Protein integration into biological membranes is a vital cellular event for all organisms. We previously reported an integration factor in the inner membrane of Escherichia coli, named MPIase (membrane protein integrase). Here we show that in contrast to previously identified integration factors that are proteins, MPIase is a glycolipid composed of diacylglycerol and a glycan chain of three acetylated aminosugars linked through pyrophosphate. Hydrolytic removal of the lipid moiety gives a soluble product with higher integration activity than that of the original MPIase. This soluble form of MPIase directly interacts with a newborn membrane protein, maintaining its integration-competent structure and allowing its post-translational integration. MPIase actively drives protein integration following chaperoning membrane proteins. We further demonstrate with anti-MPIase antibodies that MPIase is likely involved in integration in vivo. Collectively, our results suggest that MPIase, essential for membrane protein integration, is to our knowledge the first glycolipid with an enzyme-like activity.

MPIase is the first known glycolipid that is essential for membrane protein integration in the inner membrane of E. coli. Since the amount of natural MPIase available for analysis is limited and it contains structural heterogeneity, precisely designed synthetic derivatives are promising tools for further elucidation of its membrane protein integration mechanism. Thus, we synthesized the minimal unit of MPIase, a trisaccharyl pyrophospholipid termed mini-MPIase-3, and its derivatives. Integration assays revealed that the chemically synthesized trisaccharyl pyrophospholipid possesses significant activity, indicating that it includes the essential structure for membrane integration. Structure-activity relationship studies demonstrated that the number of trisaccharide units and the 6- O-acetyl group on N-acetylglucosamine contribute to efficient integration. Furthermore, anchoring in the membrane by a lipid moiety was essential for the integration. However, the addition of phosphorylated glycans devoid of the lipid moiety in the assay solution modulated the integration activity of MPIase embedded in liposomes, suggesting an interaction between phosphorylated glycans and substrate proteins in aqueous solutions. The prevention of protein aggregation required the 6- O-acetyl group on N-acetylglucosamine, a phosphate group at the reducing end of the glycan, and a long glycan chain. Taken together, we verified the mechanism of the initial step of the translocon-independent pathway in which a membrane protein is captured by a glycan of MPIase, which maintains its structure to be competent for integration, and then MPIase integrates it into the membrane by hydrophobic interactions with membrane lipids.