Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

Abstract

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.

The Arabidopsis Chloroplast stromal N-terminome; complexities of N-terminal protein maturation and stability

“…The lack of NME for just RBCS is likely due to the presence of a bulky residue (Lys) immediately after the Met. It should be noted that Lys methylation has been observed for several Arabidopsis chloroplast proteins downstream of their mature Nti Alban et al, 2014). Lys-14 of RBCL has been shown in pea (Pisum sativum) to be (tri)methylated (Houtz et al, 2008).…”

Posttranslational Modifications of FERREDOXIN-NADP+ OXIDOREDUCTASE in Arabidopsis Chloroplasts

“…Acetylation and methylation of chloroplast proteins have been identified recently (Houtz et al, 1992;Finkemeier et al, 2011;Wu et al, 2011;Mininno et al, 2012;Alban et al, 2014). Therefore, the acetylation and methylation states of the four membrane-bound AtFNR forms were analyzed.…”

Posttranslational Modifications of Chloroplast Proteins: An Emerging Field

“…A recent study has identified 23 chloroplast methylproteins (Alban et al, 2014), including the previously described ALDOLASE isoforms (Mininno et al, 2012) and PLASTID RIBOSOMAL PROTEIN L11 (Yamaguchi and Subramanian, 2000). Four out of these 23 proteins contained one or more methylated Arg residues, while 17 proteins had Lys methylation.…”

“…RBCL and ATP SYNTHASE b-subunit were methylated at both Lys and Arg residues (Alban et al, 2014). In addition to Rubisco and FRU-1,6-BISPHOSPHATE ALDOLASE, five additional proteins functioning in CO 2 assimilation were methylated (Alban et al, 2014). Interestingly, chloroplast protein methylation is, at least partly, induced by light (Niemi et al, 1990).…”

“…Four out of these 23 proteins contained one or more methylated Arg residues, while 17 proteins had Lys methylation. RBCL and ATP SYNTHASE b-subunit were methylated at both Lys and Arg residues (Alban et al, 2014). In addition to Rubisco and FRU-1,6-BISPHOSPHATE ALDOLASE, five additional proteins functioning in CO 2 assimilation were methylated (Alban et al, 2014).…”