Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Mar 4;291(10):5270-7.
doi: 10.1074/jbc.M115.709428. Epub 2016 Jan 11.

The N-terminal Acetyltransferase Naa10/ARD1 Does Not Acetylate Lysine Residues

Robert S Magin  1 , Zachary M March  2 , Ronen Marmorstein  3
Affiliations

The N-terminal Acetyltransferase Naa10/ARD1 Does Not Acetylate Lysine Residues

Robert S Magin et al. J Biol Chem. .

Abstract

The N-terminal acetyltransferase NatA is a heterodimeric complex consisting of a catalytic subunit (Naa10/ARD1) and an auxiliary subunit (Naa15). NatA co-translationally acetylates the N termini of a wide variety of nascent polypeptides. In addition, Naa10 can act independently to posttranslationally acetylate a distinct set of substrates, notably actin. Recent structural studies of Naa10 have also revealed the molecular basis for N-terminal acetylation specificity. Surprisingly, recent reports claim that Naa10 may also acetylate lysine residues of diverse targets, including methionine sulfoxide reductase A, myosin light chain kinase, and Runt-related transcription factor 2. Here we used recombinant proteins to reconstitute and assess lysine acetylation events catalyzed by Naa10 in vitro. We show that there is no difference in lysine acetylation of substrate proteins with or without Naa10, suggesting that the substrates may be acetylated chemically rather than enzymatically. Together, our data argue against a role for Naa10 in lysine acetylation.

Keywords: ARD1; NAT; Naa10p; acetyl-CoA; acetylation; acetyltransferase; chemical acetylation; posttranslational modification (PTM); transcription factor.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
The structure of NATs is incompatible with lysine acetylation. A, superposition of the Gcn5 KAT-histone H3 peptide complex (dark and light blue, respectively) with the Naa10 N-terminal peptide complex (yellow and wheat, respectively). The proteins are shown as cartoon and the peptide ligands are shown as sticks. Naa10 is shown with its β6-β7 loop in orange. B, superposition of NAT/N-terminal peptide complexes with Naa10 (yellow), Naa40p (salmon) Naa50p (green), and the NAT from Sulfolobus solfataricus (ssNAT, purple).
FIGURE 2.
FIGURE 2.
Naa10 does not acetylate peptides or proteins corresponding to reported lysine substrates. Recombinant human Naa10 was used for these experiments. A, radioactive filter binding assay of peptide acetylation in the presence or absence of Naa10. An N-terminal actin peptide was used as a positive control. B, radioactive TCA precipitation assay of protein acetylation with Runx2 and MSRA in the presence of absence of Naa10. The experiments were carried out in triplicate, and error bars are indicated. CPM, counts per minute.
FIGURE 3.
FIGURE 3.
The kinetic profiles of Naa10 against MSRA and Runx2 are indicative of chemical acetylation. A–C, radioactive assay against (A) N-terminal actin peptide, (B) MSRA, and (C) Runx2 at increasing concentrations of acetyl-CoA in the presence (circles) and absence (squares) of Naa10. The experiments were carried out in triplicate, and error bars are indicated. CPM, counts per minute.
FIGURE 4.
FIGURE 4.
Western blotting detection of acetylated MSRA and Runx2 shows independence of Naa10. A and B, Western blotting analyses using two different antibodies from (A) Cell Signaling Technology (catalog no. 9681) and (B) ImmuneChem (catalog no. ICP0380) in the absence or presence of acetyl-CoA and Naa10 with MSRA and Runx2 as substrates. The hMOF KAT with histone H3/H4 substrate was used as a positive control. Coomassie staining of the gel is shown at the bottom.
FIGURE 5.
FIGURE 5.
NatA does not acetylate peptides or proteins corresponding to reported lysine substrates. Recombinant human Naa10/Naa15 (NatA) was used for these experiments. A, radioactive filter binding assay of peptide acetylation in the presence or absence of Naa10. An N-terminal SASE peptide was used as a positive control as reported previously (9). B, radioactive TCA precipitation assay of protein acetylation with Runx2 and MSRA in the presence of absence of Naa10. The experiments were carried out in triplicate, and error bars are indicated.
FIGURE 6.
FIGURE 6.
Naa10 and NatA do not interact with MSRA or Runx2. A, GST pulldown assays of Naa10 using GST tagged Runx2 and MSRA. Coomassie staining of SDS-PAGE is shown for both the input (left) and elution (right). Corresponding migrating proteins in the gels are labeled. B, GST pulldown assays of Naa10/Naa15 (NatA) using GST-tagged Runx2 and MSRA. The experiment was carried out as described in A. C, as a positive control, pulldowns of NatA were carried out using GST-tagged Naa50. Asterisks indicate free hydrolyzed GST.
FIGURE 7.
FIGURE 7.
The lysines in MSRA and Runx2 are found in proximity to basic residues that could facilitate chemical acetylation. A and B, crystal structures of (A) bovine MSRA and (B) murine Runx1 (90% identity to Runx2), highlighting the lysines within these proteins reported to be acetylated by Naa10 (yellow) as well as proximal basic residues (salmon).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Verdin E., and Ott M. (2015) 50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond. Nat. Rev. Mol. Cell Biol. 16, 258–264 - PubMed
    1. Starheim K. K., Gevaert K., and Arnesen T. (2012) Protein N-terminal acetyltransferases: when the start matters. Trends Biochem. Sci. 37, 152–161 - PubMed
    1. Choudhary C., Kumar C., Gnad F., Nielsen M. L., Rehman M., Walther T. C., Olsen J. V., and Mann M. (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325, 834–840 - PubMed
    1. Forte G. M., Pool M. R., and Stirling C. J. (2011) N-terminal acetylation inhibits protein targeting to the endoplasmic reticulum. PLoS Biol. 9, e1001073. - PMC - PubMed
    1. Scott D. C., Monda J. K., Bennett E. J., Harper J. W., and Schulman B. A. (2011) N-terminal acetylation acts as an avidity enhancer within an interconnected multiprotein complex. Science 334, 674–678 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources