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

Abstract

The 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 eight out of ten tested Chlamydomonas chloroplast transit peptides, significant additional sequence stretches past the cleavage site are required to enable efficient chloroplast import of heterologous cargo proteins. Analysis of Chlamydomonas cTPs with known cleavage sites and 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

“…6A), and significantly longer than mTPs (t-test, p=0.0017) that are on average 37 residues long. The difference is even greater than shown here given that many cTPs require a contribution from post-cleavage site residues for successful targeting (Bionda et al, 2010;Caspari, 2022). Consistently, cp-set HA-RAMPs (in green, Fig.…”

“…Both of these features are genuine markers of chloroplast localization: in Chlamydomonas, the single cup-shaped chloroplast has a reliable morphology, and the dip in chlorophyll fluorescence at the base of the chloroplast is a well-established marker of the pyrenoid, a proteinaceous chloroplast sub-compartment that contains a large majority of Rubisco (Fig. S2B) (Mackinder et al, 2016;Caspari et al, 2017;Mackinder et al, 2017;Caspari, 2022). While small proteins like Venus can enter the pyrenoid, most thylakoid membranes are excluded, leading to the observed decrease in chlorophyll autofluorescence in this spot.…”

“…While small proteins like Venus can enter the pyrenoid, most thylakoid membranes are excluded, leading to the observed decrease in chlorophyll autofluorescence in this spot. Venus channel signal emanating from the pyrenoid is thus a useful visual guide to true chloroplast localization of the fluorescent reporter (Caspari, 2022). Note that due to cTP-C containing a Rubisco-binding motif (Meyer et al, 2020), Venus accumulation in the Rubisco microcompartment, the pyrenoid, is particularly pronounced in constructs carrying this element.…”

“…Here, we find that the nature of the C-terminus does matter: the more unstructured cTP-C enabled chloroplast targeting more often and more effectively than mTP-C, which contains a long predicted amphipathic helix. The need to contain unstructured sequences (von Heijne and Nishikawa, 1991) as well as the increased length of cTPs, even extending beyond the cleavage site in many cases (Bionda et al, 2010;Caspari, 2022), could be mechanistically related to the import system (Fig. 7A): whereas mitochondrial import makes use of the proton gradient to power uptake of positively charged presequences (Martin et al, 1991;Garg and Gould, 2016), energized chloroplast import requires the cTP to stretch across both TOC and TIC and contact the translocation motor (Chotewutmontri et al, 2017;Richardson et al, 2018;Nakai, 2018).…”

“…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;Bhushan et al, 2006;Chotewutmontri et al, 2012;Chotewutmontri and Bruce, 2015;Köhler et al, 2015;Chotewutmontri et al, 2017;Lee et al, 2019).…”