Chepuri V, Gennis R B
Department of Biochemistry, University of Illinois, Urbana 61801.
J Biol Chem. 1990 Aug 5;265(22):12978-86.
The cytochrome o terminal oxidase complex is a component of the aerobic respiratory chain of Escherichia coli. This enzyme catalyzes the oxidation of ubiquinol-8 to ubiquinone-8 within the cytoplasmic membrane and the concomitant reduction of O2 to H2O. The hydropathy profiles of the deduced amino acid sequences suggest that all five of the gene products of the cyo operon contain multiple membrane-spanning helical segments. The goal of this work was to obtain experimental evidence for the topology of the five gene products in the cytoplasmic membrane by using the technique of gene fusions. A number of random gene fusions were generated in vitro encoding hybrid proteins in which the amino-terminal portion was provided by the subunit of interest and the carboxyl-terminal portion by one of two sensor proteins, alkaline phosphatase lacking its signal sequence or beta-galactosidase. Results obtained are self-consistent, and topological models are proposed for all of the five gene products encoded by the cyo operon. Based on the sequence similarities with subunits of the aa3-type cytochrome c oxidases, the experimental evidence obtained here can be used to infer topological models for the mitochondrial encoded subunits of the eukaryotic cytochrome c oxidases.
细胞色素 o 末端氧化酶复合体是大肠杆菌有氧呼吸链的一个组成部分。该酶催化细胞质膜内泛醇 -8 氧化为泛醌 -8，并伴随将 O₂ 还原为 H₂O。推导的氨基酸序列的亲水性图谱表明，cyo 操纵子的所有五个基因产物都含有多个跨膜螺旋片段。这项工作的目标是通过基因融合技术获得这五个基因产物在细胞质膜中拓扑结构的实验证据。在体外产生了许多随机基因融合体，编码杂合蛋白，其中氨基末端部分由感兴趣的亚基提供，羧基末端部分由两种传感蛋白之一提供，即缺乏信号序列的碱性磷酸酶或β - 半乳糖苷酶。获得的结果是自洽的，并为 cyo 操纵子编码的所有五个基因产物提出了拓扑模型。基于与 aa3 型细胞色素 c 氧化酶亚基的序列相似性，此处获得的实验证据可用于推断真核细胞色素 c 氧化酶线粒体编码亚基的拓扑模型。
