Anionic lipids stimulate Sec-independent insertion of a membrane protein lacking charged amino acid side chains

We have investigated the influence of the different lipid classes of Escher ichia coli on Sec-independent membrane protein insertion, using an assay in which a mutant of the single-spanning Pf3 coat protein is biosynthetically inserted into liposomes. It was found that phosphatidylethanolamine and ot her non-bilayer lipids do not have a significant effect on insertion. Surpr isingly, the anionic lipids phosphatidylglycerol and cardiolipin stimulate N-terminal translocation of the protein, even though it has no charged amin o acid side chains. This novel effect is general for anionic lipids and dep ends on the amount of charge on the lipid headgroup. Since the N-terminus o f the protein is at least partially positively charged due to a helix dipol e moment, apparently negatively charged lipids can stimulate translocation of slightly positively charged protein segments in a direction opposite to the positive-inside rule. A mechanism is proposed to explain these results.