Structure and function of cyanobacterial Omp85 reveal Toc159/Toc90 as a chloroplast protein import pore

Chloroplast protein-import channel Toc75 evolved from a cyanobacterial outer-membrane Omp85 protein, despite all known Omp85 proteins functioning in export. Chlamydomonas Toc75 assembles with Toc90M, the membrane-spanning domain of Toc90 (equivalent of higher-plant Toc159) into a binocular-shaped complex, but how this architecture arose and functions in import remain unknown. Here, cryo-electron microscopy and mutational analysis of a cyanobacterial Omp85 reveal a lidded 16-β-strand barrel and three POTRA-domains involved in assembling outer-membrane proteins. Structural comparisons show that the Toc75–Toc90M complex mirrors an Omp85–substrate assembly intermediate, and Toc90M can indeed serve as a bacterial Omp85 substrate. While canonical Omp85 systems release substrates after assembly, Toc75 evolved a basic patch in its barrel to anchor Toc90M as a stable lateral pore through which precursor proteins are imported. Our findings indicate that a bacterial export system was converted into a chloroplast import channel by locking a substrate in place to provide an import conduit. This requirement for a stable assembly also supports that additional parts of the ancestral complex, including Omp85 POTRA-domains and Tic236 orthologs, were recruited to facilitate chloroplast protein import. Chloroplasts evolved from a cyanobacterial ancestor. Here authors show a cyanobacterial protein export system was transformed into a chloroplast protein import channel by gaining a specific patch to lock a substrate in place to provide the import conduit.