Background

Membrane proteins and presecretory proteins, synthesized in the cytosol, are integrated into and translocated across biomembranes, respectively, to be localized at their destinations and to express their functions. Cells possess systems to integrate membrane proteins into and translocate presecretory proteins across biomembranes, these proteins being commonly conserved from bacteria to higher eukaryotes.

In E. coli, a model organism, some integration factors were identified through genetic approaches, these factors including translocon SecYEG (Newitt and Bernstein, 1998), and signal recognition particle (SRP) and its receptor SR (Ulbrandt et al., 1997), and membrane protein insertase/chaperone YidC (Samuelson et al., 2000). These genetic studies were complemented by biochemical approaches. The molecular mechanisms underlying membrane protein integration have been studied extensively using an in vitro system, in which the protein integration reactions were reproduced in test tubes.

Inverted membrane vesicles (INV) can be prepared by disrupting E. coli cells with a French press, followed by sucrose gradients (Alami et al., 2002). The outside surface of INV corresponds to the cytoplasmic face of the inner membrane. Therefore, presecretory proteins are translocated into the lumen of INV. Membrane proteins, synthesized outside of INV using the cell-free translation system, are integrated into INV. The roles of the above-mentioned factors have been analyzed using INV prepared from the respective mutant cells (Koch et al., 1999; Koch and Muller, 2000). However, since most of the factors are essential for cell growth, depletion of each of the factors causes unexpected pleiotropic effects. To exclude such secondary effects, a reconstitution system has been developed using purified factors. Proteoliposomes can be prepared by mixing membrane proteins, solubilized and purified in a detergent solution, and phospholipids, followed by removal of the detergent. The reconstituted proteoliposomes are essentially the same as INV and thus they can be directly used in the integration assay in vitro. On the other hand, disordered spontaneous integration of membrane proteins into liposomes formed of phospholipids is a serious problem, since such spontaneous integration is an in vitro artefact that does not reflect the reaction in vivo. We have solved this problem by introducing diacylglycerol (DAG) into the liposomes (Nishiyama et al., 2006; Kawashima et al., 2008). As a consequence, DAG blocked spontaneous integration at the physiological content, which enabled reconstitution of membrane protein integration. By using such a reconstitution system we identified a glycolipid named MPIase (Membrane Protein Integrase) as an essential factor for membrane protein integration (Nishiyama et al., 2006; Nishiyama et al., 2010). Based on the observation that MPIase catalyzes membrane protein integration (Nishiyama et al., 2010), we proposed that MPIase is a “glycolipozyme” (Nishiyama et al., 2012). We also determined the MPIase structure, in which a glycan chain composed of 9~11 repeats of a unit of three N-acetylated amino sugars, GlcNAc, ManNAcA and Fuc4NAc, is connected to DAG through a pyrophosphate linker (Nishiyama et al., 2012). Thus, membrane protein integration can be reproduced by introducing lipid components such as DAG and MPIase into reconstituted proteoliposomes.

Recently, we found another problem when reconstituting proteoliposomes. DAG is not only solubilized by a series of detergents such as dodecyl maltoside (DDM) and dodecyl phosphocholine (DPC), but also forms wax-like complexes with them (Sasaki et al., 2019). Since these wax-like complexes induce the spontaneous integration, the detergents must be removed completely from the reconstituted proteoliposomes. On the other hand, DDM and DPC are frequently used to purify the SecYEG complex (Collinson et al., 2001) and YidC (Stiegler et al., 2011; Welte et al., 2012). Therefore, the membrane components should be properly reconstituted without the formation of the unexpected complexes that induce spontaneous integration.

In this protocol, we report a method for reconstituting proteoliposomes containing SecYEG and YidC, but free of the complex of DAG and DDM/DPC. To achieve this, SecYEG and/or YidC were first reconstituted into DAG-free proteoliposomes, followed by membrane fusion with liposomes containing DAG and MPIase by freezing-thawing-sonication, which yielded unilamellar proteoliposomes (Sasaki et al., 2019). Such proteoliposomes are ready for the membrane integration assay. This assay system includes a Pure System, a reconstituted translation system, to in vitro synthesize substrate membrane proteins (Nishiyama et al., 2010; Shimizu et al., 2001). The integration activity was determined by analyzing the membrane protected fragments (MPF) (Koch et al., 1999). The MPFs generated upon protease digestion after the integration reaction reflect the membrane integration.