ACTIVE TRANSPORT OF MALTOSE IN ESCHERICHIA COLI

小分子在生物膜上的主动传输 是所有细胞的基本属性。运输蛋白必须 (一)具体识别基材；(二)从一侧移动基材 以及(Iii)耦合能量源 底物运动。这项研究的长期目标 本建议书中描述的内容是了解这些步骤及其 在分子水平上的调控。要做到这一点，一种分子遗传 大肠杆菌Will麦芽糖转运系统的分析 被执行。该体系由周质麦芽糖组成。 结合蛋白(MBP)和三种膜蛋白MALF、G、K 是传输所需的，并与MARF和MALG交互 蛋白质。MARF和MALG蛋白也包含一个门控 底物识别部位。Malk蛋白有一个核苷酸 结合折叠，并与许多ATP具有广泛的序列相似性 参与多种生物功能的结合蛋白。一些人 这些相关蛋白包括MDR P-糖蛋白，它们是 参与肿瘤细胞的多药耐药。突变 扰动MBP与MALF和MALG的相互作用将被隔离 并进行了测序。影响MALF和MALG基因的突变 底物识别位置的可达性将是 通过DNA测序定位。此信息将确定哪些 区域形成控制对衬底的访问的栅极 识别站点。核苷酸上的三磷酸腺苷结合作用 将对Malk蛋白的结合折叠进行评估。运输 ATP结合不再发生的有缺陷的Malk突变体将 被研究。这些突变体的返回体重新获得运输 活动将被隔离并确定其特征。试图取代 Malk与其他结构相似的蛋白质的功能将是 制造。编码可以取代Malk的其他蛋白质的基因 将会被确认。影响MALK基因的突变 马尔克的监管职能将被隔离。这些措施包括 使MALK代谢内源性的能力丧失的突变 正规子的诱导者和构成正规子系统的突变 抵抗PTS的葡萄糖专一性EIII的抑制 系统。预计这些实验将导致 详细了解重要的

The active transport of small molecules across biological membranes is a fundamental property of all cells. Transport proteins must (i) specifically recognize substrates, (ii) move them from one side of the membrane to the other and (iii) couple a source of energy to substrate movement. The long term objective of the research described in this proposal is to understand these steps and their regulation at the molecular level. To do this a molecular genetic analysis of the maltose transport system of Escherichia coli will be carried out. This system is composed of a periplasmic maltose binding-protein (MBP), and three membrane proteins MalF,G,K. MBP is required for transport and interacts with the MalF and MalG proteins. The MalF and MalG proteins also contain a gated substrate recognition site. The MalK protein has a nucleotide binding fold and shares extensive sequence similarity with many ATP binding proteins involved in diverse biological functions. Some of these related proteins include the mdr P-glycoproteins that are involved in tumor cell multiple drug resistance. Mutations which perturb the interaction of MBP with MalF and MalG will be isolated and sequenced. Mutations in the malF and malG genes that affect the accessibility of the substrate recognition site will be localized by DNA sequencing. This information will determine which regions form the gate that controls access to the substrate recognition site. The role of ATP binding at the nucleotide binding fold of the MalK protein will be evaluated. Transport defective malK mutants in which ATP binding no longer occurs will be studied. Revertants of these mutants that regain transport activity will be isolated and characterized. Attempts to replace the MalK function with other proteins of similar structure will be made. Genes which encode the other proteins that can replace MalK will be identified. Mutations in the malK gene that affect the regulatory functions of MalK will be isolated. These include mutations that abolish the ability of MalK to metabolize endogenous inducers of the mal regulon and mutations that make the mal system resistant to inhibition by the glucose-specific EIII of the PTS system. It is anticipated that these experiments will result in detailed knowledge of the important