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Morgane Mininno

Methylation of chloroplastic proteins in Arabidopsis thaliana

Published on 16 September 2014

Thesis presented September​ 16, 2014

Protein methylation is a post-translational modification that regulates dynamically the function of many proteins involved in essentials cellular proc​esses. With the exception of histones, the function of protein methylation is still largely unknown. The aim of my PhD thesis project was to investigate the role of protein methylation in chloroplasts, from the molecular to the whole plant level.
During this work we edited the first methylproteome from Arabidopsis thaliana chloroplasts. This methylproteome consists of 23 proteins belonging to essential metabolic processes such as photosynthesis and biogenesis of plastids (translation, protein import, division). Also in silico analysis allowed the identification of nine chloroplastic protein methyltransferases. We have shown by in vitro methylation assays that, among these enzymes, PRMA-like and PPKMT2 are able to methylate the ribosomal protein L11 and the β-subunit of ATP synthase, respectively. Our data provide a useful resource to build the enzymes/substrates relationships that govern protein methylation in chloroplasts. Deciphering this network is a crucial step towards understanding the role of protein methylation in these organelles.
The basal complex associated with the plastid RNA polymerase PEP is composed of twelve proteins including the predicted protein lysine methyltransferase PTAC14. We showed that the ptac14 null mutation is lethal in Arabidopsis. At the molecular level, the absence of PTAC14 is responsible for a drastic decrease in the transcriptional activity of the PEP polymerase. This defect irreversibly alters the differentiation of plastids that are unable to produce thylakoid structures. Thus the ptac14 mutant is characterized by an albino phenotype and is unable to grow in photoautotrophic conditions. These data indicate that PTAC14 plays an essential role in regulating transcription of the plastid genome. Our first attempts to identify the substrate of PTAC14, which is presumably present in the basal PEP complex, were unsuccessful. Alternatives are proposed to achieve this goal.
Methylation of the large subunit of Rubisco by a protein lysine methyltransferase named LSMT is limited to a few plant species. The role of this modification is still unknown. LSMT- like enzymes are found in all plants and likely display species-specific substrate specificity. In Arabidopsis, we showed that AtLSMT-like does not methylate Rubisco but fructose 1,6- bisphosphate aldolases, chloroplastic enzymes that are involved in the Calvin cycle and glycolysis. Aldolases are also methylated in all species we examined. Trimethylation of aldolases does not alter their kinetic properties or stability, or their oligomerization state. However, the identification of aldolases as methylproteins in Arabidopsis and other plant species suggests that protein methylation may have a role in the control of carbon metabolism in chloroplasts.

Methylation, chloroplast, methylproteome, PTAC14, LSMT-like