Properties of Nat4, an Nα-Acetyltransferase of Saccharomyces cerevisiae That Modifies N Termini of Histones H2A and H4

N-terminal acetylation is one of the most common protein modifications in eukaryotes, occurring on approximately 50% of yeast soluble proteins and about 80 to 90% of mammalian proteins (10, 13, 18). Eukaryotic cytosolic proteins initiate with methionine, which is cleaved from nascent chains of most proteins. Subsequently, N-terminal acetylation occurs on certain proteins either containing or lacking the methionine residue (10, 13, 18, 25, 27). Proteins susceptible to N-terminal acetylation have a variety of different N-terminal sequences, with no simple consensus motifs and with no dependence on a single type of residue (25, 27). Earlier workers attempted to determine the N-acetylation rules, and some distinguishing features were found in the sequences, mainly between residues 1 and 10 and fewer features between residues 16 and 24 and residues 30 and 40, but the precise nature of these features was not determined (2). Proteins with serine and alanine termini are the most frequently acetylated, and these residues, along with methionine, glycine, and threonine, account for over 95% of the N-terminal acetylated residues. However, proteins having these amino acids at their N termini are not always acetylated and none of these N-terminal residues guarantees acetylation, indicating that the enzymes recognize some structural characteristics of the N-terminal portion in addition to a particular amino acid at the N terminus (25). N-terminal acetylation of proteins is catalyzed by N-terminal acetyltransferases (NATs) that transfer acetyl groups from acetyl coenzyme A to termini of α-amino groups. We have established that Saccharomyces cerevisiae contains the three major NATs, NatA, NatB, and NatC, with each having different catalytic subunits, Ard1, Nat3, and Mak3, respectively, and with each acting on a different group of proteins (Table ​(Table1)1) (28, 29). As previously summarized (21, 24, 25), 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; subclasses of proteins with Met-Ile, Met-Leu, Met-Trp, or Met-Phe termini are acetylated by NatC. TABLE 1. The five types of yeast N-terminal acetyltransferases NatA activity requires two subunits, the catalytic subunit Ard1p and the auxiliary subunit Nat1 (22). nat1 and ard1 mutants were unable to N-terminally acetylate in vivo the same subset of 24 normally acetylated proteins, including those with Ala and Ser termini (1). In addition to lacking NAT activity, both nat1-Δ and ard1-Δ mutants exhibited slower growth, derepression of the silent mating-type gene HMLα, and failure to enter Go, due in part to the lack of acetylation of Sir3 and Orc1 (15, 45). Overexpression of both Ard1 and Nat1 subunits are required for increased NAT activity in vivo (22), and both interact with each other to form an active complex in vitro (23). A putative N-terminal acetyltransferase, Nat5, homologous to catalytic subunits Ard1, Nat3, and Mak3, was shown to copurify with NatA at about a 1:1:1 ratio (14). Although the substrates of Nat5 have not been identified, the nat5-Δ strain produces a phenotype that is distinct from those found in NatA, NatB, NatC, and NatD mutants (see below), and Nat5 is not required for NatA activity, suggesting that Nat5 forms a distinct NAT, which we have denoted NatE (Table ​(Table11). NatB acetyltransferase is composed of at least two subunits, the catalytic subunit Nat3 (29) and the auxiliary subunit Mdm20 (16), which also is required for acetylation of the NatB substrates (30, 38). The corresponding deletion mutants have similar phenotypes, including slow growth, temperature and osmotic sensitivity, calcium and caffeine sensitivity, deficiency in utilization of nonfermentable carbon sources, reduced mating efficiency, sensitivity to antimitotic drugs, and susceptibility to DNA-damaging agents (30). NatC contains three subunits, Mak3, Mak10, and Mak31 (34). Tercero et al. (41) described the MAK3 gene, which encodes the catalytic subunit of NatC and is required for the N-terminal acetylation of the viral major coat protein, gag, with an Ac-Met-Leu-Arg-Phe terminus (42). We demonstrated that each of the Mak3, Mak10, and Mak31 subunits are required for acetylation of the NatC-type sequences in vivo (26). In addition, all three deletion strains had similar phenotypes, including slower growth on nonfermentable carbon sources at elevated temperature. Recently, an additional NAT, Nat4 (NatD), was shown to acetylate the N termini of highly conserved histones H2A and H4, which have Ser-Gly-Gly-Lys-Gly and Ser-Gly-Arg-Gly-Lys N termini, respectively (39). Song et al. (39) also demonstrated that Nat4 acetylates in vitro a 23-amino-acid-long synthetic peptide corresponding to the N terminus of histone H4, but not an H3 peptide, which is not normally acetylated, nor a adrenocorticotropin peptide, which is an NatA substrate. However no obvious nat4-Δ phenotype was reported (39). So far, there is no evidence that Nat4 acetylates any other yeast protein, as judged by two-dimensional (2D) gel analysis of the soluble proteins prepared from normal and nat4-Δ mutant strains (39). We have designated this NAT activity as NatD, to be consistent with our established nomenclature. In this work we investigated the NatD requirements for N-terminal acetylation and uncovered nat4-Δ phenotypes. Surprisingly, NatD differs from the other NATs in its requirement for an extended N-terminal region for efficient acetylation. Mutant forms of iso-1-cytochrome c with extensions with as many as 23 amino acid residues corresponding to the N terminus of H4 histone were only partially acetylated by Nat4. In contrast, only five or fewer N-terminal amino acid replacements were necessary to acetylate mutant forms of iso-1-cytochrome c (iso-1) by NatA, NatB, or NatC. For example, iso-1 with eight N-terminal amino acids corresponding to the N-terminal region of H2B histone was sufficient for acetylation by NatA (29). In addition, we have investigated the site of action of NatD in the cell and demonstrated that Nat4 is a cytoplasmic protein that colocalizes with mono- and polyribosomes in a sucrose density gradient. Also, we have uncovered nat4-Δ phenotypes by using a variety of special media, and these phenotypes include sensitivity to 3-aminotriazol (3-AT), benomyl, salt, and thiabendazole (TBZ). Interestingly, nat4-Δ showed a synthetic growth defect in the strain with an altered histone H4 protein having K5R K8R K12R replacements and therefore lacking acetylated lysine residues in the N-terminal region.