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 May;10(5):M110.004580. doi: 10.1074/mcp.M110.004580. Epub 2011 Mar 7.


The impact of N(α)-terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N(α)-terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N(α)-acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are γ- and β-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both γ- and β-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore, NatA or hNaa10p may function as a post-translational actin N(α)-acetyltransferase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Acetyltransferases / biosynthesis
  • Acetyltransferases / chemistry*
  • Actins / chemistry
  • Amino Acid Sequence
  • Cell Line
  • Cloning, Molecular
  • Enzyme Assays
  • Humans
  • N-Terminal Acetyltransferase A
  • N-Terminal Acetyltransferase E
  • Peptide Library*
  • Polyribosomes / chemistry
  • Protein Processing, Post-Translational*
  • Proteome / chemistry*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry*
  • Substrate Specificity


  • Actins
  • Peptide Library
  • Proteome
  • Recombinant Proteins
  • Acetyltransferases
  • N-Terminal Acetyltransferase A
  • NAA10 protein, human
  • N-Terminal Acetyltransferase E
  • NAA50 protein, human