ACTIVE TRANSPORT OF MALTOSE IN ESCHERICHIA COLI

本申请中提出的研究的长期目标是 了解ATP依赖的主动转运机制。广泛的 E。麦芽糖运输系统的生化和遗传分析。 正在追查的是大肠杆菌。这一运输系统由一个 周质麦芽糖结合蛋白(MBP)和三种细胞膜 组件、MARF、MALG和MARK。这些异构体与一种 显示ATP底物的1F：1G：2K[FGK2]的化学计量比 依赖于ATPase活性。麦芽糖亚基具有广泛的序列 与一个不同的例子同源，cftr，这是在 囊性纤维化，并可能在假单胞菌的定植中发挥作用； 大肠杆菌溶血素输出子的hylB亚基；以及P-糖蛋白 导致许多癌症的多药耐药表型 细胞。具体目标是：(1)确定 MBP和FGK2复合体通过定点交联的使用 以及对不再发生相互作用的突变体的分析。 (2)描述导致MalF和MalG突变的后果 在MBP非依赖性转运和改变的跨膜信号传递中。 MBP存在和不存在时的底物结合和ATPase活性 将会被测量。(3)识别FGK中的底物识别位点 通过与麦芽糖的光活性类似物进行光交联制得络合物。在……里面 在改变的特异性中添加核苷酸序列的改变 FGK复合体获得转运能力的突变体 将测定一种新的底物--乳糖。(4)确定 麦芽蛋白亚基ATPase活性与其活性的关系 与MALF和MALG亚基相互作用的研究 MALK突变，消除复制的MALK-MALK基因上的ATP结合。 此外，Malk突变可以纠正未偶联的MALF和 将分离出麦芽G突变株。(5)遗传方法将是 为研究FGK复合体的物理排列而开发的。 这些措施包括使用人工转座子TnSnip，它 在基因中引入翻译终止和重新启动信号。 MALF和MALG的N-端和C-端片段的能力 干扰FGK复合体活性或组装的蛋白质 将会受到考验。(6)确定负性的分子基础 麦芽蛋白亚基表现出转录调控。C端子 麦芽蛋白亚基的区域将被单独表达并进行检测 监管活动。

The long term goal of the research proposed in this application is to understand the mechanism of ATP-dependent active transport. An extensive biochemical and genetic analysis of the maltose transport system of E. coli is being pursued. This transport system is composed of a periplasmic maltose-binding protein, MBP; and three cytoplasmic membrane components, MalF, MalG, and MalK. These form a heterotetramer with a stoichiometry of 1F:1G:2K[FGK2], which exhibits substrate of ATP- dependent ATPase activity. The MalK subunit shares extensive sequence homology with a variety example, CFTR, which is the altered protein in cystic fibrosis and may play a role in colonization y Pseudomonas; the hylB subunit of the hemolysin exporter of E. coli; and the P-glycoprotein responsible for the multiple-drug resistance phenotype of many cancer cells. The specific aims are to: (1) Identify regions of contact between MBP and the FGK2 complex through the use of site-directed crosslinking and the analysis of mutants in which the interaction no longer occurs. (2) Characterize the consequences of MalF and malG mutations that result in MBP-independent transport and altered transmembrane signalling. Substrate binding and ATPase activity in the presence and absence of MBP will be measured. (3) Identify substrate recognition sites in the FGK complex by photocrosslinking with a photoactive analog of maltose. In addition the nucleotide sequence alterations in altered specificity mutants in which the FGK complex has acquired the ability to transport a novel substrate, lactose will be determined. (4) Determine the relationship between the ATPase activity of the MalK subunit and its interaction with the MalF and MalG subunits by studying the effects of malK mutations that eliminate ATP binding on a duplicated malK-malK gene. In addition malK mutations that correct defects in uncoupled malF and malG mutants strains will be isolated. (5) Genetic methods will be developed for studying the physical arrangement of the FGK complex. These include the use of an artificial transposon, Tnsnip which introduces translation termination and re-start signals within a gene. The ability of N-terminal and C-terminal fragments of the MalF and MalG proteins to interfere with the activity or assembly of the FGK complex will be tested. (6) Determine the molecular basis for the negative transcriptional regulation exhibited by the MalK subunit. The C-terminal region of the MalK subunit will be expressed separately and tested for regulatory activity.