First Things First: Vital Protein Marks by N-Terminal Acetyltransferases

The identification of the first membrane-associated NAT, Naa60/NatF, and the first chloroplast NAT, Naa70/NatG, established new modes of the NAT machinery in their capacity to acetylate transmembrane and lumenal chloroplast proteins, respectively. The structure of the NatA complex revealed molecular determinants for substrate-specific acetylation, including the significant impact of the auxiliary Naa15 on the specificity of the catalytic Naa10. Nt-acetylation has been shown to regulate protein complex stoichiometry through the Ac/N-end rule pathway, which has also been connected to hypertension. Further, a link was established between Nt-acetylation and global protein folding. Nt-acetylation has been found to play essential roles in A. thaliana drought-stress and immune responses, in C. elegans development and metabolism, as well as in human diseases. N-terminal (Nt) acetylation is known to be a highly abundant co-translational protein modification, but the recent discovery of Golgi- and chloroplast-resident N-terminal acetyltransferases (NATs) revealed that it can also be added post-translationally. Nt-acetylation may act as a degradation signal in a novel branch of the N-end rule pathway, whose functions include the regulation of human blood pressure. Nt-acetylation also modulates protein interactions, targeting, and folding. In plants, Nt-acetylation plays a role in the control of resistance to drought and in regulation of immune responses. Mutations of specific human NATs that decrease their activity can cause either the lethal Ogden syndrome or severe intellectual disability and cardiovascular defects. In sum, recent advances highlight Nt-acetylation as a key factor in many biological pathways. N-terminal (Nt) acetylation is known to be a highly abundant co-translational protein modification, but the recent discovery of Golgi- and chloroplast-resident N-terminal acetyltransferases (NATs) revealed that it can also be added post-translationally. Nt-acetylation may act as a degradation signal in a novel branch of the N-end rule pathway, whose functions include the regulation of human blood pressure. Nt-acetylation also modulates protein interactions, targeting, and folding. In plants, Nt-acetylation plays a role in the control of resistance to drought and in regulation of immune responses. Mutations of specific human NATs that decrease their activity can cause either the lethal Ogden syndrome or severe intellectual disability and cardiovascular defects. In sum, recent advances highlight Nt-acetylation as a key factor in many biological pathways. N-terminal acetylation (Nt-acetylation), also called Nα-acetylation (see Glossary and Figure 1), is a protein modification that is poorly represented in textbooks and, as such, many scientists are unaware that 80% of all human proteins receive an acetyl group at their N-terminus. Owing to several recent advances in the field, however, there is now a growing interest in this protein modification. Several recent contributions shed light on the molecular mechanisms through which the N-terminal acetyltransferases (NATs) exert their important biological functions. This review aims at advancing our current understanding of the protein, cellular, and physiological consequences of Nt-acetylation based on a multitude of recent reports. Consequently, it also highlights the fact that Nt-acetylation can no longer be viewed as an automatic negligible modification, but instead emerges as a crucial component in many biological pathways. Nt-acetylation has been established as a highly abundant protein modification in eukaryotic cells [1Starheim K.K. et al.Protein N-terminal acetyltransferases: when the start matters.Trends Biochem. Sci. 2012; 37: 152-161Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 2Arnesen T. et al.Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8157-8162Crossref PubMed Scopus (382) Google Scholar, 3Goetze S. et al.Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster.PLoS Biol. 2009; 7: e1000236Crossref PubMed Scopus (132) Google Scholar, 4Bienvenut W.V. et al.Comparative large scale characterization of plant versus mammal proteins reveals similar and idiosyncratic N-alpha-acetylation features.Mol. Cell Proteomics. 2012; 11 (M111.015131)Crossref PubMed Scopus (118) Google Scholar]. In fact, it is so common that it is reasonably safe to presume that your favorite protein is probably subject to this modification. The determinant for undergoing Nt-acetylation is mainly the identity of the first two amino acids at the N-terminus. Several NATs (NatA to NatF in humans) collectively catalyze Nt-acetylation of a majority (80%) of the different types of N-termini occurring in the proteome, resulting in the Nt-acetylome (Figure 2A) . NatA has specificity towards A-, S-, T-, V-, C-, and sometimes G-starting N-termini, whose initiator methionine (iMet) has been removed by methionine amino peptidases (MetAPs) [2Arnesen T. et al.Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8157-8162Crossref PubMed Scopus (382) Google Scholar, 5Mullen J.R. et al.Identification and characterization of genes and mutants for an N-terminal acetyltransferase from yeast.EMBO J. 1989; 8: 2067-2075Crossref PubMed Scopus (244) Google Scholar]. NatD also acts within the iMet-processed group; however, it is far more selective because it has only been shown to Nt-acetylate histones H2A and H4, making its contribution to the Nt-acetylome negligible [6Song O.K. et al.An N-alpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A.J. Biol. Chem. 2003; 278: 38109-38112Crossref PubMed Scopus (96) Google Scholar, 7Hole K. et al.The human N-alpha-acetyltransferase 40 (hNaa40p/hNatD) is conserved from yeast and N-terminally acetylates histones H2A and H4.PloS ONE. 2011; 6: e24713Crossref PubMed Scopus (83) Google Scholar]. Those N-termini that retain the iMet are modified by NatB, NatC, NatE, and NatF. In this group NatB acetylates Met-'Asx/Glx'-type N-termini (MD-, ME-, MN-, and MQ-starting) [8Polevoda B. et al.Identification and specificities of N-terminal acetyltransferases from Saccharomyces cerevisiae.EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (174) Google Scholar, 9Van Damme P. et al.N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 12449-12454Crossref PubMed Scopus (128) Google Scholar] whereas NatC, NatE, and NatF act on Met-'hydrophobic/amphipathic'-type N-termini (ML-, MI-, MF-, MY-, and MK-starting) [10Tercero J.C. Wickner R.B. MAK3 encodes an N-acetyltransferase whose modification of the L-A gag NH2 terminus is necessary for virus particle assembly.J. Biol. Chem. 1992; 267: 20277-20281Abstract Full Text PDF PubMed Google Scholar, 11Polevoda B. Sherman F. NatC Nalpha-terminal acetyltransferase of yeast contains three subunits, Mak3p, Mak10p, and Mak31p.J. Biol. Chem. 2001; 276: 20154-20159Crossref PubMed Scopus (75) Google Scholar, 12Evjenth R. et al.Human Naa50p (Nat5/San) displays both protein N alpha- and N epsilon-acetyltransferase activity.J. Biol. Chem. 2009; 284: 31122-31129Crossref PubMed Scopus (75) Google Scholar, 13Starheim K.K. et al.Knockdown of human N alpha-terminal acetyltransferase complex C leads to p53-dependent apoptosis and aberrant human Arl8b localization.Mol. Cell Biol. 2009; 29: 3569-3581Crossref PubMed Scopus (80) Google Scholar, 14Van Damme P. et al.Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of N(alpha)-acetyltransferases and point to hNaa10p as the post-translational actin N(alpha)-acetyltransferase.Mol. Cell Proteomics. 2011; 10 (M110.004580)Crossref Scopus (111) Google Scholar, 15Van Damme P. et al.NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation.PLoS Genet. 2011; 7: e1002169Crossref PubMed Scopus (126) Google Scholar]. The recently identified plant NatG acetylates M-, A-, S-, T-starting N-termini, however it lacks a human ortholog [16Dinh T.V. et al.Molecular identification and functional characterization of the first Nalpha-acetyltransferase in plastids by global acetylome profiling.Proteomics. 