Summary
We asked what peptide features govern targeting to the mitochondria versus the chloroplast using antimicrobial peptides as a starting point. This approach was inspired by the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell, to which organelle progenitors became resistant. To explore the molecular changes required to convert antimicrobial into targeting peptides, we expressed a set of 13 antimicrobial peptides in Chlamydomonas reinhardtii. Peptides were systematically modified to test distinctive features of mitochondrial and chloroplast targeting peptides, and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as cargo protein. Mitochondrial targeting can be achieved by some unmodified antimicrobial peptide sequences. Targeting to both organelles is improved by replacing Lysines with Arginines. Chloroplast targeting is enabled by the presence of flanking unstructured sequences, additional constraints consistent with chloroplast endosymbiosis having occurred in a cell that already contained mitochondria. If indeed targeting peptides evolved from antimicrobial peptides, required modifications imply a temporal evolutionary scenario with an early exchange of cationic residues, and a late acquisition of chloroplast specific motifs.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We made changes in previous figures 2, 3, 4 and 6 and corresponding captions, and interchanged the order of figures 5 and 6. We adapted corresponding parts in the Results section (lines 138-150, 163-170, 187-197, 208-221, 223-227, 230- 245, 253-258, 265-268, 271-274, 287-333, 335-345), and we moved most of the in silico analysis into supplementary text. Furthermore, we altered parts of the Discussion section (lines 369-379, 381-408, 487- 18th of November 2022 489) and the Abstract (lines 33-36) to more clearly emphasize the novelty of our contributions to the field of organelle targeting. Finally, we improved the quantification of Venus localization in our imaging approach, adding a new supplementary figure (now figure S2), corresponding supplementary text, a corresponding Materials and Methods paragraph (lines 545-559) a new supplementary table (Table S3) and modified Fig. S3 to S20 related to quantification analysis. The latter changes represented a significant research effort, for which we benefited from the input of Chris O Law, who is now co-author of our revised version. We updated author contributions to reflect this. We have also updated the data availability statement, reflecting the fact that we have made our plasmids and relevant strains available to the community through the Chlamydomonas resource centre.