Converting antimicrobial into targeting peptides reveals key features governing protein import into mitochondria and chloroplasts

Abstract

We experimentally challenged 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 such antimicrobial peptides into bona fide organelle-targeting peptides, we expressed a set of 13 antimicrobial peptides of various origins in the green alga Chlamydomonas reinhardtii that serves as a model for both mitochondrial and chloroplast import. The peptides were modified to match 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. We present a temporal evolutionary scenario that emphasizes the early contribution of exchanging Lysines with Arginines in the sequence of the antimicrobial peptide, the evolution of a processing site followed by the addition of unstructured sequence and protein interaction sites that allow the selective targeting to the chloroplast.

Transit Peptides Often Require Downstream Unstructured Sequence for Efficient Chloroplast Import in Chlamydomonas reinhardtii

“…Based on the position of cleavage sites, algal cTPs have been noted in the past to be much shorter than their counterparts in vascular plants ( Franzén et al, 1990 ). Yet the requirement for post-cleavage site residues implies that the amino-terminal sequences contributing to targeting in Chlamydomonas are in fact longer than recognized to date, with sequence elements that play a role in import present past the cleavage site just as in short plant cTPs ( Caspari et al, 2021 ). With lengths of 56 and 60 residues, respectively, the import competent RBCA-cTP + 23 and RBCS-cTP + 23 constructs mirror the ~55–60 amino acid requirement of Arabidopsis cTPs ( Bionda et al, 2010 ).…”

“…For quantification of epifluorescence images, ten cells per construct and their chloroplasts (based on chlorophyll autofluorescence) were outlined manually in Fiji version 1.53q ( Schindelin et al, 2012 ), areas and mean Venus channel intensities recorded, and cytoplasmic intensities inferred. Immunoblotting was performed as described elsewhere ( Caspari et al, 2021 ). For disorder analysis, Chlamydomonas cTPs with characterized cleavage sites were obtained from the literature ( Terashima et al, 2011 ; Tardif et al, 2012 ) and IUPRED3 (short) disorder scores obtained using the webserver ( Erdos et al, 2021 ).…”

“…Relatively uncharged in plant cTPs ( Chotewutmontri et al, 2012 ), the presence of multiple arginines in mTP N-termini was found to prevent chloroplast import ( Lee et al, 2019 ). In Chlamydomonas, cTP N-termini are less uncharged and more unstructured, but retain key physico-chemical attributes ( Caspari et al, 2021 ). Hsp70-binding sites where shown to be present in cTP N-termini and be important for import ( Chotewutmontri and Bruce, 2015 ).…”

“…Hsp70-binding sites where shown to be present in cTP N-termini and be important for import ( Chotewutmontri and Bruce, 2015 ). The central part of cTPs is overall positively charged due to the presence of arginines ( Caspari et al, 2021 ). While generally unstructured ( von Heijne and Nishikawa, 1991 ), this part carries amphipathic helix signatures ( Garrido et al, 2020 ) and may fold into such helices upon contact with a membrane ( Bruce, 2000 ).…”

“…A different set of receptors, notably Toc159 and Toc34, also bind cTPs directly. Semi-conserved FGLK-motifs have been suggested as cis-elements that may mediate TOC-interactions ( Chotewutmontri et al, 2017 ), although the F may be less strictly required in Chlamydomonas ( Razzak et al, 2017 ; Caspari et al, 2021 ). Thus, plenty of evidence exists that chloroplast targeting, in the sense of differential protein sorting, is encoded within the cleavable cTP.…”