Identification and characterization of stable membrane protein complexes

Abstract

Many membrane proteins exist as oligomers. Such oligomers play an important role in a broad variety of cellular processes such as ion transport, energy transduction, osmosensing and cell wall synthesis. We developed an electrophoresis-based method of identifying oligomeric membrane proteins that are stable in SDS-based electrophoresis but become dissociated upon exposure to small alcohols. When this method was applied to a preparation of E.coli inner membrane vesicles 58 different proteins were detected. These proteins included both integral and peripheral membrane proteins with a large variety of different functions. Three proteins were selected for further studies. Studies on the preprotein translocase SecA indicated that this protein is associated with the translocase as a dimer, a subject under dispute in the scientific literature. For the cell wall synthesis enzyme MurG it was found that it occurs as an oligomer on the inner membrane, most likely in the form of a homotetramer. Finally, the mechanosensitive channel of small conductance MscS was found to dissociate upon exposure to membrane-active alcohols in a manner independent of the lipid environment. Several mechanisms are offered to explain the ability of small alcohols to disrupt membrane protein oligomers. Furthermore the method presented here is compared to the commonly employed method to detect such oligomers, Blue-Native PAGE. While BN-PAGE typically identifies protein complexes that are dissociated by SDS, alcohol-induced dissociation targets a subproteome of exceptionally stable complexes: those that are stable in SDS, but become dissociated by small alcohols. Overall, our method can be seen as complementary to the traditional BN-PAGE method. Based on the data obtained for SecA and MurG we present a hypothesis for peripheral membrane protein oligomerisation: Oligomeric peripheral membrane proteins may occur in different forms that display different behaviour on SDS-PAGE gel, migrating as monomers and as oligomers respectively. The monomeric form may either be monomeric in-vivo or become dissociated from an oligomer by exposure to SDS. The oligomeric form on gel, however, is unaffected by SDS but becomes dissociated when it is exposed to TFE. We propose that this oligomeric form originates from peripheral membrane proteins that interact with another membrane component and that it is this interaction which protects them from dissociation by SDS, possibly by inducing a different conformation.