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

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)。对这一过程的适当调控是至关重要的，因为许多遗传性疾病，包括囊性纤维化和肾上腺脑白质营养不良，都是这个无处不在的超家族中不同蛋白质功能障碍的结果。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空间。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相似。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，可以激活或失活蛋白质，具体取决于修饰的位点。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。*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Ycf1p的活性是如何控制的，这将适用于超级家族的其他成员。到目前为止，很少有人研究这类重要蛋白质的调控机制。我们将研究单个Ycf1p蛋白片段的相互作用，这些蛋白片段参与传递从利用ATP到毒素运输的信息，并结合辅助蛋白。我们将进一步研究生化修饰如何改变Ycf1p的结构和相互作用。最后，我们将进行实验以获得Ycf1p的3D图片，这将产生关于Ycf1p中具有整个超级家族共享的最小架构的独特区域的组织的许多受欢迎的信息。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是共享共同结构和机制特征的蛋白质超家族的一部分，我们的Ycf1p研究将提供关于这类蛋白质的信息，这些信息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 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.**