ACTIVE TRANSPORT OF MALTOSE IN ESCHERICHIA COLI

小分子穿过生物膜的主动运输 是所有细胞的基本属性 转运蛋白必须 (i)特异性识别基板，（ii）从一侧移动它们 以及（iii）将能量源耦合到 基板移动。 研究的长期目标 本提案中描述的是了解这些步骤及其 在分子水平上的调节。 要做到这一点， 对大肠杆菌麦芽糖转运系统的分析将 执行。 该系统由周质麦芽糖组成， 结合蛋白（MBP）和三种膜蛋白MalF、G、K. MBP 是运输所必需的，并与MalF和MalG相互作用 proteins. MalF和MalG蛋白还含有门控的 底物识别位点。 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 参与多种生物学功能的结合蛋白。 一些 包括mdr P-糖蛋白， 参与肿瘤细胞的多药耐药。 的突变 干扰MBP与MalF和MalG的相互作用将被分离 和序列测定 malF和malG基因的突变， 底物识别位点的可接近性将是 通过DNA测序定位。 这些信息将决定 区域形成控制对衬底的访问的栅极 识别位点。 ATP在核苷酸结合中的作用 将评估MalK蛋白的结合倍数。 运输 其中不再发生ATP结合的缺陷malK突变体将 被研究。 这些突变体的回复突变体， 活动将被隔离和表征。 尝试更换 MalK与其他类似结构蛋白功能将被 进行了 编码其他可以取代MalK的蛋白质的基因 将被识别。 malK基因的突变会影响 MalK的调节功能将被分离。 这些包括 消除MalK代谢内源性代谢物的能力的突变 mal调节子的诱导物和使mal系统 对PTS的葡萄糖特异性EIII抑制具有抗性 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