A transit peptide-like sorting signal at the C terminus directs the Bienertia sinuspersici preprotein receptor Toc159 to the chloroplast outer membrane.

Although Toc159 is known to be one of the key GTPase receptors for selective recognition of chloroplast preproteins, the mechanism for its targeting to the chloroplast surface remains unclear. To compare the targeting of these GTPase receptors, we identified two Toc159 isoforms and a Toc34 from Bienertia sinuspersici, a single-cell C₄ species with dimorphic chloroplasts in individual chlorenchyma cells. Fluorescent protein tagging and immunogold studies revealed that the localization patterns of Toc159 were distinctive from those of Toc34, suggesting different targeting pathways. Bioinformatics analyses indicated that the C-terminal tails (CTs) of Toc159 possess physicochemical and structural properties of chloroplast transit peptides (cTPs). These results were further confirmed by fluorescent protein tagging, which showed the targeting of CT fusion proteins to the chloroplast surface. The CT of Bs Toc159 in reverse orientation functioned as a cleavable cTP that guided the fluorescent protein to the stroma. Moreover, a Bs Toc34 mutant protein was retargeted to the chloroplast envelope using the CTs of Toc159 or reverse sequences of other cTPs, suggesting their conserved functions. Together, our data show that the C terminus and the central GTPase domain represent a novel dual domain-mediated sorting mechanism that might account for the partitioning of Toc159 between the cytosol and the chloroplast envelope for preprotein recognition.