title: N-terminal acetylome analysis reveals the specificity of Naa50 (Nat5) and suggests a kinetic competition between N-terminal acetyltransferases and methionine aminopeptidases.
abstract: Cotranslational N-terminal (Nt-) acetylation of nascent polypeptides is mediated by N-terminal acetyltransferases (NATs). The very N-terminal amino acid sequence largely determines whether or not a given protein is Nt-acetylated. Currently, there are six distinct NATs characterized, NatA-NatF, in humans of which the in vivo substrate specificity of Naa50 (Nat5)/NatE, an alternative catalytic subunit of the human NatA, so far remained elusive. In this study, we quantitatively compared the Nt-acetylomes of wild-type yeast S. cerevisiae expressing the endogenous yeast Naa50 (yNaa50), the congenic strain lacking yNaa50, and an otherwise identical strain expressing human Naa50 (hNaa50). Six canonical yeast NatA substrates were Nt-acetylated less in yeast lacking yNaa50 than in wild-type yeast. In contrast, the ectopically expressed hNaa50 resulted, predominantly, in the Nt-acetylation of N-terminal Met (iMet) starting N-termini, including iMet-Lys, iMet-Val, iMet-Ala, iMet-Tyr, iMet-Phe, iMet-Leu, iMet-Ser, and iMet-Thr N-termini. This identified hNaa50 as being similar, in its substrate specificity, to the previously characterized hNaa60/NatF. In addition, the identification, in yNaa50-lacking yeast expressing hNaa50, of Nt-acetylated iMet followed by a small residue such as Ser, Thr, Ala, or Val, revealed a kinetic competition between Naa50 and Met-aminopeptidases (MetAPs), and implied that Nt-acetylated iMet followed by a small residue cannot be removed by MetAPs, a deduction supported by our in vitro data. As such, Naa50-mediated Nt-acetylation may act to retain the iMet of proteins of otherwise MetAP susceptible N-termini and the fraction of retained and Nt-acetylated iMet (followed by a small residue) in such a setting would be expected to depend on the relative levels of ribosome-associated Naa50/NatA and MetAPs.
authors.affiliation: Department of Medical Protein Research, VIB, Ghent, Belgium.|Department of Biochemistry, Ghent University, Ghent, Belgium.
authors.forename: Petra
authors.initials: P
authors.lastname: Van Damme
authors.affiliation: Department of Molecular Biology, University of Bergen, Bergen, Norway.|Department of Clinical Science, University of Bergen, Bergen, Norway.
authors.forename: Kristine
authors.initials: K
authors.lastname: Hole
authors.affiliation: Department of Medical Protein Research, VIB, Ghent, Belgium.|Department of Biochemistry, Ghent University, Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.affiliation: Department of Molecular Biology, University of Bergen, Bergen, Norway.|Department of Surgery, Haukeland University Hospital, Bergen, Norway.
authors.forename: Thomas
authors.initials: T
authors.lastname: Arnesen
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D063212:N-Terminal Acetyltransferase D / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D063213:N-Terminal Acetyltransferase E / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D008715:Methionine; D063212:N-Terminal Acetyltransferase D; D063213:N-Terminal Acetyltransferase E; C520714:NAA50 protein, human; C540857:NAT5 protein, S cerevisiae; D000626:Aminopeptidases; C081311:METAP2 protein, human; D063208:Methionyl Aminopeptidases
keywords: Acetylation; Methionine aminopeptidase; N-terminal acetyltransferase; N-terminomics; NAT; Naa50; Nt-acetylome