Kampfenkel K, Braun V
University of Tübingen, Germany.
J Biol Chem. 1993 Mar 15;268(8):6050-7.
The ExbB protein together with the ExbD and TonB proteins is involved in energy-coupled transport across the outer membrane of Escherichia coli. To understand this unusual process it is required to determine the subcellular location of ExbB and its transmembrane arrangement. Using ExbB-beta-lactamase fusion proteins as reporters for a periplasmic versus a cytoplasmic location of the fusion sites, and accessibility of ExbB in spheroplasts and cell lysates to aminopeptidase K, trypsin, and proteinase K, we arrived at a model of ExbB topology in the cytoplasmic membrane. Starting with the N terminus in the periplasm ExbB contains three transmembrane segments (residues 16-39, 128-155, 162-194) a small periplasmic loop and two large portions in the cytoplasm. Two of the 18 fusion proteins studied, ExbB34-beta-lactamase and ExbB41-beta-lactamase, conferred a high ampicillin resistance. Protease experiments revealed a high respectively low percentage of the molecules in a reverse transmembrane orientation. Both proteins were lacking positive charges at the inner side of the cytoplasmic membrane which determine the orientation of transmembrane segments.
ExbB蛋白与ExbD和TonB蛋白一起参与大肠杆菌外膜的能量偶联转运。为了解这一不同寻常的过程，需要确定ExbB的亚细胞定位及其跨膜排列。使用ExbB-β-内酰胺酶融合蛋白作为融合位点周质与细胞质定位的报告分子，以及原生质球和细胞裂解物中ExbB对氨肽酶K、胰蛋白酶和蛋白酶K的可及性，我们得出了ExbB在细胞质膜中的拓扑模型。从周质中的N端开始，ExbB包含三个跨膜区段（第16 - 39位、128 - 155位、162 - 194位残基）、一个小的周质环和细胞质中的两个大部分。所研究的18种融合蛋白中的两种，ExbB34-β-内酰胺酶和ExbB41-β-内酰胺酶，赋予了高氨苄青霉素抗性。蛋白酶实验显示，反向跨膜取向的分子比例分别很高或很低。这两种蛋白在细胞质膜内侧都缺乏决定跨膜区段取向的正电荷。
