N ␣ -terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys-and Val-N termini. NatA is composed of subunits encoded by yARD1 and yNAT1 in yeast and hARD1 and hNAT1 in humans. A yeast ard1-⌬ nat1-⌬ strain was phenotypically complemented by hARD1 hNAT1, suggesting that yNatA and hNatA are similar. However, heterologous combinations, hARD1 yNAT1 and yARD1 hNAT1, were not functional in yeast, suggesting significant structural subunit differences between the species. Proteomics of a yeast ard1-⌬ nat1-⌬ strain expressing hNatA demonstrated that hNatA acts on nearly the same set of yeast proteins as yNatA, further revealing that NatA from humans and yeast have identical or nearly identical specificities. Nevertheless, all NatA substrates in yeast were only partially N-acetylated, whereas the corresponding NatA substrates in HeLa cells were mainly completely N-acetylated. Overall, we observed a higher proportion of N-terminally acetylated proteins in humans (84%) as compared with yeast (57%). N-acetylation occurred on approximately one-half of the human proteins with Met-Lys-termini, but did not occur on yeast proteins with such termini. Thus, although we revealed different N-acetylation patterns in yeast and humans, the major NAT, NatA, acetylates the same substrates in both species.Ard1 ͉ COFRADIC ͉ N-terminal acetylation ͉ Nat1 ͉ NatA P rotein N ␣ -terminal acetylation (here referred to as Nacetylation) is one of the most common covalent modifications of eukaryotic proteins, in which an acetyl group is transferred from acetyl-CoA to the ␣-amino group of protein N-terminal residues. N-acetylation occurs cotranslationally on nascent polypeptide chains and almost all N-acetylations in Saccharomyces cerevisiae are catalyzed by 1 of 3 major Nterminal acetyltransferase (NAT) complexes, NatA, NatB or NatC, consisting of catalytic subunits Ard1p, Nat3p, and Mak3p, respectively, and 1 or more auxiliary subunits (1). Yeast NatA, the major and best studied NAT, is composed of the catalytic subunit Ard1p in complex with Nat1p (2). Nat1p is responsible for anchoring Ard1p to the ribosome, thus facilitating cotranslational N-acetylation (3). Both subunits are required for optimal acetyltransferase activity and yeast strains lacking either one of the subunits display the same phenotypes, indicating that both genes are also functionally linked (4). The yeast NatA, NatB and NatC complexes differ in their substrate specificities. NatA substrates represent by far the largest group and contain proteins with Ser-, Ala-, Gly-, Val-, Cys-or Thr-N termini, whereas NatB and NatC act on diff...

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The two cotranslational processes, cleavage of N-terminal methionine residues and N-terminal 1 acetylation, are by far the most common modifications, occurring on the vast majority of eukaryotic proteins. Studies with the yeast Saccharomyces cerevisiae revealed three N-terminal acetyltransferases, NatA, NatB, and NatC, that acted on groups of substrates, each containing degenerate motifs. Orthologous genes encoding the three N-terminal acetyltransferases and the patterns of N-terminal acetylation suggest that eukaryotes generally use the same systems for N-terminal acetylation. The biological significance of this N-terminal modification varies with the particular protein, with some proteins requiring acetylation for function, whereas others do not. Methionine CleavageCleavage of N-terminal methionine residues is by far the most common modification, occurring on the vast majority of proteins. Proteins from prokaryotes, mitochondria, and chloroplasts initiate with formylmethionine, whereas proteins from the cytosol of eukaryotes initiate with methionine. The formyl group is usually removed from prokaryotic proteins by a deformylase, resulting in methionine at N termini. The methionine at N termini is cleaved from nascent chains of most prokaryotic and eukaryotic proteins. Results with altered iso-1-cytochromes c from yeast (1) were the basis for the hypothesis that methionine is cleaved from penultimate residues having radii of gyration of 1.29 Å or less (glycine, alanine, serine, cysteine, threonine, proline, and valine) (2), a hypothesis that was confirmed from the results of a complete set of altered iso-1 having all possible amino acids at the penultimate position (3). A similar pattern of cleavage also was observed with prokaryotic systems in vivo (4, 5) and in vitro (6, 7), and other eukaryotic systems in vivo (8, 9) and in vitro (10). Only minor differences were observed between the quantitative results obtained in vivo with yeast iso-

