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膜蛋白超家族的成员。通过利用被称为细胞的能量流通的ATP的能量，这些蛋白质主动地将溶质运入和运出细胞，调节其他蛋白质的功能，或者打开毛孔或通道，允许带电物种通过。这些使用三磷酸腺苷的膜蛋白有一个保守的分子结构，包含两个嵌入在细胞包膜中的区域(称为膜)和两个位于细胞内的区域(称为细胞质)。由于许多遗传性疾病，包括囊性纤维化和肾上腺脑白质营养不良，都是由这个无处不在的超家族中不同蛋白质的功能障碍引起的，因此对这些使用ATP的蛋白质的适当调节是必不可少的。 Ycf1p将毒素从细胞的一部分(细胞质)运输到细胞内也发现的膜结合的隔室。通过这种方式，Ycf1p通过隔离有毒分子来为细胞排毒。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产生耐药性的多药耐药蛋白有相似之处。Ycf1p和类Ycf1p蛋白除了具有整个超家族所共有的最小结构外，还具有一个独特的结构区域。Ycf1p行动以一种复杂的方式进行监管。Ycf1p在细胞中的生化修饰导致在特定的位点上增加一个带电基团，可以激活或停用该蛋白，具体取决于修饰的位点。Ycf1p中特定化学键的断裂会影响所运输的毒素类型。Ycf1p还与许多其他蛋白质结合，这些蛋白质调节Ycf1p的运输功能或将其他功能归因于Ycf1p。 这项建议描述了基于生化、生物物理和细胞生物学的实验，旨在深入了解Ycf1p活性是如何控制的，这将适用于超家族的其他成员。到目前为止，很少有人研究这类重要蛋白质的调控机制。我们将进行旨在确定Ycf1p的高分辨率结构的研究，该结构将产生关于该蛋白质中与整个超家族共享的最小结构相关的唯一区域的组织信息。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。这项拟议的研究将阐明Ycf1p活性受到控制的一些复杂机制。由于Ycf1p是具有共同结构和机制特征的蛋白质超家族的一部分，我们的Ycf1p研究将产生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.