Assembling of AcrB Trimer in Cell Membrane
Assembling of AcrB Trimer in Cell Membrane
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DOI：
10.1016/j.jmb.2012.06.036
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发表时间：
2012-10-12
影响因子：
5.6
通讯作者：
Wei, Yinan
中科院分区：
文献类型：
--
作者：
Lu, Wei;Chai, Qian;Wei, Yinan
许多膜蛋白以低聚物的形式存在并发挥作用，但单体如何在细胞膜中齐聚仍然知之甚少。AcrB是一种专性的高三聚体。我们先前发现，单个亚基的折叠先于齐聚。折叠后，单个AcrB亚基必须定位并相互作用，才能二聚化并最终三聚化。目前尚不清楚AcrB三聚化是否是遵循“偶然相遇和随机组装”机制的自发过程。换言之，目前尚不清楚单体亚单位在共翻译插入并折叠到细胞膜后是否会自由扩散，以相互寻找。通过使用具有不同融合标签的AcrB变体、二硫化物捕获和活性测量的四组实验，我们在这里证明了在同一大肠杆菌细胞中共表达的AcrB变体在体内确实共同组装成杂交三聚体。然而，实验测量的联合组装水平与随机组装得出的计算结果不一致。多聚体结构在蛋白质翻译过程中的潜在作用以及由此产生的聚集效应被讨论为对观察到的AcrB亚基在体内组装中的偏差的潜在解释。我们的结果为细胞膜中专性同源低聚膜蛋白的动态组装和平衡过程提供了新的见解。(C)2012爱思唯尔有限公司。保留所有权利。
Many membrane proteins exist and function as oligomers, but how monomers oligomerize in the cell membrane remains poorly understood. AcrB is an obligate homo-trimer. We previously found that the folding of individual subunit precedes oligomerization. Following folding, individual AcrB subunits must locate and interact with each other in order to dimerize and eventually trimerize. It has been unclear if AcrB trimerization is a spontaneous process following the "chance encounter and random assembling" mechanism. In other words, it is currently unknown whether monomeric subunits diffuse freely to "search" for each other after they are co-translationally inserted and folded into the cell membrane. Using four sets of experiments exploiting AcrB variants with different fusion tags, disulfide trapping, and activity measurement, here we showed that AcrB variants co-expressed in the same Escherichia coli cell did co-assemble into hybrid trimers in vivo. However, the level of co-assembly measured experimentally was not consistent with calculations derived from random assembling. The potential role of the polysome structure during protein translation and the resultant clustering effect were discussed as a potential explanation for the observed bias in AcrB subunit assembling in vivo. Our results provide new insights into the dynamic assembling and equilibration process of obligate homo-oligomeric membrane proteins in the cell membrane. (C) 2012 Elsevier Ltd. All rights reserved.