2015; 15: 2426-2435Crossref PubMed Scopus (67) Google Scholar]. Although proteomic technologies are incapable of directly defining the Nt-acetylation status of the entire proteome, an estimate of the total Nt-acetylation events is possible by extrapolation of proteomics data to all Swiss-Prot entries (Figure 2, based on [17Aksnes H. et al.An organellar Nα-acetyltransferase, Naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.Cell Rep. 2015; 10: 1362-1374Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]). Through this approach, it is calculated that the NAT family Nt-acetylates 80% of the human proteome (Figure 2A). This amount relates to both full and partial Nt-acetylation because many proteins exist as both Nt-acetylated and unacetylated variants. Hence it is very likely that a given protein, for example your favorite protein, is subject to Nt-acetylation. In determining the likelihood of a particular protein being Nt-acetylated based on its two first amino acids one can make use of the chart in Figure 2B where the size of subgroups within each NAT substrate class is shown as well as the frequency of Nt-acetylation events within the subgroups. If the N-terminus of the protein in question starts with alanine, which is in the NatA substrate class, there is a 95% chance that it is Nt-acetylated, but if it starts with valine there is a 80% chance that its N-terminus is unacetylated. NatB is distinguished in this sense because it has near 100% coverage of the Met-'Asx/Glx'-type N-termini. The combined action of NatC, E and F within the Met-'hydrophobic/amphipathic'-group will probably undergo further refinement in substrate specificity-profiling because it acts only on ∼75% of the sequence-predicted substrates. A recent study on NatF revealed that it has selectivity for membrane proteins and that the NatF substrate category is enriched for transmembrane proteins [17Aksnes H. et al.An organellar Nα-acetyltransferase, Naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.Cell Rep. 2015; 10: 1362-1374Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. This study was also the first to investigate the membrane-bound part of the Nt-acetylome, which was found to be equally large in the amount of proteins Nt-acetylated, although these were typically Nt-acetylated to a lesser degree, meaning that there were more partial Nt-acetylation events. The eukaryotic NAT machinery known to date is composed of seven NATs (NatA–NatG) (Figure 3) of which five (NatA–NatE) are present in Saccharomyces cerevisiae (reviewed in [18Aksnes H. et al.Molecular, cellular, and physiological significance of N-terminal acetylation.Int. Rev. Cell Mol. Biol. 2015; 316: 267-305Crossref PubMed Scopus (76) Google Scholar, 19Rathore O.S. et al.Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms.Sci. Rep. 2016; 6: 21304Crossref PubMed Scopus (36) Google Scholar]), whereas multicellular eukaryotes have a sixth NAT, NatF [15Van Damme P. et al.NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation.PLoS Genet. 2011; 7: e1002169Crossref PubMed Scopus (126) Google Scholar, 17Aksnes H. et al.An organellar Nα-acetyltransferase, Naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.Cell Rep. 2015; 10: 1362-1374Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar], and in addition in the plant Arabidopsis thaliana a seventh NAT, NatG [16Dinh T.V. et al.Molecular identification and functional characterization of the first Nalpha-acetyltransferase in plastids by global acetylome profiling.Proteomics. 2015; 15: 2426-2435Crossref PubMed Scopus (67) Google Scholar] has been identified. The eukaryotic NAT machinery is more complex than that of prokaryotes, which only have three known NATs. It is not well understood why eukaryotic cells have evolved different NATs. NAT diversification could conceivably have co-evolved with the increasing complexity of the proteome. However, based on the currently identified enzymes, NATs did not diverge during the evolution of eukaryotes in which proteome complexity vastly increased [19Rathore O.S. et al.Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms.Sci. Rep. 2016; 6: 21304Crossref PubMed Scopus (36) Google Scholar]. The more complex eukaryotic NAT machinery entails a specialization towards different substrate groups that could provide a more flexible system in terms of regulation. In fact, some examples of upstream regulation of NATs have recently been uncovered (discussed in following sections). The recently discovered NATs, NatF targeting transmembrane proteins and NatG targeting chloroplast proteins, are clear examples of specialization, and likely exist owing to a need for particular subgroups of these substrate types to be Nt-acetylated. NAT activity typically requires a NAT complex in which the catalytic transferase subunit joins up to two auxiliary subunits that may mediate ribosome anchoring and in some cases contribute to substrate specificity (Figure 3A) [20Gautschi M. et al.The yeast N(alpha)-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides.Mol. Cell Biol. 2003; 23: 7403-7414Crossref PubMed Scopus (171) Google Scholar, 21Liszczak G. et al.Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.Nat. Struct. Mol. Biol. 2013; 20: 1098-1105Crossref PubMed Scopus (112) Google Scholar, 22Polevoda B. et al.Yeast N(alpha)-terminal acetyltransferases are associated with ribosomes.J. Cell Biochem. 2008; 103: 492-508Crossref PubMed Scopus (75) Google Scholar]. The NatA complex is formed by the catalytic subunit Naa10 and the auxiliary subunit Naa15 [5Mullen J.R. et al.Identification and characterization of genes and mutants for an N-terminal acetyltransferase from yeast.EMBO J. 1989; 8: 2067-2075Crossref PubMed Scopus (244) Google Scholar, 23Arnesen T. et al.Identification and characterization of the human ARD1–NATH protein acetyltransferase complex.Biochem. J. 2005; 386: 433-443Crossref PubMed Scopus (147) Google Scholar]. Naa50 is also attached to the NatA complex together with HYPK (Huntingtin-interacting protein K) [20Gautschi M. et al.The yeast N(alpha)-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides.Mol. Cell Biol. 2003; 23: 7403-7414Crossref PubMed Scopus (171) Google Scholar, 24Williams B.C. et al.Two putative acetyltransferases, san and deco, are required for establishing sister chromatid cohesion in Drosophila.Curr. Biol. 2003; 13: 2025-2036Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 25Arnesen T. et al.The chaperone-like protein HYPK acts together with NatA in cotranslational N-terminal acetylation and prevention of Huntingtin aggregation.Mol. Cell Biol. 2010; 30: 1898-1909Crossref PubMed Scopus (94) Google Scholar]. Naa50 also forms part of the NatE complex (together with Naa15 and Naa10), which displays distinct substrate specificity and a distinct depletion phenotype compared to NatA [12Evjenth R. et al.Human Naa50p (Nat5/San) displays both protein N alpha- and N epsilon-acetyltransferase activity.J. Biol. Chem. 2009; 284: 31122-31129Crossref PubMed Scopus (75) Google Scholar, 14Van Damme P. et al.Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of N(alpha)-acetyltransferases and point to hNaa10p as the post-translational actin N(alpha)-acetyltransferase.Mol. Cell Proteomics. 2011; 10 (M110.004580)Crossref Scopus (111) Google Scholar, 20Gautschi M. et al.The yeast N(alpha)-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides.Mol. Cell Biol. 2003; 23: 7403-7414Crossref PubMed Scopus (171) Google Scholar, 21Liszczak G. et al.Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.Nat. Struct. Mol. Biol. 2013; 20: 1098-1105Crossref PubMed Scopus (112) Google Scholar, 26Hou F. et al.The acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshin-independent manner.J. Cell Biol. 2007; 177: 587-597Crossref PubMed Scopus (64) Google Scholar]. NatB is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25 [27Polevoda B. et al.Nat3p and Mdm20p are required for function of yeast NatB Nalpha-terminal acetyltransferase and of actin and tropomyosin.J. Biol. Chem. 2003; 278: 30686-30697Crossref PubMed Scopus (91) Google Scholar, 28Starheim K.K. et al.Identification of the human N(alpha)-acetyltransferase complex B (hNatB): a complex important for cell-cycle progression.Biochem. J. 2008; 415: 325-331Crossref PubMed Scopus (81) Google Scholar]. The catalytic subunit of NatC is Naa30, which associates with two noncatalytic subunits, Naa35 and Naa38, of which Naa35 mediates ribosomal association, whereas the role of Naa38 is less well described [11Polevoda B. Sherman F. NatC Nalpha-terminal acetyltransferase of yeast contains three subunits, Mak3p, Mak10p, and Mak31p.J. Biol. Chem. 2001; 276: 20154-20159Crossref PubMed Scopus (75) Google Scholar, 13Starheim K.K. et al.Knockdown of human N alpha-terminal acetyltransferase complex C leads to p53-dependent apoptosis and aberrant human Arl8b localization.Mol. Cell Biol. 2009; 29: 3569-3581Crossref PubMed Scopus (80) Google Scholar, 22Polevoda B. et al.Yeast N(alpha)-terminal acetyltransferases are associated with ribosomes.J. Cell Biochem. 2008; 103: 492-508Crossref PubMed Scopus (75) Google Scholar]. For NatD (Naa40), NatF (Naa60), and NatG (Naa70) no auxiliary subunit has been identified so far (Figure 3A). Interestingly, a recent study revealed that an N-terminal segment of Naa40, which is unique compared to the other catalytic Naas, might play a role that is analogous to the ribosomal-binding auxiliary subunits found in the other NATs [29Magin R.S. et al.The molecular basis for histone H4- and H2A-specific amino-terminal acetylation by NatD.Structure. 2015; 23: 332-341Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. Most NATs are localized in the cytosol (Figure 3B). NatA–NatE are associated with ribosomes, where they perform co-translational Nt-acetylation (Figure 3A) [20Gautschi M. et al.The yeast N(alpha)-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides.Mol. Cell Biol. 2003; 23: 7403-7414Crossref PubMed Scopus (171) Google Scholar, 22Polevoda B. et al.Yeast N(alpha)-terminal acetyltransferases are associated with ribosomes.J. Cell Biochem. 2008; 103: 492-508Crossref PubMed Scopus (75) Google Scholar]. In addition, some catalytic subunits may localize to the nucleus [7Hole K. et al.The human N-alpha-acetyltransferase 40 (hNaa40p/hNatD) is conserved from yeast and N-terminally acetylates histones H2A and H4.PloS ONE. 2011; 6: e24713Crossref PubMed Scopus (83) Google Scholar, 17Aksnes H. et al.An organellar Nα-acetyltransferase, Naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.Cell Rep. 2015; 10: 1362-1374Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 23Arnesen T. et al.Identification and characterization of the human ARD1–NATH protein acetyltransferase complex.Biochem. J. 2005; 386: 433-443Crossref PubMed Scopus (147) Google Scholar] and Naa10 and Naa50 also display NAT activity in the absence of the ribosome-anchoring subunit, Naa15, but with altered substrate specificities [12Evjenth R. et al.Human Naa50p (Nat5/San) displays both protein N alpha- and N epsilon-acetyltransferase activity.J. Biol. Chem. 2009; 284: 31122-31129Crossref PubMed Scopus (75) Google Scholar, 14Van Damme P. et al.Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of N(alpha)-acetyltransferases and point to hNaa10p as the post-translational actin N(alpha)-acetyltransferase.Mol. Cell Proteomics. 2011; 10 (M110.004580)Crossref Scopus (111) Google Scholar, 17Aksnes H. et al.An organellar Nα-acetyltransferase, Naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.Cell Rep. 2015; 10: 1362-1374Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 21Liszczak G. et al.Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.Nat. Struct. Mol. Biol. 2013; 20: 1098-1105Crossref PubMed Scopus (112) Google Scholar]. Given that these NATs also exist in nonribosomal forms, this suggests that NATs might also act post-translationally [14Van Damme P. et al.Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of N(alpha)-acetyltransferases and point to hNaa10p as the post-translational actin N(alpha)-acetyltransferase.Mol. Cell Proteomics. 