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Acetylation on amino groups is a common modification seen in proteins across various organisms. This process involves N-terminal acetyltransferases (NATs) and lysine acetyltransferases (KATs) that transfer acetyl groups from acetyl-Coenzyme A (acetyl-CoA) to the N-terminal amino groups and to the side chains of lysine residues, respectively. In the case of plants, the majority of plastid proteins are imported from the cytosol and are subject to cleavage of their N-terminal signal peptides. The plastid proteins that are encoded internally are processed at their N-termini by various aminopeptidases. It has been observed that many of the newly formed N-termini and the lysine residues on the side chains of proteins undergo acetylation. However, the specific NATs and KATs responsible for these acetylation processes remain largely unidentified. In recent studies, specifically in the model plant Arabidopsis thaliana, eight plastid-localized and dual-specific acetyltransferases (GNATs) have been discovered (Bienvenut et al. 2020). Especially, the large subunit of Rubisco which is plastid encoded, undergoes many posttranslational modifications. The initiator methionine gets deformylated and removed. Subsequently, the second amino acid at the N-terminus gets cleaved of, revealing a proline. Multiple studies found the Pro3 N-terminus of RbcL acetylated (Zybailov et al. 2008; Rowland et al. 2015; Soh et al. 2020). Beside of this up to 19 ε-lysine acetylation sites were found on RbcL (Hartl et al. 2017; Finkemeier et al. 2011).
We employed three N-terminal CoA-conjugated peptide probes (CoA-N) to selectively enrich active N-terminal acetyltransferases (NATs) from plant extracts based on substrate preferences. Additionally, HPLC based activity assays with synthetic fluorophore-labelled peptides mimicking RbcL N-terminal sequences were used to identify plastid-localized NATs and lysine acetyltransferases (KATs) capable of modifying Rubisco. Additionally, lysine acetylome profiling of two gnat7 knockout lines and co-immunoprecipitation of GNAT7-GFP from Arabidopsis leaf material revealed regulated lysine acetylation sites on Calvin-Benson-Bassham cycle enzymes. Notably, three enzymes, previously linked to N-terminal modifications of RbcL, were significantly enriched in the co-immunoprecipitation, indicating their potential involvement in a modification pathway.