛋白，这些蛋白调节Ycf1p的转运功能或将其他功能赋予Ycf1p。本提案描述了基于生化、生物物理和细胞生物学的实验，旨在深入了解如何控制Ycf1p活性，这将适用于超家族的其他成员。迄今为止，很少有研究解决这类重要蛋白质的调节机制。我们将进行旨在确定Ycf1p的高分辨率结构的研究，这将产生有关该蛋白中与整个超家族共享的最小结构相关的独特区域的组织信息。我们将研究生化修饰如何改变Ycf1p的结构和相互作用。最后，我们确定了Ycf1p的哪些部分与辅助蛋白结合，以及生化修饰如何改变这些相互作用。这项研究将阐明控制Ycf1p活性的一些复杂机制。由于Ycf1p是一个大的蛋白质超家族的一部分，具有共同的结构和机制特征，我们的Ycf1p研究将获得关于这类蛋白质的信息，这些蛋白质在整个生物学中都是保守的。

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 these ATP-using proteins 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 membrane protein superfamily and shares similarity 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 the protein depending on the site modified. Breaking of a specific chemical bond in Ycf1p affects the types of toxins transported. 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 the mechanism of regulation in this important class of proteins. We will conduct studies aimed at determining a high-resolution structures of Ycf1p that will yield information about the organization of the unique region in this protein in relation to the minimum architecture shared by the entire superfamily. We will study how biochemical modifications change the structure and interactions of Ycf1p. Finally, we determine which parts of Ycf1p bind accessory proteins, and how biochemical modifications alter those interactions. 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.