N-terminal acetylation can occur cotranslationally on the initiator methionine residue or on the penultimate residue if the methionine is cleaved. We investigated the three N-terminal acetyltransferases (NATs), Ard1p/ Nat1p, Nat3p and Mak3p. Ard1p and Mak3p are significantly related to each other by amino acid sequence, as is Nat3p, which was uncovered in this study using programming alignment procedures. Mutants deleted in any one of these NAT genes were viable, but some exhibited diminished mating efficiency and reduced growth at 37°C, and on glycerol and NaClcontaining media. The three NATs had the following substrate specificities as determined in vivo by examining acetylation of 14 altered forms of iso-1-cytochrome c and 55 abundant normal proteins in each of the deleted strains: Ard1p/Nat1p, subclasses with Ser-, Ala-, Gly-and Thr-termini; Nat3p, Met-Glu-and MetAsp-and a subclass of Met-Asn-termini; and Mak3p subclasses with Met-Ile-and Met-Leu-termini. In addition, a special subclass of substrates with Ser-GluPhe-, Ala-Glu-Phe-and Gly-Glu-Phe-termini required all three NATs for acetylation.

Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln-N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

NatB N␣-terminal acetyltransferase of Saccharomyces cerevisiae acts cotranslationally on proteins with MetGlu-or Met-Asp-termini and subclasses of proteins with Met-Asn-and Met-Met-termini. NatB is composed of the interacting Nat3p and Mdm20p subunits, both of which are required for acetyltransferase activity. The phenotypes of nat3-⌬ and mdm20-⌬ mutants are identical or nearly the same and include the following: diminished growth at elevated temperatures and on hyperosmotic and nonfermentable media; diminished mating; defective actin cables formation; abnormal mitochondrial and vacuolar inheritance; inhibition of growth by DNAdamaging agents such as methyl methanesulfonate, bleomycin, camptothecin, and hydroxyurea; and inhibition of growth by the antimitotic drugs benomyl and thiabendazole. The similarity of these phenotypes to the phenotypes of certain act1 and tpm1 mutants suggests that such multiple defects are caused by the lack of acetylation of actin and tropomyosins. However, the lack of acetylation of other unidentified proteins conceivably could cause the same phenotypes. Furthermore, unacetylated actin and certain N-terminally altered actins have comparable defective properties in vitro, particularly actin-activated ATPase activity and sliding velocity.The two cotranslational processes, cleavage of N-terminal methionine residues and N-terminal acetylation, are by far the most common modifications, occurring on the vast majority of proteins. Eukaryotic cytosolic proteins initiate with methionine that is cleaved from nascent chains of most proteins. Subsequently, N-terminal acetylation occurs on certain of the proteins, either containing or lacking the methionine residue. This N-terminal acetylation occurs on over one-half of soluble eukaryotic proteins but seldom on prokaryotic proteins (1-3).N-terminal acetylation of proteins is catalyzed by N-terminal acetyltransferases (NATs) 1 that transfer acetyl groups from acetyl-CoA to termini of ␣-amino groups. We have established that Saccharomyces cerevisiae contains three major NATs, NatA, NatB, and NatC, with catalytic subunits Ard1p, Nat3p, and Mak3p, respectively, and that each is required for acetylating different groups of proteins (4, 5). As summarized previously (4, 5), subclasses of proteins with Ser-, Ala-, Gly-, or Thr-termini are acetylated by NatA; proteins with Met-Glu-or Met-Asp-termini and subclasses of proteins with Met-Asn-and Met-Met-termini are acetylated by NatB; and subclasses of proteins with Met-Ile, Met-Leu-, Met-Trp-, or Met-Phe-termini are acetylated by NatC. In addition, a special subclass of NatA substrates with Ser-Glu-, Ser-Asp-, Ala-Glu-, or Gly-Glu-termini, designated NatAЈ, are only partially acetylated in nat3-⌬ or mak3-⌬ mutants (6).In regard to the present study, special emphasis should be made of the NatB substrates, some of which were previously identified by using two-dimensional gel electrophoresis of proteins from normal and nat3-⌬ strains, including the following: actin (Act1p); the small subunit of ribonucleotide...

Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatAmediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis.

We have introduced a consistent nomenclature for the various subunits of the NatA-NatE Nterminal acetyltransferases from yeast, humans and other eukaryotes.