Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

Abstract

1AbstractThe N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for nine out of ten tested Chlamydomonas chloroplast transit peptides, additional sequence past the cleavage site is required to enable chloroplast targeting. Using replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.

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

“…By contrast, most cTPs are significantly longer (p=0.0017). The difference is even greater than shown here given that many cTPs require a contribution from post-cleavage site residues for successful targeting 5,18,19 . Consistently, cp-set HA-RAMPs (in green, Fig.…”

“…Thus HA-RAMPs that intrinsically contained relatively unstructured N-termini proved most able to support chloroplast targeting even in the absence of cTP-N. The need to contain unstructured sequence 8 as well as the increased length of cTPs, even extending beyond the cleavage site in many cases 5,18,19 , appear mechanistically related to the import system. Whereas mitochondrial import makes use of the proton gradient across the inner membrane to power uptake of positively charged presequences 34,35 , energized chloroplast import requires the cTP to be elongated enough to reach across both TOC and TIC and contact the translocation motor 20,36 .…”

“…Indeed mTP-C are more structured, containing a long predicted amphipathic helix. The need to contain unstructured sequences 13 as well as the increased length of cTPs, even extending beyond the cleavage site in many cases 10, 21, 22 , appear mechanistically related to the import system (Fig. 7a): whereas mitochondrial import makes use of the proton gradient to power uptake of positively charged presequences 37, 38 , energized chloroplast import requires the cTP to stretch across both TOC and TIC and contact the translocation motor 2, 39, 40 .…”

“…While targeting information is usually contained within mTP sequences upstream of the cleavage site, targeting by cTPs shorter than ca. 60 amino acids often requires downstream unstructured sequence stretches in the N-terminal domain of the mature protein 18,19 . Besides the amphipathic helical module and the C-terminal cleavage module shared between mTPs et cTPs, it has been argued that distinct features at their N-termini conferred organelle specificity to each set of targeting peptides 6,16,[20][21][22][23][24][25][26] .…”

“…While targeting information is usually contained within mTP sequences upstream of the cleavage site, targeting by cTPs shorter than ca. 60 amino acids often requires downstream unstructured sequence stretches in the N-terminal domain of the mature protein (Bionda et al, 2010; Caspari, 2022). Besides the amphipathic helical module and the C-terminal cleavage module shared between mTPs and cTPs, it has been argued that distinct features at their N-termini conferred organelle specificity to each set of targeting peptides (von Heijne et al, 1989; Ivey and Bruce, 2000; Ivey et al, 2000; Bhushan et al, 2006; Chotewutmontri et al, 2012; Chotewutmontri and Bruce, 2015; Köhler et al, 2015; Chotewutmontri et al, 2017; Lee et al, 2019).…”