Opsins are phospholipid scramblases in all domains of life.

Opsins are highly abundant retinal proteins in the membranes of photoheterotrophic bacteria. However, some microbial genomes encode an opsin but lack the gene for the final enzyme in retinal synthesis. To account for this paradox, we hypothesized that bacterial opsins play a role in membrane structure and/or biogenesis independent of their potential for light-driven signaling or proton pumping. After purifying actinorhodopsin from a cell-free expression system and from Escherichia coli membranes upon overexpression, we demonstrated both in vitro and in silico that actinorhodopsin from Nanopelagicus ca. is a phospholipid scramblase, serving in its pentameric state as a retinal-independent phospholipid diffusion channel. Phospholipid headgroups move along a transbilayer path between actinorhodopsin protomers to equilibrate lipid content in the inner and outer leaflets. Two profound activities, membrane biosynthesis and capture of light energy, are thus facilitated by one ancient bacterial polypeptide. Light-dependent activity and light-independent phospholipid scrambling are shared functions of eukaryotic, archaeal, and bacterial rhodopsins.IMPORTANCECells are surrounded by membranes that concentrate metabolites and protect cellular contents. Most biomembranes are phospholipid bilayers, in which the phospholipids of each leaflet orient their greasy tails inward and polar groups outward. Bilayer biogenesis depends on phospholipids synthesized on the cytofacial side of the membrane reorienting to the extracellular membrane leaflet. This reorientation requires proteins, termed scramblases, and it was shown that rhodopsins-7-helix photoactive membrane proteins bound to the cofactor retinal-from organisms as widely divergent as mammals and archaea possess scramblase activity. Now we conclusively demonstrate, using purified proteins in laboratory membranes as well as computational approaches, that bacterial rhodopsins are also phospholipid scramblases. This work is important because it highlights a surprising commonality among bacteria, archaea, and eukaryotes and because it shows that rhodopsins-ancient proteins found in the last universal common ancestor-manifest two seemingly unrelated biochemical functions in one protein.