Lipochaperoning folding of water-soluble functional membrane protein in genetically and biosynthetically tuned Escherichia coli

Hydrophobic membrane proteins are widely believed to require lipid bilayers for proper folding, collapsing or aggregating in aqueous environments. Using lactose permease (LacY) as a model membrane protein, we show instead that folding <i>in vivo</i> can be optimized for unusual specific molecular interactions with particular phospholipids during functional folding and final localization. Co-overproducing LacY with lysophosphatidic acid (LPA), a normally short-lived intermediate in phospholipid biosynthesis, unexpectedly yields a water-soluble, native LacY in the cytoplasm. This approach obviates SIMPLEx and QTY protein redesign strategies, enabling soluble production of unmodified membrane proteins <i>in vivo</i>. Although surplus LacY spills from the cytoplasmic leaflet together with LPA into cytoplasm, LPA-driven activation of only principal ClsA and buildup of cardiolipin (CL) ensure the SecYEG translocon capacity for proper membrane insertion of LacY. These findings challenge current models of membrane protein biogenesis and provide new opportunities to study membrane proteins in both membrane and aqueous contexts.