The insertion of membrane proteins requires proteinaceous complexes in the cytoplasm, the membrane, and the lumen of organelles. Most of the required complexes have been described, while the components for insertion of β‐barrel‐type proteins into the outer membrane of chloroplasts remain unknown. The same holds true for the signals required for the insertion of β‐barrel‐type proteins. At present, only the processing of Toc75‐III, the β‐barrel‐type protein of the central chloroplast translocon with an atypical signal, has been explored in detail. However, it has been debated whether Toc75‐V/ outer envelope protein 80 (OEP80), a second protein of the same family, contains a signal and undergoes processing. To substantiate the hypothesis that Toc75‐V/OEP80 is processed as well, we reinvestigated the processing in a protoplast‐based assay as well as in native membranes. Our results confirm the existence of a cleavable segment. By protease protection and pegylation, we observed intermembrane space localization of the soluble N‐terminal domain. Thus, Toc75‐V contains a cleavable N‐terminal signal and exposes its polypeptide transport‐associated domains to the intermembrane space of plastids, where it likely interacts with its substrates.

The insertion of organellar membrane proteins with the correct topology requires the following: First, the proteins must contain topogenic signals for translocation across and insertion into the membrane. Second, proteinaceous complexes in the cytoplasm, membrane, and lumen of organelles are required to drive this process. Many complexes required for the intracellular distribution of membrane proteins have been described, but the signals and components required for the insertion of plastidic β-barrel-type proteins into the outer membrane are largely unknown. The discovery of common principles is difficult, as only a few plastidic β-barrel proteins exist. Here, we provide evidence that the plastidic outer envelope β-barrel proteins OEP21, OEP24, and OEP37 from pea (Pisum sativum) and Arabidopsis thaliana contain information defining the topology of the protein. The information required for translocation of pea proteins across the outer envelope membrane is present within the six N-terminal β-strands. This process requires the action of TOC (translocon of the outer chloroplast membrane). After translocation into the intermembrane space, β-barrel proteins interact with TOC75-V, as exemplified by OEP37 and P39, and are integrated into the membrane. The membrane insertion of plastidic β-barrel proteins is affected by mutation of the last β-strand, suggesting that this strand contributes to the insertion signal. These findings shed light on the elements and complexes involved in plastidic β-barrel protein import.