The proper conformation and orientation of membrane protein integration in cells is an important biological event. Interestingly, a new factor named MPIase (membrane protein integrase) was proven essential in this process in Escherichia coli, besides proteinaceous factors, such as Sec translocons and an insertase YidC. A combination of spectroscopic analyses and synthetic work has revealed that MPIase is a glycolipid despite its enzyme-like activity. MPIase has a long glycan chain comprised of repeating trisaccharide units, a pyrophosphate linker, and a diacylglycerol anchor. In order to determine the mechanism of its activity, we synthesized a trisaccharyl pyrophospholipid termed mini-MPIase-3, a minimal unit of MPIase, and its derivatives. A significant activity of mini-MPIase-3 indicated that it involves an essential structure for membrane protein integration. We also analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate protein using physicochemical methods. The structure-activity relationship studies demonstrated that the glycan part of MPIase prevents the aggregation of substrate proteins, and the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles for interactions with substrate proteins. MPIase serves at an initial step in the Sec-independent integration, whereas YidC, proton motive force, and/or SecYEG cooperatively function(s) with MPIase at the following step in vivo. Furthermore, depletion of the biosynthetic enzyme demonstrated that MPIase is crucial for membrane protein integration and cell growth. Thus, we elucidated new biological functions of glycolipids using a combination of synthetic chemistry, biochemistry, physicochemical measurements, and molecular-biological approaches.
Keywords: Anti-aggregation; Biomembrane; Biosynthesis; Chaperone; Glycolipid; Intermolecular interaction; Membrane protein integration; Synthetic ligand; Translocon-independent integration.
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