Role of a bacterial glycolipid in Sec-independent membrane protein integration

Abstract Non-proteinaceous components in membranes regulate membrane protein integration cooperatively with proteinaceous translocons. An endogenous glycolipid in the Escherichia coli membrane called membrane protein integrase (MPIase) is one such component. Here, we focused on the Sec translocon-independent pathway and examined the mechanisms of MPIase-facilitated protein integration using physicochemical techniques. We determined the membrane integration efficiency of a small hydrophobic protein using solid-state nuclear magnetic resonance, which showed good agreement with that determined by the integration assay using an in vitro translation system. The observed integration efficiency was strongly correlated with membrane physicochemical properties measured using fluorescence techniques. Diacylglycerol, a trace component of E. coli membrane, reduced the acyl chains mobility in the core region and inhibited the integration, whereas MPIase increased the mobility and restored the integration. We observed the electrostatic intermolecular interactions between MPIase and the side chain of basic amino acids in the protein, suggesting that the negatively charged pyrophosphate of MPIase attracts the positively charged residues of a protein near the membrane surface, which acts as a trigger during the early stage of integration. Thus, this study demonstrated the ingenious approach of MPIase to support membrane integration of proteins by using its unique molecular structure in various ways.