GALLEX, a measurement of heterologous association of transmembrane helices in a biological membrane
GALLEX, a measurement of heterologous association of transmembrane helices in a biological membrane
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DOI：
10.1074/jbc.m206287200
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发表时间：
2003-01-31
影响因子：
4.8
通讯作者：
Engelman, DM
中科院分区：
文献类型：
--
作者：
Schneider, D;Engelman, DM
虽然各种各样的双杂交系统可用于测量可溶性蛋白质的相互作用，相关的方法是显着较少开发的膜蛋白相互作用的测量。在这里，我们提出了一个双杂交系统，遵循异源二聚化的膜蛋白在大肠杆菌内膜。该方法是基于两个具有不同DNA结合特异性的莱克萨DNA结合结构域对报告基因活性的抑制。当与跨膜结构域偶联时，通过抑制β-半乳糖苷酶合成报告异二聚体缔合。发现LexA-跨膜嵌合蛋白正确地插入膜中，并且获得可重复的信号，测量野生型和突变型血型糖蛋白A跨膜螺旋的同源二聚化以及异源二聚化。的GALLEX数据进行了比较，最近获得的数据通过其他方法和讨论的一般情况下，单TM螺旋的异源缔合。此外，测试了α（4）和β（7）整联蛋白亚基的TM结构域之间异二聚体的形成。结果表明，同源和异源二聚化的膜蛋白可以准确地测量使用GALLEX系统。
Whereas a variety of two-hybrid systems are available to measure the interaction of soluble proteins, related methods are significantly less developed for the measurement of membrane protein interactions. Here we present a two-hybrid system to follow the heterodimerization of membrane proteins in the Escherichia coli inner membrane. The method is based on the repression of a reporter gene activity by two LexA DNA binding domains with different DNA binding specificities. When coupled to transmembrane domains, heterodimeric association is reported by repression of beta-galactosidase synthesis. The LexA-transmembrane chimeric proteins were found to correctly insert into the membrane, and reproducible signals were obtained measuring the homodimerization as well as heterodimerization of wildtype and mutant glycophorin A transmembrane helices. The GALLEX data were compared with data recently gained by other methods and discussed in the general context of heteroassociation of single TM helices. Additionally, the formation of heterodimers between the TM domains of the alpha(4) and the beta(7) integrin subunits were tested. The results show that both homo- and heterodimerization of membrane proteins can be measured accurately using the GALLEX system.