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Toc75 and OEP80 are paralogous proteins found in the Viridiplantae lineages, and appear to have evolved from a protein in the outer membrane of an ancient cyanobacterium. Toc75 is known to act as a protein translocation channel at the outer membrane of the chloroplast envelope, whereas the exact function of OEP80 is not understood. In Arabidopsis thaliana, each protein is encoded by a single gene, and both are essential for plant viability from embryonic stages onward. Sequence annotation and immunoblotting data with an antibody against its internal sequence (αOEP80(325-337)) indicated that the molecular weight of OEP80 is ca. 80 kD. Here we present multiple data to show that the size of A. thaliana OEP80 is smaller than previously estimated. First, we prepared the antibody against a recombinant protein consisting of annotated full-length A. thaliana OEP80 with an N-terminal hexahistidine tag (αOEP80(1-732)). This antibody recognized a 70-kD protein in the A. thaliana chloroplast membrane fraction which migrated faster than the His-tagged antigen and the protein recognized by the αOEP80(325-337) antibody on SDS-PAGE. Immunoprecipitation followed by LC-MS/MS analysis confirmed that the 70-kD protein was encoded by the OEP80 cDNA. Next, we performed a genetic complementation assay using embryo-lethal oep80-null plants and constructs encoding OEP80 and its variants. The results revealed that the nucleotide sequence encoding the 52 N-terminal amino acids was not required for functional expression of OEP80 and accumulation of the 70-kD protein. The data also indicated that an additional C-terminal T7 tag remained intact without disrupting the functionality of OEP80, and was not exposed to the cytoplasmic surface of the chloroplast envelope. Finally, OEP80-T7 and Toc75 showed distinct migration patterns on blue native-PAGE. This study provides molecular tools to investigate the function of OEP80, and also calls for caution in using an anti-peptide antibody.

Chloroplasts are organelles specific to photosynthetic eukaryotes that support the lives of most organisms on earth. Chloroplasts were derived from an ancient cyanobacterium by endosymbiosis, and one characteristic shared between them and extant cyanobacteria is the presence of beta-barrel proteins in the outer membrane. These integral membrane proteins are also found in the outer membranes of proteobacteria and mitochondria. In particular, a group of homologous beta-barrel proteins called BamA homologs are present in all Gram-negative bacteria and the endosymbiotic organelles, i.e., chloroplasts and mitochondria. It was recently revealed that, in both proteobacteria and mitochondria, there is a single essential BamA homolog that mediates beta-barrel protein assembly. In a chloroplast, there are two distinct BamA homologs, Toc75 and OEP80, which diverged early in the evolution of chloroplasts from their common ancestor with extant cyanobacteria. Recent genetic studies demonstrated that each of these proteins is indispensable for viability of plants although neither has been shown to be involved in beta-barrel protein assembly. Toc75 catalyzes import of nuclear-encoded precursor proteins, a process that is not required for bacteria, whereas the molecular function of OEP80 remains elusive. Establishment of a protein import apparatus was required to facilitate the transfer of genes from the endosymbiont to the host cell nucleus. Hence, we propose that the gene duplication giving rise to the two essential BamA homologs was a prerequisite for the successful conversion of the cyanobacterial endosymbiont into the chloroplast. Consequently, continued study of these two chloroplast proteins should advance our understanding of endosymbiosis and evolutionarily conserved proteins in general.