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依赖的主动运输机制。 一个广泛 麦芽糖转运系统的生化和遗传分析。 大肠杆菌感染， 该运输系统由一个 周质麦芽糖结合蛋白（MBP）和三种细胞质膜 组件MalF、MalG和MalK。 这些形成异四聚体， 化学计量为1F：1G：2K[FGK 2]，其表现出ATP的底物。 依赖ATP酶活性。 MalK亚基共享广泛的序列 与各种实例CFTR同源，CFTR是在细胞中改变的蛋白质， 囊性纤维化，并可能在定植假单胞菌中发挥作用; hylB亚单位是E.和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）确定区域之间的接触 MBP和FGK 2复合物通过使用定点交联 以及分析不再发生相互作用的突变体。 (2)表征导致的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导中。 存在和不存在MBP时的底物结合和ATP酶活性 将被衡量。 (3)鉴定FGK中的底物识别位点 通过与麦芽糖的光活性类似物光交联形成复合物。 在 此外，核苷酸序列的改变改变了特异性 突变体，其中FGK复合物已获得转运能力， 一种新的底物乳糖将被确定。 (4)确定 MalK亚基ATP酶活性与 与MalF和MalG亚基的相互作用，通过研究 malK突变消除了重复的malK-malK基因上的ATP结合。 此外，纠正非偶联malF和 将分离malG突变株。 (5)遗传学方法将是 用于研究FGK复合物的物理排列。 这些方法包括使用人工转座子Tnsnip， 在基因内引入翻译终止和重新启动信号。 MalF和MalG的N-末端和C-末端片段的能力 干扰FGK复合物的活性或组装的蛋白质 会得到考验 (6)确定阴性的分子基础 MalK亚基表现出的转录调控。 c-末端 MalK亚基的区域将被单独表达，并测试其 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.