Interaction between maltose-binding protein and the membrane-associated maltose transporter complex in Escherichia coli.
Interaction between maltose-binding protein and the membrane-associated maltose transporter complex in Escherichia coli.
复制标题
大肠杆菌中麦芽糖结合蛋白与膜相关麦芽糖转运蛋白复合物之间的相互作用。
DOI：
10.1111/j.1365-2958.1992.tb01376.x
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发表时间：
1992
影响因子：
3.6
通讯作者：
Nikaido,H
中科院分区：
文献类型：
--
作者：
Dean,DA;Hor,LI;Shuman,HA;Nikaido,H
在大肠杆菌中，麦芽糖的主动转运需要在周质中存在麦芽糖结合蛋白（MBP）和位于细胞质膜中的MalF、MalG和MalK蛋白（FGK 2）的复合物。早先，分离出了在完全不存在MBP的情况下能够在麦芽糖上生长的malForMalG突变体。当编码MBP的野生型E+等位基因被引入这些MBP非依赖性突变体中时，它们经常失去在麦芽糖上生长的能力。此外，从这些Mal-菌株开始，分离出在malE中含有抑制突变的Mal+二级突变体。在这项研究中，我们通过使用右侧外膜囊泡检查了野生型和突变型MBP与野生型和突变型FGK 2复合物的相互作用。来自MBP非依赖性突变体（malG 511）的囊泡在不存在MBP的情况下转运麦芽糖，其中KmandVmax值与在完整细胞中发现的那些相似。然而，将野生型MBP添加到这些突变体囊泡中产生了意想不到的反应。尽管雄性+malG 511细胞不能利用麦芽糖，但低浓度的野生型MBP刺激malG 511囊泡摄取麦芽糖。然而，在较高浓度的野生型MBP和麦芽糖下，麦芽糖转运到malG 511囊泡中受到严重抑制。囊泡的这种行为也反映在雄性+malG 511细胞的表型中，发现其能够从低外部浓度（1μM）转运麦芽糖，但显然不能从麦芽糖基本培养基中存在的毫摩尔浓度转运。我们发现含有MalG 511的突变体FGK 2复合物对野生型MBP的表观亲和力比野生型FGK 2复合物高得多。我们认为野生型FGK 2复合物至少存在两种构象，活性和非活性，并且配体MBP的结合将后者转化为前者。突变体复合物可能主要以对配体MBP具有更高亲和力的活性形式存在，过量麦芽糖和野生型MBP对突变体复合物的抑制可以解释为过量底物的抑制形式。
Active transport of maltose inEscherichia colirequires the presence of both maltose‐binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane. Earlier, mutants inmalForMalGwere isolated that are able to grow on maltose in the complete absence of MBP. When the wild‐typemalE+allele, coding for MBP, was introduced into these MBP‐independent mutants, they frequently lost their ability to grow on maltose. Furthermore, starting from these Mal‐strains, Mal+secondary mutants that contained suppressor mutations inmalEwere isolated. In this study, we examined the interaction of wild‐type and mutant MBPs with wild‐type and mutant FGK2 complexes by using right‐side‐out membrane vesicles. The vesicles from a MBP‐independent mutant (malG511) transported maltose in the absence of MBP, withKmandVmaxvalues similar to those found in intact cells. However, addition of wild‐type MBP to these mutant vesicles produced unexpected responses. Althoughmale+malG511cells could not utilize maltose, wild‐type MBP at low concentrations stimulated the maltose uptake bymalG511vesicles. At higher concentrations of the wild‐type MBP and maltose, however, maltose transport intomalG511vesicles became severely inhibited. This behaviour of the vesicles was also reflected in the phenotype ofmale+malG511cells, which were found to be capable of transporting maltose from a low external concentration (1μM), but apparently not from millimolar concentrations present in maltose minimal medium. We found that the mutant FGK2complex, containing MalG511, had a much higher apparent affinity towards the wild‐type MBP than did the wild‐type FGK2complex. We propose that the wild‐type FGK2complex exists in at least two conformations, active and inactive, and that the binding of the liganded MBP converts the latter into the former. The mutant complex presumably exists predominantly in the active form that has a higher affinity toward liganded MBP, and the Inhibition of the mutant complex by an excess of maltose and wild‐type MBP may be explained as a form of inhibition by excess substrate.