Knockdown of Human N -Terminal Acetyltransferase Complex C Leads to p53-Dependent Apoptosis and Aberrant Human Arl8b Localization

Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, N-5020 Bergen, Norway.
Molecular and Cellular Biology (Impact Factor: 4.78). 05/2009; 29(13):3569-81. DOI: 10.1128/MCB.01909-08
Source: PubMed


Protein Nα-terminal acetylation is one of the most common protein modifications in eukaryotic cells. In yeast, three major complexes,
NatA, NatB, and NatC, catalyze nearly all N-terminal acetylation, acetylating specific subsets of protein N termini. In human
cells, only the NatA and NatB complexes have been described. We here identify and characterize the human NatC (hNatC) complex,
containing the catalytic subunit hMak3 and the auxiliary subunits hMak10 and hMak31. This complex associates with ribosomes,
and hMak3 acetylates Met-Leu protein N termini in vitro, suggesting a model in which the human NatC complex functions in cotranslational
N-terminal acetylation. Small interfering RNA-mediated knockdown of NatC subunits results in p53-dependent cell death and
reduced growth of human cell lines. As a consequence of hMAK3 knockdown, p53 is stabilized and phosphorylated and there is a significant transcriptional activation of proapoptotic genes
downstream of p53. Knockdown of hMAK3 alters the subcellular localization of the Arf-like GTPase hArl8b, supporting that hArl8b is a hMak3 substrate in vivo.
Taken together, hNatC-mediated N-terminal acetylation is important for maintenance of protein function and cell viability
in human cells.

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    • "This observation is in accordance with the fact that in vitro recombinant hNaa60 also displayed very similar substrate specificity profiles as compared to recombinant hNaa50 [7] [46], indicative for a potential in vivo substrate redundancy between hNaa50 and hNaa60. Interestingly, the property of Nt-acetylating Met-hydrophobic N-termini is also partially shared with NatC [20] "
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    ABSTRACT: Co-translational 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. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Apr 2015 · Proteomics
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    • "N termini recognized by NatF include those starting with Met-Lys as well as other Met-starting N termini, preferentially those followed by a hydrophobic amino acid (Van Damme et al., 2011b). Previously, these N termini were thought to be acetylated by NatC (Naa30) (Polevoda et al., 1999; Starheim et al., 2009) and NatE (Naa50) (Evjenth et al., 2009; Van Damme et al., 2011a). As such, the addition of NatF to the NAT family revealed some potential redundancy among these three enzymes. "
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    ABSTRACT: N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or Nα-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Apr 2015 · Cell Reports
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    • "lation, to promote p53 association with p300, which in turn acetylates p53 in its C-terminal [Starheim et al., 2009]. The Lys residues in the C-terminal regions of histones also compete for ubiquitination, promoting nuclear export and protein degradation, while acetylation rather promotes nuclear localization and protein stability [Yang and Seto, 2008]. "
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    ABSTRACT: Proteins function is regulated by co- and post-translational modifications (CTMs and PTMs) such as phosphorylation, glycosylation and acetylation, which induce proteins to perform multiple tasks in a specified environment. Acetylation takes place post-translationally on the ε-amino group of Lys in histone proteins, allowing regulation of gene expression. Furthermore, amino group acetylation also occurs co-translationally on Ser, Thr, Gly, Met and Ala, possibly contributing to the stability of proteins. In this work, the influence of amino acids next to acetylated sites has been investigated by using MAPRes (Mining Association Patterns among preferred amino acid residues in the vicinity of amino acids targeted for post-translational modifications). MAPRes was utilized to examine the sequence patterns vicinal to modified and non-modified residues, taking into account their charge and polarity. The PTMs data was further sub-divided according to their sub-cellular location (nuclear, mitochondrial and cytoplasmic), and their association patterns were mined. The association patterns mined by MAPRes for acetylated and non-acetylated residues are consistent with the existing literature but also revealed novel patterns. These rules have been utilized to describe the acetylation and its effects on the protein structure-function relationship. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.
    Full-text · Article · Apr 2013 · Journal of Cellular Biochemistry
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