Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli

Abstract

A genetic system for directly synthesizing eukaryotic membrane proteins in Escherichia coli and assessing their ability to insert into the bacterial cytoplasmic membrane is described. The components of this system are the direct expression vector, pYZ4, and the mature β-lactamase (BlaM) cassette plasmid, pYZ5, that can be used to generate translational fusions of BlaM to any synthesized membrane protein. The β-subunit of sheep-kidney Na,K-ATPase (βNKA), a class-II plasma membrane protein, was synthesized in E. coli using pYZ4, and BlaM was fused to a normally extracellular portion of it. The fusion protein conferred ampicillin resistance on individual host cells, indicating that the BlaM portion had been translocated to the bacterial periplasm, and that, by inference, the eukaryotic plasma-membrane protein can insert into the bacterial cytoplasmic membrane. A series of 31 βNKA :: BlaM fusion proteins was isolated and characterised to map the topology of the eukaryotic plasma membrane protein with respect to the bacterial cytoplasmic membrane. This analysis revealed that the organisation of the βNKA in the E. coli cytoplasmic membrane was indistinguishable from that in its native plasma membrane.