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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We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton. Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin. We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins.