Diacylglycerol Specifically Blocks Spontaneous Integration of Membrane Proteins and Allows Detection of a Factor-assisted Integration

Abstract

We recently found that the spontaneous integration of M13 procoat is blocked by diacylglycerol (DAG) (Nishiyama, K., Ikegami, A., Moser, M., Schiltz, E., Tokuda, H., and Muller, M. (2006) J. Biol. Chem. 281, 35667-35676). Here, we demonstrate that the spontaneous integration of Pf3 coat, another membrane protein that has been thought to be integrated spontaneously into liposomes, can be blocked by DAG at physiological concentrations. Moreover, the spontaneous integration of the membrane potential-independent version of Pf3 coat (3L-Pf3 coat), which is independent of YidC, was also blocked by DAG. To clarify the mechanism by which DAG blocks spontaneous integration, we examined lipid compounds similar to DAG and DAG derivatives. The blockage of spontaneous integration was specific to DAG, as fatty acids, monoacylglycerol, and phosphatidic acids were not effective for the blockage. When the acyl chains in DAG were shortened even to octanoyl residues, it still blocked spontaneous integration, whereas diheptanoylglycerol did not block it at all. Triacylglycerol was more effective than DAG. However, the lipid A-derivative-dependent integration of M13 procoat could not be reconstituted when triacylglycerol was included in the liposomes. On the other hand, when DAG was included in the liposomes, we found that the integration of 3L-Pf3 coat was strictly dependent on the lipid A-derived integration factor. We propose that the bulky structure of DAG rather than changes in membrane curvature is essential for the blockage of spontaneous integration. We also demonstrated that the blockage of spontaneous integration by DAG is also operative in native membrane vesicles.

Lipid dependencies, biogenesis and cytoplasmic micellar forms of integral membrane sugar transport proteins of the bacterial phosphotransferase system

“…However DAG addition provides a kind of 'seal' so that II Mtl insertion is entirely dependent on the SRP and SecYEG complexes (Kawashima et al, 2008). If DAG is present in sufficient amounts in native E. coli membranes, then the experiments reported by Kawashima et al (2008) of the bilayer and micellar forms of II Glc in vitro does not prove that this occurs at an appreciable rate in vivo. Interestingly, the presence of 'soluble' SecY and FtsY has been demonstrated (Ito & Akiyama, 1991;M.…”

“…It has been found that without the minor E. coli lipid DAG, II Mtl can insert into phospholipid vesicles in vitro via a spontaneous unassisted integration process (Nishiyama et al, 2006). However DAG addition provides a kind of 'seal' so that II Mtl insertion is entirely dependent on the SRP and SecYEG complexes (Kawashima et al, 2008). If DAG is present in sufficient amounts in native E. coli membranes, then the experiments reported by Kawashima et al (2008) of the bilayer and micellar forms of II Glc in vitro does not prove that this occurs at an appreciable rate in vivo.…”

Glycolipozyme membrane protein integrase (MPIase): recent data

“…A dgkA mutant, deficient in DAG kinase, expresses up to ∼10% DAG (19,20). However, overproduction of dgkA did not affect the DAG expression, suggesting the occurrence of tight regulation as to DAG expression (17). Several genes are known to be involved in DAG biosynthesis, i.e., mdoB (22,23), pgpB (24)(25)(26), and eptB (27).…”

“…The existence of DAG has been shown; however, its precise function remains unclear. It is feasible that the in vivo role of DAG is to block deregulated spontaneous insertion (7,17). Wild-type E. coli cells express DAG at ∼1.5% as to phospholipids (17).…”

“…It is feasible that the in vivo role of DAG is to block deregulated spontaneous insertion (7,17). Wild-type E. coli cells express DAG at ∼1.5% as to phospholipids (17). A dgkA mutant, deficient in DAG kinase, expresses up to ∼10% DAG (19,20).…”