Delivery of respiratory chain enzymes into membrane mimicking systems by charge-mediated fusion

To investigate interactions of several membrane proteins, they have to be co-reconstituted into a well-defined membrane mimicking-system. We developed a method which enables the co-reconstitution of up to three membrane proteins into vesicles of different sizes. Our approach is to reconstitute membrane proteins first into liposomes and to fuse the liposomes afterwards. We found that if two proteoliposome populations either containing negatively or positively charged lipids were mixed, they rapidly undergo fusion yielding a liposome containing both membrane proteins. Using this strategy, it was possible to co-reconstitute different terminal oxidases and the E. coli ATP synthase, imitating the last step of oxidative phosphorylation. Next, we applied this technique to deliver ATP synthase, reconstituted into positively charged liposomes, into native inverted membrane vesicles of E. coli (which are negatively charged) that lack this enzyme. Upon fusion, respiratory driven ATP synthesis was restored. Our method can be used for the bottom-up approach to increase the complexity of membrane proteins in artificial cells