2011; 10 (M110.004580)Crossref Scopus (111) Google Scholar]. In fact, several internal peptides, produced by post-translational cleavage in vivo, are Nt-acetylated [30Helbig A.O. et al.Profiling of N-acetylated protein termini provides in-depth insights into the N-terminal nature of the proteome.Mol. Cell Proteomics. 2010; 9: 928-939Crossref PubMed Scopus (98) Google Scholar, 31Helsens K. et al.Bioinformatics analysis of a Saccharomyces cerevisiae N-terminal proteome provides evidence of alternative translation initiation and post-translational N-terminal acetylation.J. Proteome Res. 2011; 10: 3578-3589Crossref PubMed Scopus (44) Google Scholar] and a specific example of a protein undergoing such post-translational processing is actin [32Redman K. Rubenstein P.A. NH2-terminal processing of Dictyostelium discoideum actin in vitro.J. Biol. Chem. 1981; 256: 13226-13229Abstract Full Text PDF PubMed Google Scholar]. Of particular interest are two newly identified NATs with organellar localization (Figure 3B). NatF is associated with the Golgi apparatus facing the cytosolic side where it Nt-acetylates transmembrane proteins [17Aksnes H. et al.An organellar Nα-acetyltransferase, Naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.Cell Rep. 2015; 10: 1362-1374Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. NatG was identified as the first NAT localized inside an organelle, in this case in chloroplasts of the plant A. thaliana [16Dinh T.V. et al.Molecular identification and functional characterization of the first Nalpha-acetyltransferase in plastids by global acetylome profiling.Proteomics. 2015; 15: 2426-2435Crossref PubMed Scopus (67) Google Scholar]. These recent studies underpin the post-translational nature of Nt-acetylation and finally connect this modification to cellular organelles. Structural information on the NATs is currently emerging as the crystal structures of several Nα-acetyltransferases and NAT complexes have been solved. The recently developed bisubstrate analog-based Nα-acetyltransferase inhibitors [33Foyn H. et al.Design, synthesis, and kinetic characterization of protein N-terminal acetyltransferase inhibitors.ACS Chem. Biol. 2013; 8: 1121-1127Crossref PubMed Scopus (35) Google Scholar] have been useful in some of these structural studies. A crystal structure with both subunits of the NatA complex (Naa10 and Naa15) has been solved (Figure 4A) [21Liszczak G. et al.Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.Nat. Struct. Mol. Biol. 2013; 20: 1098-1105Crossref PubMed Scopus (112) Google Scholar]. The Naa15 subunit consists of 37 α-helices arranged into 13 TPR (tetratricopeptide repeat) motifs [21Liszczak G. et al.Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.Nat. Struct. Mol. Biol. 2013; 20: 1098-1105Crossref PubMed Scopus (112) Google Scholar]. These are conserved motifs composed of sequences of 34 amino acids that generally serve as protein–protein interaction motifs [34Das A.K. et al.The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein–protein interactions.EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (709) Google Scholar]. Some of the TPR motifs in Naa15 are involved in Naa10 binding [21Liszczak G. et al.Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.Nat. Struct. Mol. Biol. 2013; 20: 1098-1105Crossref PubMed Scopus (112) Google Scholar], and it is likely that they are also crucial for the associations of Naa15 with Naa50, HYPK, and the ribosome (Figure 3A). The overall tertiary structure of Naa15 forms a ring-like structure with a cavity into which Naa10 binds (Figure 4A). The Naa15–Naa10 binding is mainly conducted by a large hydrophobic interface as well as by a few hydrogen bonds [21Liszczak G. et al.Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.Nat. Struct. Mol. Biol. 2013; 20: 1098-1105Crossref PubMed Scopus (112) Google Scholar]. The catalytic subunits of NATs belong to the GCN5-related N-acetyltransferase (GNAT) superfamily together with some of the lysine acetyltransferases (KATs). All the NATs share the structural GNAT-domain (Figure 4B), which is an evolutionarily conserved characteristic of NATs, as shown by structural analyses of the bacterial NAT RimI [35Vetting M.W. et al.Crystal structure of RimI from Salmonella typhimurium LT2, the GNAT responsible for N(alpha)-acetylation of ribosomal protein S18.Protein Sci. 2008; 17: 1781-1790Crossref PubMed Scopus (64) Google Scholar] and the NAT from the archaea Sulfolobus solfataricus [36Liszczak G. Marmorstein R. Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 14652-14657Crossref PubMed Scopus (35) Google Scholar]. The GNAT domain consists of a central acetyl-CoA binding motif (Q/RxxGxG/A) flanked by four α-helices and seven β-sheet segments [16Dinh T.V. et al.Molecular identification and functional characterization of the first Nalpha-acetyltransferase in plastids by global acetylome profiling.Proteomics. 2015; 15: 2426-2435Crossref PubMed Scopus (67) Google Scholar, 21Liszczak G. et al.Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.Nat. Struct. Mol. Biol. 2013; 20: 1098-1105Crossref PubMed Scopus (112) Google Scholar, 29Magin R.S. et al.The molecular basis for histone H4- and H2A-specific amino-terminal acetylation by NatD.Structure. 2015; 23: 332-341Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 37Liszczak G. et al.Structure of a ternary Naa50p (NAT5/SAN) N-terminal acetyltransferase complex reveals the molecular basis for substrate-specific acetylation.J. Biol. Chem. 2011; 286: 37002-37010Crossref PubMed Scopus (71) Google Scholar, 38Stove S.I. et al.Crystal structure of the Golgi-associated human Nα-acetyltransferase 60 reveals the molecular determinants for substrate-specific acetylation.Structure. 2016; 24: 1044-1056Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. The acetyl-CoA binding domain is formed by several secondary elements (α3 and β2, β3, and β4) [37Liszczak G. et al.Structure of a ternary Naa50p (NAT5/SAN) N-terminal acetyltransferase complex reveals the molecular basis for substrate-specific acetylation.J. Biol. Chem. 2011; 286: 37002-37010Crossref PubMed Scopus (71) Google Scholar]. Substrate binding and the catalytic bi-bi reaction occur in a semi-open cavity that harbors both acetyl-CoA and the first 4–5 amino acids of the N-terminal peptide (Figure 4B,C). Substrate specificity is assured by an N-terminal helix-to-helix loop and a C-terminal β-hairpin loop (Figure 4B) [21Liszczak G. et al.Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.Nat. Struct. Mol. Biol. 2013; 20: 1098-1105Crossref PubMed Scopus (112) Google Scholar, 37Liszczak G. et al.Structure of a ternary Naa50p (NAT5/SAN) N-terminal acetyltransferase complex reveals the molecular basis for substrate-specific acetylation.J. Biol. Chem. 2011; 286: 37002-37010Crossref PubMed Scopus (71) Google Scholar]. This C-terminal β-hairpin in NATs has an extended loop compared to KATs of the same superfamily, restricting the substrate specificity to α-amino groups [39Magin R.S. et al.The N-terminal acetyltransferase Naa10/ARD1 does not acetylate lysine residues.J. Biol. Chem. 2016; 291: 5270-5277Crossref PubMed Scopus (36) Google Scholar, 40Li Y. et al.Hat2p recognizes the histone H3 tail to specify the acetylation of the newly synthesized H3/H4 heterodimer by the Hat1p/Hat2p complex.Genes Dev. 2014; 28: 1217-1227Crossref PubMed Scopus (30) Google Scholar]. Thus, these recent structural advances and enzymatic investigations reveal that it is improbable that NATs can acetylate ɛ-amino groups on lysine residues [39Magin R.S. et al.The N-terminal acetyltransferase Naa10/ARD1 does not acetylate lysine residues.J. Biol. Chem. 2016; 291: 5270-5277Crossref PubMed Scopus (36) Google Scholar], as suggested for Naa10 and other NATs by previous in vitro studies [41Shin S.H. et al.Arrest defective 1 regulates the oxidative stress response in human cells and mice by acetylating methionine sulfoxide reductase A.Cell Death Dis. 2014; 5: e1490Crossref PubMed Scopus (25)