GALLEX, a measurement of heterologous association of transmembrane helices in a biological membrane.
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 wild-type 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.
Schneider
Dirk
D
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Engelman
Donald M
DM
D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002462:Cell Membrane / Q000648:ultrastructure*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D017930:Genes, Reporter; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D010957:Plasmids; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000737:chemistry*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein