PROTEIN EXPORT BY AN ACCESSORY PROTEIN-SPECIFIED PATHWAY

大肠杆菌素V是大肠杆菌分泌的少数蛋白质之一， coli细胞。 大肠杆菌素V的排泄需要两种产物 辅助基因cvaA和cvaB。 CvaB是同源的 参与能量依赖性运输的膜蛋白 各种分子，包括蛋白质、肽、药物、离子和 多糖 这些蛋白质的同源性的特征在于： 存在几个潜在的跨膜区域和高度 保守的亲水性ATP结合结构域，并且可能具有共同的机制 来运输不同的分子。 该项目的目标是 了解蛋白质通过辅助蛋白输出的机制- 特异性途径，可能与一般SecA/SecY显著不同 出口通道。 目的是从生物化学上阐明 这一出口途径的机制，以补充并行遗传研究。 我们将开发一种体外系统， 大肠杆菌素V，其具有非典型的N-末端靶向序列， 在出口过程中被切断。 大肠杆菌素V的前体将在 T7-聚合酶指导的转录/翻译系统。 是 从遗传学研究中预计，活跃的系统将需要 cvaA和cvaB基因的产物。 CvaA和CvaB蛋白的作用 将通过使用缺乏一个或多个这些辅助基因的突变体进行评估。 蛋白质，并通过使用针对肽的特定区域的抗体。 CvaB的ATP结合结构域与 P-糖蛋白将被ATP类似物标记，并被位点特异性改变。 突变及其在CvaB功能中的作用将被检查。 我们将 确定易位的要求并评估SecA， SecY/PrlA、一般信号肽酶和ATP参与了这一过程。 为了与一般蛋白质易位的要求进行比较，我们 将使用N-末端CvaC靶向序列39的融合物的前体 氨基酸残基）连接到碱性磷酸酶的成熟区，和/或 OmpA。 用双官能试剂进行化学交联，然后 免疫沉淀和膜部分溶解将用于 表征辅助蛋白特异性输出途径。 膜 囊泡将用于测试大肠杆菌素V输出系统是否能够 进行依赖能源的毒品出口。 这些研究将阐明CvaB介导的大肠杆菌素V的作用机制 运输，并可能适用于探讨多药机制 阻力

Colicin V is one of the few proteins that are secreted out of Escherichia coli cells. The excretion of Colicin V requires the products of two accessory genes, cvaA and cvaB. The CvaB is a member of homologous membrane proteins that are involved in the energy-dependent transport of various kinds of molecules, including proteins, peptides, drugs, ions and polysaccharides. The homologies of these proteins are characterized by the presence of several potential membrane-spanning regions and a highly conserved hydrophilic ATP-binding domain, and may share a common mechanism for transporting different molecules. The objective of this project is to understand the mechanism of protein export via the accessory protein- specified pathway, which may differ markedly from the general SecA/SecY export pathway. The aim is to elucidate biochemically the molecular mechanism of this export pathway to complement concurrent genetic studies. We will develop an in vitro system for the specific translocation of Colicin V, which has an atypical N-terminal targeting sequence that is cleaved during export. The precursor of Colicin V will be synthesized in a T7-polymerase-directed transcription/translation system. It is anticipated from genetic studies that the active system will require the products of cvaA and cvaB genes. The roles of the CvaA and CvaB proteins will be assessed by using mutants devoid of one or more of these accessory proteins, and by using antibodies against specific regions of the peptides. The ATP-binding domain of CvaB that shares extensive homology with that of P-glycoprotein will be labeled by ATP analogs and changed by site-specific mutagenesis and its role in CvaB function will be examined. We will determine the requirements for the translocation and assess whether SecA, SecY/PrlA, general signal peptidases and ATP are involved in the process. For comparison to the requirements of general protein translocation, we will use precursors of fusions of N-terminal CvaC targeting sequence 39 amino acid residues) to the mature region of alkaline phosphatase and/or OmpA. Chemical crosslinking with bifunctional reagents, followed by immunoprecipitation, and partial solubilization of membrane will be used to characterize the accessory protein-specified export pathway. Membrane vesicles will be used to test whether Colicin V export system is capable of carrying out energy-dependent drug export. These studies should elucidate the mechanism of CvaB-mediated Colicin V transport, and may be adapted to explore the mechanism of multi-drug resistance.