Bioinformatics Analysis of a Saccharomyces cerevisiae N-Terminal Proteome Provides Evidence of Alternative Translation Initiation and Post-Translational N-Terminal Acetylation

Abstract

Initiation of protein translation is a well-studied fundamental process, albeit high-throughput and more comprehensive determination of the exact translation initiation sites (TIS) was only recently made possible following the introduction of positional proteomics techniques that target protein N-termini. Precise translation initiation is of crucial importance, as truncated or extended proteins might fold, function, and locate erroneously. Still, as already shown for some proteins, alternative translation initiation can also serve as a regulatory mechanism. By applying N-terminal COFRADIC (combined fractional diagonal chromatography), we here isolated N-terminal peptides of a Saccharomyces cerevisiae proteome and analyzed both annotated and alternative TIS. We analyzed this N-terminome of S. cerevisiae which resulted in the identification of 650 unique N-terminal peptides corresponding to database annotated TIS. Furthermore, 56 unique N(α)-acetylated peptides were identified that suggest alternative TIS (MS/MS-based), while MS-based evidence of N(α)-acetylation led to an additional 33 such peptides. To improve the overall sensitivity of the analysis, we also included the 5' UTR (untranslated region) in-frame translations together with the yeast protein sequences in UniProtKB/Swiss-Prot. To ensure the quality of the individual peptide identifications, peptide-to-spectrum matches were only accepted at a 99% probability threshold and were subsequently analyzed in detail by the Peptizer tool to automatically ascertain their compliance with several expert criteria. Furthermore, we have also identified 60 MS/MS-based and 117 MS-based N(α)-acetylated peptides that point to N(α)-acetylation as a post-translational modification since these peptides did not start nor were preceded (in their corresponding protein sequence) by a methionine residue. Next, we evaluated consensus sequence features of nucleic acids and amino acids across each of these groups of peptides and evaluated the results in the context of publicly available data. Taken together, we present a list of 706 annotated and alternative TIS for yeast proteins and found that under normal growth conditions alternative TIS might (co)occur in S. cerevisiae in roughly one tenth of all proteins. Furthermore, we found that the nucleic acid and amino acid features proximate to these alternative TIS favor either guanine or adenine nucleotides following the start codon or acidic amino acids following the initiator methionine. Finally, we also observed an unexpected high number of N(α)-acetylated peptides that could not be related to TIS and therefore suggest events of post-translational N(α)-acetylation.

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

“…The latter indicate alternative translation events or posttranslational Nt-acetylation (19,(27)(28)(29). Of the protein N termini, 414 (25%) were identified in all three N terminomes.…”

A Saccharomyces cerevisiae Model Reveals In Vivo Functional Impairment of the Ogden Syndrome N-Terminal Acetyltransferase NAA10 Ser37Pro Mutant

“…Further, besides the database annotated N termini identified, these datasets also holds numerous N termini not compliant with the current rules for N-terminal modifications; either no iMet is present and/or the NAT rules of Nt-acetylation do not match the canonical Nt-acetylation patterns (54). Various of these N termini belonging to the NatA type class of N termini and display a NatA dependence of Nt-acetylation, hinting to the fact that these N termini could represent either post-translational Nt-acetylation events (54), co-translational acting (di-)aminopeptidase activity, non-cognate (non-AUG) co-translational translation initiation events (55) or simply wrong database annotations.…”

“…Of the 1,608 N termini, 1212 N termini were in compliance with the rules of Ntacetylation (53) and initiator methionine (iMet) processing (supplemental Table S1). Of these, 1056 started at position 1 or 2 (1011 proteins), whereas 156 started beyond position 2 (145 proteins), the latter indicative of alternative translation initiation (54). 396 N termini did not comply with the rules of Nt-acetylation and iMet processing and thus indicate posttranslational Nt-acetylation (54) or non-AUG translation initiation (55) among others.…”

Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress

“…1). Notably, N ␣ -acetylation can occur co-or posttranslationally, alters protein stability, and is typically irreversible (17,18). In contrast, N ε -acetylation can modify protein structure and function and typically is reversible by a deacetylase (Fig.…”