Increased expression of the bacterial glycolipid MPIase is required for efficient protein translocation across membranes in cold conditions.

Protein integration into and translocation across biological membranes are vital events for organismal survival and are fundamentally conserved among many organisms. Membrane protein integrase (MPIase) is a glycolipid that drives membrane protein integration into the cytoplasmic membrane in Escherichia coli MPIase also stimulates protein translocation across the membrane, but how its expression is regulated is incompletely understood. In this study, we found that the expression level of MPIase significantly increases in the cold (<25 °C), whereas that of the SecYEG translocon does not. Using previously created gene-knockout E. coli strains, we also found that either the cdsA or ynbB gene, both encoding rate-limiting enzymes for MPIase biosynthesis, is responsible for the increase in the MPIase expression. Furthermore, using pulse-chase experiments and protein integration assays, we demonstrated that the increase in MPIase levels is important for efficient protein translocation, but not for protein integration. We conclude that MPIase expression is required to stimulate protein translocation in cold conditions and is controlled by cdsA and ynbB gene expression.

The bacterial protein YidC accelerates MPIase-dependent integration of membrane proteins.

Bacterial membrane proteins are integrated into membranes through the concerted activities of a series of integration factors, including membrane protein integrase (MPIase). However, how MPIase activity is complemented by other integration factors during membrane protein integration is incompletely understood. Here, using inverted inner-membrane vesicle and reconstituted (proteo)liposome preparations from Escherichia coli cells, along with membrane protein integration assays and the PURE system to produce membrane proteins, we found that anti-MPIase IgG inhibits the integration of both the Sec-independent substrate 3L-Pf3 coat and the Sec-dependent substrate MtlA into E. coli membrane vesicles. MPIase-depleted membrane vesicles lacked both 3L-Pf3 coat and MtlA integration, indicating that MPIase is involved in the integration of both proteins. We developed a reconstitution system in which disordered spontaneous integration was precluded, which revealed that SecYEG, YidC, or both, are not sufficient for Sec-dependent and -independent integration. Although YidC had no effect on MPIase-dependent integration of Sec-independent substrates in the conventional assay system, YidC significantly accelerated the integration when the substrate amounts were increased in our PURE system-based assay. Similar acceleration by YidC was observed for MtlA integration. YidC mutants with amino acid substitutions in the hydrophilic cavity inside the membrane were defective in the acceleration of the Sec-independent integration. Of note, MPIase was up-regulated upon YidC depletion. These results indicate that YidC accelerates the MPIase-dependent integration of membrane proteins, suggesting that MPIase and YidC function sequentially and cooperatively during the catalytic cycle of membrane protein integration.

Intermolecular Interactions between a Membrane Protein and a Glycolipid Essential for Membrane Protein Integration.

Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in Escherichia coli is mediated by proteinaceous factors, such as Sec translocons and an insertase YidC. Additionally, a glycolipid named MPIase (membrane protein integrase), composed of a long sugar chain and pyrophospholipid, was proven essential for membrane protein integration. We reported that a synthesized minimal unit of MPIase possessing only one trisaccharide, mini-MPIase-3, involves an essential structure for the integration activity. Here, to elucidate integration mechanisms using MPIase, we analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate, the Pf3 coat protein, using physicochemical methods. Surface plasmon resonance (SPR) analyses revealed the importance of a pyrophosphate for affinity to the Pf3 coat protein. Compared with mini-MPIase-3, natural MPIase showed faster association and dissociation due to its long sugar chain despite the slight difference in affinity. To focus on more detailed MPIase substructures, we performed docking simulations and saturation transfer difference-nuclear magnetic resonance. These experiments yielded that the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles leading to interactions with the Pf3 coat protein. The high affinity of MPIase to the hydrophobic region and the basic amino acid residues of the protein was suggested by docking simulations and proven experimentally by SPR using protein mutants devoid of target regions. These results demonstrated the direct interactions of MPIase with a substrate protein and revealed detailed mechanisms of membrane protein integration.

Biosynthesis of glycolipid MPIase (membrane protein integrase) is independent of the genes for ECA (enterobacterial common antigen).

MPIase (membrane protein integrase) is an essential glycolipid that drives protein integration into the inner membrane of E. coli, while glycolipid ECA (enterobacterial common antigen) is a major component at the surface of the outer membrane. Irrespective of the differences in molecular weight, subcellular localization and function in cells, the glycan chains of the two glycolipids are similar, since the repeating unit comprising the glycan chains is the same. A series of biosynthetic genes for ECA, including ones for the corresponding nucleotide sugars, have been identified and extensively characterized. In this study, we found that knockouts as to the respective genes for ECA biosynthesis can grow in the minimum medium with the normal expression level of MPIase, indicating that MPIase can be biosynthesized de novo without the utilization of any compounds generated through ECA biosynthesis. Conversely, ECA was expressed normally upon MPIase depletion. From these results, we conclude that the biosynthetic genes for MPIase and ECA are independent.

Cholesterol blocks spontaneous insertion of membrane proteins into liposomes of phosphatidylcholine.

Spontaneous insertion of membrane proteins into liposomes formed from Escherichia coli polar phospholipids is blocked by diacylglycerol (DAG) at a physiological level. We found that cholesterol also blocks this spontaneous insertion, although a much larger amount is necessary for sufficient blockage. Reversely, sphingomyelin enhanced the spontaneous insertion. DAG at a physiological level was found not to block spontaneous insertion into liposomes formed from phosphatidylcholine (PC), while non-physiologically high concentrations of DAG reduced it. On the other hand, cholesterol blocked the spontaneous insertion into PC liposomes at a physiological level, explaining that both PC and cholesterol are absent in E. coli. While sphingomyelin did not enhance spontaneous insertion into PC liposomes, the effect of cholesterol on blockage of spontaneous insertion was dominant over that of sphingomyelin, suggesting that cholesterol functions as a blocker of disordered spontaneous insertion in eukaryotic cells. Lower amount of cholesterol was necessary to block spontaneous insertion into ER-mimic liposomes, explaining that ER membranes contain less amount of cholesterol. These results also explain that cholesterol, but not DAG, is involved in blockage of spontaneous insertion in eukaryotic cells, since DAG plays an important role as a second messenger in signal transduction.

Syntheses and Activities of the Functional Structures of a Glycolipid Essential for Membrane Protein Integration.

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.