The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40S). Intriguingly, although upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40p (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Furthermore, Naa40L was shown to be N-terminally myristoylated and to interact with N-myristoyltransferase 1 (NMT1), implicating NMT1 in steering Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context-dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.

Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes.

N-terminal acetylation (Nt-acetylation) catalyzed by conserved N-terminal acetyltransferases or NATs embodies a modification with one of the highest stoichiometries reported for eukaryotic protein modifications to date. Comprising the catalytic N-alpha acetyltransferase (NAA) subunit NAA10 plus the ribosome anchoring regulatory subunit NAA15, NatA represents the major acetyltransferase complex with up to 50% of all mammalian proteins representing potential substrates. Largely in consequence of the essential nature of NatA and its high enzymatic activity, its experimentally confirmed mammalian substrate repertoire remained poorly charted. In this study, human NatA knockdown conditions achieving near complete depletion of NAA10 and NAA15 expression resulted in lowered Nt-acetylation of over 25% out of all putative NatA targets identified, representing an up to 10-fold increase in the reported number of substrate N-termini affected upon human NatA perturbation. Besides pointing to less efficient NatA substrates being prime targets, several putative NatE substrates were shown to be affected upon human NatA knockdown. Intriguingly, next to a lowered expression of ribosomal proteins and proteins constituting the eukaryotic 48S preinitiation complex, steady-state levels of protein N-termini additionally point to NatA Nt-acetylation deficiency directly impacting protein stability of knockdown affected targets.

Protein N(α)-terminal acetylation (Nt-acetylation) is considered one of the most common protein modification in eukaryotes, and 80-90% of all soluble human proteins are modified in this way, with functional implications ranging from altered protein function and stability to translocation potency amongst others. Nt-acetylation is catalyzed by N-terminal acetyltransferases (NATs), and in yeast five NAT types are identified and denoted NatA-NatE. Higher eukaryotes additionally express NatF. Except for NatD, human orthologues for all yeast NATs are identified. yNatD is defined as the catalytic unit Naa40p (Nat4) which co-translationally Nt-acetylates histones H2A and H4. In this study we identified and characterized hNaa40p/hNatD, the human orthologue of the yeast Naa40p. An in vitro proteome-derived peptide library Nt-acetylation assay indicated that recombinant hNaa40p acetylates N-termini starting with the consensus sequence Ser-Gly-Gly-Gly-Lys-, strongly resembling the N-termini of the human histones H2A and H4. This was confirmed as recombinant hNaa40p Nt-acetylated the oligopeptides derived from the N-termini of both histones. In contrast, a synthetically Nt-acetylated H4 N-terminal peptide with all lysines being non-acetylated, was not significantly acetylated by hNaa40p, indicating that hNaa40p catalyzed H4 N(α)-acetylation and not H4 lysine N(ε)-acetylation. Also, immunoprecipitated hNaa40p specifically Nt-acetylated H4 in vitro. Heterologous expression of hNaa40p in a yeast naa40-Δ strain restored Nt-acetylation of yeast histone H4, but not H2A in vivo, probably reflecting the fact that the N-terminal sequences of human H2A and H4 are highly similar to each other and to yeast H4 while the N-terminal sequence of yeast H2A differs. Thus, Naa40p seems to have co-evolved with the human H2A sequence. Finally, a partial co-sedimentation with ribosomes indicates that hNaa40p co-translationally acetylates H2A and H4. Combined, our results strongly suggest that human Naa40p/NatD is conserved from yeast. Thus, the NATs of all classes of N-terminally acetylated proteins in humans now appear to be accounted for.

N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or Nα-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.

The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15. Biochemical data further demonstrate a reduced catalytic capacity and an impaired interaction between hNaa10 S37P and Naa15 as well as Naa50 (NatE), another interactor of the NatA complex. N-Terminal acetylome analyses revealed a decreased acetylation of a subset of NatA and NatE substrates in Ogden syndrome cells, supporting the genetic findings and our hypothesis regarding reduced Nt-acetylation of a subset of NatA/NatE-type substrates as one etiology for Ogden syndrome. Furthermore, Ogden syndrome fibroblasts display abnormal cell migration and proliferation capacity, possibly linked to a perturbed retinoblastoma pathway. N-Terminal acetylation clearly plays a role in Ogden syndrome, thus revealing the in vivo importance of N-terminal acetylation in human physiology and disease.