We have reported that mechanosensitive channels of large conductance (MscL) reconstituted in cell-sized asymmetric lipid-protein vesicles containing phospholipids in the outer leaflet and amphiphilic protein (oleosin) in the inner leaflet can transport molecules in response to changes in the lateral membrane pressure. MscL was activated on the lipid-oleosin vesicles without negatively charged lipids, suggesting that negatively charged amino acids in the hydrophilic regions of oleosin at around N and C terminals interact with MscL. In this study, the activity of MscL was controlled by the modifications of the charged amino acids in hydrophilic regions of oleosin in the asymmetric lipid-oleosin vesicle membranes. First, two types of mutated oleosins with all negatively charged amino acids and all positively charged amino acids were prepared by modifying the hydrophilic regions of oleosin at both terminals. Three types of asymmetric lipid-oleosin vesicles containing each oleosin, such as normal oleosin, both negatively charged oleosin, and both positively charged oleosin were formed. Next, we confirmed the orientation of MscL inserted in each asymmetric vesicle. As a result, the orientation of MscL insertion into all types of asymmetric vesicles was at N and C terminals of MscL on the inner leaflet. Finally, to confirm the effects of negatively charged amino acids of oleosin for MscL activation, we observed calcein influx into each asymmetric vesicle via MscL. As a result, the amount of calcein influx into the asymmetric vesicles containing both positively charged oleosin was lower than that containing normal and both negatively charged oleosin, indicating that negatively charged amino acids in the hydrophilic regions of oleosin contribute to MscL activation.
Baba et al. (Sun,) studied this question.