Mokhonov Vladislav V, Mokhonova Ekaterina I, Akama Hiroyuki, Nakae Taiji
Department of Molecular Life Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara 259-1193, Japan.
Biochem Biophys Res Commun. 2004 Sep 17;322(2):483-9. doi: 10.1016/j.bbrc.2004.07.140.
The tripartite xenobiotic-antibiotic transporter of Pseudomonas aeruginosa consists of the inner membrane transporter (e.g., MexB, MexY), the periplasmic membrane-fusion-protein (e.g., MexA, MexX), and the outer membrane channel protein (e.g., OprM). These subunits were assumed to assemble into a transporter unit during export of the substrates. However, subunit interaction and their specificity in native form remained to be elucidated. To address these important questions, we analyzed the role of the individual subunits for the assembly of MexAB-OprM by pull-down assay tagging only one of the subunits. We found stable MexA-MexB-OprM complex without chemical cross-linking that withstand all purification procedures. Results of bi-partite interactions analysis showed tight association between MexA and OprM in the absence of MexB, whereas the expression systems lacking MexA failed to co-purify MexB or OprM. None of the heterologous subunit combinations such as MexA+MexY(his)+OprM and MexX+MexB(his)+OprM showed interaction. These results implied that the membrane fusion protein is central to the tripartite xenobiotic transporter assembly.
铜绿假单胞菌的三方异生素 - 抗生素转运体由内膜转运蛋白（如MexB、MexY）、周质膜融合蛋白（如MexA、MexX）和外膜通道蛋白（如OprM）组成。这些亚基被认为在底物输出过程中组装成一个转运体单元。然而，亚基相互作用及其天然形式的特异性仍有待阐明。为了解决这些重要问题，我们通过仅标记其中一个亚基的下拉分析来分析各个亚基在MexAB - OprM组装中的作用。我们发现无需化学交联即可形成稳定的MexA - MexB - OprM复合物，并且该复合物能经受所有纯化程序。二元相互作用分析结果表明，在没有MexB的情况下，MexA和OprM之间紧密结合，而缺乏MexA的表达系统无法共纯化MexB或OprM。诸如MexA + MexY(his) + OprM和MexX + MexB(his) + OprM等异源亚基组合均未显示出相互作用。这些结果表明膜融合蛋白对于三方异生素转运体的组装至关重要。
