Abstract
Alternative translation initiation is a widespread event in biology that can shape multiple protein forms or proteoforms from a single gene. However, the respective contribution of alternative translation to protein complexity remains largely enigmatic. By complementary ribosome profiling and N-terminal proteomics (i.e., riboproteogenomics), we provide clear-cut evidence for ~90 N-terminal proteoform pairs shaped by (alternative) translation initiation in Arabidopsis thaliana. Next to several cases additionally confirmed by directed mutagenesis, identified alternative protein N-termini follow the enzymatic rules of co-translational N-terminal protein acetylation and initiator methionine removal. In contrast to other eukaryotic models, N-terminal acetylation in plants cannot generally be considered as a proxy of translation initiation because of its posttranslational occurrence on mature proteolytic neo-termini (N-termini) localized in the chloroplast stroma. Quantification of N-terminal acetylation revealed differing co- vs. posttranslational N-terminal acetylation patterns. Intriguingly, our data additionally hints to alternative translation initiation serving as a common mechanism to supply protein copies in multiple cellular compartments, as alternative translation sites are often in close proximity to cleavage sites of N-terminal transit sequences of nuclear-encoded chloroplastic and mitochondrial proteins. Overall, riboproteogenomics screening enables the identification of (differential localized) N-terminal proteoforms raised upon alternative translation.