The Evolutionary Flip of Chloroplast Omp85 Proteins

A mutual feature of the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts, is the presence of ß-barrel proteins. Insertion and assembly of these proteins is catalyzed by the Outer membrane protein of 85kDA (Omp85) family in a seemingly conserved process. The common setup of all members of this family is an N-terminal region composed one or more polypeptide transport-associated (POTRA) domains and a C-terminal ß-barrel domain. In plants, two essential but phylogenetically distinct Omp85 proteins are present in the chloroplast outer envelope, namely Toc75-III and Toc75-V. While Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, which is the insertion of β-barrel proteins into the outer membrane, Toc75-III, evolved to the translocation pore of the TOC complex responsible for pre-protein import. Current models of β-barrel biogenesis and preprotein translocation assume a conserved topology of all Omp85 proteins with the POTRA domain exposed to periplasm or the intermembrane space, respectively. By self assembly GFP-based in vivo topology studies we demonstrate a cytoplasmic exposure of the POTRA domains of both Toc75-III and Toc75-V. In constrast to this observation we show that the mitochondrial Sam50 retained the proposed topology of its bacterial ancestor. These findings explain many experimental observations and enforce a re-evaluation of current models of β-barrel biogenesis and TOC complex function.