Energy Conversion Organelles N-terminus proteome: A better understanding of organelle protein maturation – eNergiome

Subcellular fractionation provides mitochondria and purified chloroplasts further analysed by large scale mass spectrometry to improve our knowledge of the N- terminal of the proteins involved in the organelles metabolisms. Whereas we characterization hundreds of transit peptide cleavage sites, it was also possible to identify and quantify some modifications such as proteins N- terminal acetylation that appears to be a major modification in the chloroplast stroma .

The first results have uncovered the N-terminal status of hundreds of mitochondrial and chloroplast proteins. Thus , N- terminal acetylation, clearly characterised in the chloroplast organelle, is absent in human mitochondria. In addition, the mechanisms of N-terminal processing of the proteins generate a large number of coexisting proteoforms . This diversity could play a key role in protein degradation regulation.

Although hudreds of N-termini have been already collected, we are still improving our knowledge and collecting information on the N -terminal of the organelle proteins in Humans and Arabidopsis. Considering the data already obtained, combined with samples being processed , we will have a representative panel of mature proteins N-terminus of these organelles that will be used to develop more reliable predictions tools than the current ones. This approach should improve our understanding of the fate of the organelle proteins .

Charactereisation of the first chloroplast specific acetylase :
TV Dinh et al. Molecular identification and functional characterization of the first Na - acetyltransferase in plastids by global acetylome profiling . Proteomics. 2015 .
Characterization of several hundred mature N- termini of human mitochondrial proteome :
Vaca Jacome SA et al. N- terminome analysis of the human mitochondrial proteome . Proteomics. 2015 .

Unlike other cell sub-compartments, energy converting organelles (ECO) such as mitochondria for all eukaryotes and both the mitochondria (Mt) and the chloroplasts (Cp) in plants, chlorophytes and few other organisms, are endosymbiotic compartments of bacterial origin. Both organelles retain a minor part of their original genetic material, about ~110 genes (in case of the plant Cp) that directly codes for ~86 proteins in Cp and ~10-30 in the Mt. This is in contrast with the ~2500-3000 organelle-targeted proteins (Cp) are encoded by the nuclear genome of the host cells and subsequently imported into organelles. For most of these nuclear coded proteins, a transit sequence is used to target them to the correct compartment, i.e., the Mt or the Cp and sometimes both localizations. The targeting sequence is cleaved during the import and other maturation phenomena could also occur such as protein N-terminal acetylation in Cp as recently highlighted. Although protein import mechanisms in the ECO are known and well described in the literature, the exact location of the transit peptide cleavage site and possible post-translational modifications decorating such polypeptides are far less understood and described. Although a few prediction tools are available to propose possible sub-cellular localization and the prediction of transit peptide cleavage site, their results are generally considered to be, at least, imperfect. The mechanisms of protein import in Cp and Mt, and their associated “co-import” posttranslational modifications are essential to understand the composition, dynamics and homeostasis of the organelles proteomes. Organelle proteomes accumulate indeed extremely large amounts of protein material. In addition, as recombinant protein production in plant Cp is a crucial economical challenge and numerous diseases are also associated to the Mt, a better knowledge of the imported protein maturation in the ECO is crucial. The stake of this proposed project is to improve our knowledge in this field using the skills of three research teams able to perform this investigation that requires sample preparation, mass spectrometry analysis, computational treatment, and biochemical investigations for a final validation of the results.
First, this project will require the experimental characterization of the N-termini of the mature proteins involved in these ECO using large scale proteomics approaches. Second, based on a repository system, an advanced database management will provide the required structure and basis for the development of a new generation of prediction tools. Such a database will also be enriched with information available in existing databases much more oriented to protein localization or activity such as AT-CHLORO, MitoRes, PPDB or NextProt. These collected data will be shared with other curated databases such as UniprotKB and at the disposal of the biologists’ community at large. Third, experimental data highlight that some nuclear coded Mt and Cp proteins encounter multiples cleavage sites for the cleavage of the transit peptide. This project plans to determine and to quantify the distribution of such cleavages to determine the yield of cleavage at each position using targeted SRM mass spectrometry analyses. Finally, some proteins not yet described to target into ECO have been characterized repeatedly with starting sequences downstream of the N-terminus predicted from the genomic sequence. Using histology or fusion proteins combined with confocal-microscopy, we will determine the exact localization of these proteins allowing the identification of a new set of proteins targeting these organelles. To conclude, this project should provide important clues to better understand protein maturation in the ECO and to compare differences/similarities between human and plant mitochondria, a question that have never been tackled before at such a scale.