Abstract
After a nascent chain of a membrane protein emerges from the ribosomal tunnel, the protein is integrated into the cell membrane. This process is controlled by a series of proteinaceous molecular devices, such as signal recognition particles and Sec translocons. In addition to these proteins, we discovered two endogenous components regulating membrane protein integration in the inner membrane of Escherichia coli. The integration is blocked by diacylglycerol (DAG), whereas the blocking is relieved by a glycolipid named membrane protein integrase (MPIase). Here, we investigated the influence of these integration-blocking and integration-promoting factors on the physicochemical properties of membrane lipids via solid-state NMR and fluorescence measurements. These factors did not have destructive effects on membrane morphology because the membrane maintained its lamellar structure and did not fuse in the presence of DAG and/or MPIase at their effective concentrations. We next focused on membrane flexibility. DAG did not affect the mobility of the membrane surface, whereas the sugar chain in MPIase was highly mobile and enhanced the flexibility of membrane lipid headgroups. Comparison with a synthetic MPIase analog revealed the effects of the long sugar chain on membrane properties. The acyl chain order inside the membrane was increased by DAG, whereas the increase was cancelled by the addition of MPIase. MPIase also loosened the membrane lipid packing. Focusing on the transbilayer movement, MPIase reduced the rapid flip-flop motion of DAG. On the other hand, MPIase could not compensate for the diminished lateral diffusion by DAG. These results suggest that by manipulating the membrane lipids dynamics, DAG inhibits the protein from contacting the inner membrane, whereas the flexible long sugar chain of MPIase increases the opportunity for interaction between the membrane and the protein, leading to membrane integration of the newly formed protein.
Highlights
It is important for membrane proteins to be correctly integrated into biomembranes for their proper functioning
To clarify the effect of two membrane protein integration factors, DAG and membrane protein integrase (MPIase), on the membrane, we first investigated the membrane morphology of liposomes prepared from EPL in the absence and presence of DAG and/or natural MPIase
Dynamic light scattering measurement and the observation of cryo-transmission electron microscope images showed that EPL liposomes in each sample formed single large unilamellar vesicles (LUVs), regardless of the presence of DAG and/or mini-MPIase-3, and no significant difference in the average size of liposomes was observed between each sample (Fig. S3)
Summary
It is important for membrane proteins to be correctly integrated into biomembranes for their proper functioning. In the inner membrane of Escherichia coli, many membrane proteins are integrated into the membrane with the help of signal recognition particle (SRP), its receptor, and the Sec translocon complex (Sec/SRP-dependent membrane integration pathway) [1,2,3] (Fig. 1 a). In the Sec/SRP-independent process, it was previously thought that these membrane proteins spontaneously integrate into the membrane. 3L-Pf3 coat protein, which is a mutant version of bacteriophage Pf3 coat protein and has been used as a model substrate, was reported to integrate spontaneously into liposomes composed only of a commercially available mixture of polar lipids extracted from E. coli (EPL) (https:// avantilipids.com/). It became evident that the Sec/SRP-dependent and -independent membrane integration into EPL liposomes cannot be reproduced in a precise manner. Even Sec/SRP-dependent proteins such as mannitol permease spontaneously integrated into membranes in the absence of SRP receptor, SRP, or SecYEG. The addition of a physiological concentration of DAG (2–3 weight percentage (wt%) of EPL) into EPL liposomes completely inhibited
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