INVIVO ANALYSIS OF INTEGRATION OF MEMBRANE-PROTEINS IN ESCHERICHIA-COLI
INVIVO ANALYSIS OF INTEGRATION OF MEMBRANE-PROTEINS IN ESCHERICHIA-COLI
复制标题
DOI：
10.1111/j.1365-2958.1991.tb02154.x
复制
发表时间：
1991-09-01
影响因子：
3.6
通讯作者：
AKIYAMA, Y
中科院分区：
文献类型：
--
作者：
ITO, K;AKIYAMA, Y
通过脉冲标记大肠杆菌细胞，研究了膜蛋白在体内的整合过程，并根据标记蛋白对碱提取的抗性评估其在脂质双分子层上的整体锚定。为了进行本实验，我们改进了碱法提取大肠杆菌蛋白的条件，并改进了免疫沉淀方法，使其能够有效地检测整体膜蛋白。检测脉冲标记的完整膜蛋白，包括乳糖渗透酶（LacY）、SecY、细胞色素o亚基II和先导肽酶，发现它们都在碱不溶部分，表明这些蛋白的膜整合在野生型细胞中迅速发生。然而，当从多拷贝质粒中过量合成LacY时，在碱溶部分中发现了相当大的比例，这表明LacY在碱中的溶解度不是蛋白质的固有特性，并表明LacY依赖于一些有限的细胞因子来实现膜整合。未整合的LacY物种通过碱性蔗糖梯度缓慢沉积。与sec+细胞相比，secY24突变体细胞在较低的过量生产水平下积累了较高比例的未整合LacY分子。野生型细胞中LacY的过量产生抑制了β -内酰胺酶的加工（输出），但对OmpA和OmpF没有抑制作用。这些结果被解释为LacY的整合取决于多种细胞成分，其中一种成分也涉及β -内酰胺酶的输出。
The in vivo process of membrane protein integration was studied by pulse-labelling Escherichia coli cells, and assessing integral anchoring of labelled proteins to the lipid bilayer based on their resistance to alkali extraction. To conduct this experiment, conditions for extracting E. coli proteins with alkali were refined, and the immunoprecipitation procedures were improved to allow effective detection of integral membrane proteins. Examination of pulse-labelled, integral membrane proteins, including lactose permease (LacY), SecY, cytochrome o subunit II and leader peptidase revealed that all were in the alkali-insoluble fraction, indicating that membrane integration of these proteins takes place rapidly in wild-type cells. However, when LacY was synthesized in excess from a multicopy plasmid, significant proportions were found in the alkali-soluble fraction, indicating that the solubility in alkali is not an intrinsic property of the protein, and suggesting that LacY depends on some limited cellular factor for membrane integration. The unintegrated species of LacY sedimented slowly through an alkaline sucrose gradient. The secY24 mutant cells accumulated higher proportions of unintegrated LacY molecules at lower levels of overproduction than the sec+ cells. LacY overproduction in wild-type cells was found to inhibit processing (export) of beta-lactamase but not of OmpA and OmpF. These results are interpreted to mean that integration of LacY depends on multiple cellular components, one of which is also involved in export of beta-lactamase.