Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions.
Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions.
复制标题
使用嵌套缺失方法创建 mtlA-phoA 融合体，对大肠杆菌甘露醇渗透酶进行膜拓扑分析。
DOI：
10.1073/pnas.88.21.9603
复制
发表时间：
1991
影响因子：
11.1
通讯作者：
Jacobson,GR
中科院分区：
文献类型：
--
作者：
Sugiyama,JE;Mahmoodian,S;Jacobson,GR
大肠杆菌甘露醇通透酶催化D-甘露醇的伴随转运和磷酸化。这种68-kDa蛋白质由参与甘露醇结合和转运的膜结合N-末端结构域和负责甘露醇磷酸化的C-末端胞质结构域组成。二级结构预测方法表明，N-末端的一半的通透跨越膜约7倍的α-螺旋段，但这些数据不能最终预测的结构。我们已经使用mtlA（编码通透酶）和'phoA（编码缺乏其信号序列的碱性磷酸酶）之间的基因融合来进一步研究甘露醇通透酶的拓扑结构。最初，通过使用λ TnphoA载体并将λ phoA体外克隆到mtlA中天然存在的限制性位点中来构建融合体。然而，前一种方法在我们的载体系统中产生了插入“热点”的严重问题，而后一种方法受到有用限制性位点数量的限制。因此，我们开发了一种用于产生mtlA-phoA融合的嵌套缺失方法。phoA首先从编码膜结合的一半通透酶的mtlA部分的下游克隆。然后用适当的限制酶和核酸外切酶III处理该构建体以产生随机融合。通过这些方法构建的大于40个不同的融合克隆的分析提供了强有力的证据，六个跨膜区域的甘露醇通透酶与三个相对较短的周质环和两个大的细胞质环中的膜结合的一半的蛋白质。
The Escherichia coli mannitol permease catalyzes the concomitant transport and phosphorylation of D-mannitol. This 68-kDa protein consists of a membrane-bound, N-terminal domain involved in mannitol binding and translocation and a C-terminal, cytoplasmic domain responsible for mannitol phosphorylation. Secondary-structure prediction methods suggest that the N-terminal half of the permease spans the membrane approximately seven times in alpha-helical segments, but these data cannot conclusively predict the structure. We have used gene fusions between mtlA (encoding the permease) and 'phoA (encoding alkaline phosphatase lacking its signal sequence) to further investigate the topology of the mannitol permease. Initially, fusions were constructed by using a lambda TnphoA vector and in vitro cloning of 'phoA into naturally occurring restriction sites in mtlA. However, the former method gave severe problems with insertion "hot-spots" in our vector systems, and the latter method was limited by the number of useful restriction sites. Therefore, we developed a nested-deletion method for creating mtlA-phoA fusions. 'phoA was first cloned downstream from the part of mtlA encoding the membrane-bound half of the permease. This construct was then treated with the appropriate restriction enzymes and with exonuclease III to create random fusions. An analysis of greater than 40 different fusion clones constructed by these methods provides strong evidence for six membrane-spanning regions in the mannitol permease with three relatively short periplasmic loops and two large cytoplasmic loops in the membrane-bound half of the protein.