Dynamics of post‐translational modifications and protein stability in the stroma of Chlamydomonas reinhardtii chloroplasts

Abstract

The proteome of any system is a dynamic entity dependent on the intracellular concentration of the entire set of expressed proteins. In turn, this whole protein concentration will be reliant on the stability/turnover of each protein as dictated by their relative rates of synthesis and degradation. In this study, we have investigated the dynamics of the stromal proteome in the model organism Chlamydomonas reinhardtii by characterizing the half-life of the whole set of proteins. 2-DE stromal proteins profiling was set up and coupled with MS analyses. These identifications featuring an average of 26% sequence coverage and eight non-redundant peptides per protein have been obtained for 600 independent samples related to 253 distinct spots. An interactive map of the global stromal proteome, of 274 distinct protein variants is now available on-line at http://www.isv.cnrs-gif.fr/gel2dv2/. N-α-terminal-Acetylation (NTA) was noticed to be the most frequently detectable post-translational modification, and new experimental data related to the chloroplastic transit peptide cleavage site was obtained. Using this data set supplemented with series of pulse-chase experiments, elements directing the relationship between half-life and N-termini were analyzed. Positive correlation between NTA and protein half-life suggests that NTA could contribute to protein stabilization in the stroma.

Comparative Large Scale Characterization of Plant versus Mammal Proteins Reveals Similar and Idiosyncratic N-α-Acetylation Features

“…Although chloroplast imported proteins have been previously described to be acetylated after the cleavage of the cTP (38,42,47), our current study gives for the first time an idea of the extent of this modification in this organelle with more than 220 characterized proteins. Although some previous studies (38,42) suggest that the NAA is a post-translational modification on a limited number of chloroplast imported protein, the present results clearly highlight the high occurrence of this modification in the chloroplast compartment. Because these polypeptides are imported and processed to remove the cTP during their chloroplast translocation, it appears that the acetylation of these neo-N termini is a widespread protein post-translational modification in the chloroplast.…”

“…These cTP lengths are not significantly different and are comparable with previously published results (42). It is noteworthy that the cTP length determined recently for C. reinhardtii was ϳ37 Ϯ 14 (38), highlighting some important differences, suggesting potential divergences between chlorophytic species and their chloroplast import mechanism.…”

“…Because such NAA occurs at the neo-N terminus uncovered after the cleavage of the chloroplast transit peptide (cTP), this modification is clearly post-translational. Although a few similar cases have been described before (38,42,(47)(48)(49), NAA of chloroplast proteins appears to be a widespread modification in this organelle that has never been described before to this extent. Substrate specificity appears to be close to the cytosolic NatA complex, suggesting a dedicated Nat occurring in this organelle.…”

“…In this context, although large scale studies start to give a better picture of the N-␣-acetylome for a limited number of species, 200 -300 NAAed substrates have been described until now in higher plants (38,(41)(42)(43). Moreover, only one Nat catalytic subunit, i.e.…”

“…Our main goal was to identify kingdom-specific differences, if any. For this purpose, we improved and used a strategy for the N terminus peptides enrichment based on strong cation exchange-liquid chromatography (SCX-LC) and tandem mass spectrometry (MS/MS), allowing simultaneous identification of both the protein and the associated modifications (6,7,9,11,18,38,45,46). Our investigation provides the characterization of 1072 N terminus peptides related to 1007 proteins for the A. thaliana samples and 727 N terminus peptides over 715 proteins for the human sample.…”