Elsevier

Gene

Volume 371, Issue 2, 26 April 2006, Pages 291-295
Gene

Cloning and characterization of hNAT5/hSAN: An evolutionarily conserved component of the NatA protein N-α-acetyltransferase complex

https://doi.org/10.1016/j.gene.2005.12.008Get rights and content

Abstract

The human hARD1–NATH complex, cotranslationally acetylating the α-amino groups of proteins, was recently described. In S. cerevisiae and D. melanogaster this NatA complex contains a third subunit, Nat5p or San, respectively. Based on phylogenetic analyses and database searches, we here describe the human homologue, hNAT5, of these proteins. RT-PCR experiments demonstrated that hNat5 mRNA was expressed in several human cell lines. The candidacy of hNAT5 as a third subunit of the hARD1–NATH complex was investigated using anti-NATH or anti-hARD1 in co-immunoprecipitation experiments followed by Mass Spectrometry analysis of tryptic peptides. Oligopeptides specific for hNAT5 were identified. This verified the expression of endogenous hNAT5 protein in human cells and also identified hNAT5 as a NATH and hARD1 interacting partner. hNAT5 localized to the cytoplasm in accordance with hNAT5–hARD1–NATH complexes playing a role in cotranslational N-α-acetylation. Sequence alignment revealed a high degree of similarity of the NAT5 protein between species supporting its conserved role as a part of the complex. The predicted acetyltransferase domain within hNAT5 suggested that this protein, like hARD1, is an enzymatically active component. In summary, we present the first description of the human homologue of Nat5p/San, hNAT5, the third component of the human NatA N-α-acetyltransferase complex.

Introduction

There are two distinct groups of protein acetyltransferases. Of these, the best characterized acetyltransferases acetylate ε-amino groups of lysine residues of histones, transcription factors and other proteins. The second group, the N-α-acetyltransferases, cotranslationally acetylate the N-termini of nascent polypeptides in a majority of all eukaryotic proteins (reviewed in (Polevoda and Sherman, 2003a)). In yeast, the latter group of enzymes has been characterized. Three major complexes responsible for cotranslational N-α-acetylation have been described. NatA, containing the Ard1p and Nat1p subunits, acetylate proteins with ser-, thr-, ala- and gly-termini after the initial met has been cleaved off (Mullen et al., 1989, Park and Szostak, 1992, Polevoda et al., 1999). The catalytically active subunit is Ard1p, while Nat1p probably has a role in substrate binding and positioning of the complex on the ribosomes (Gautschi et al., 2003). NatB and NatC contain specific subunits and acetylate substrates with met-termini (Polevoda et al., 2003, Polevoda and Sherman, 2001). Recently, a third component, Nat5p, of the NatA complex was identified. In yeast, Nat5p, stably interacted with Nat1p and Ard1p and its sequence suggest that it is a catalytic subunit of the complex (Gautschi et al., 2003). However, no natural substrates of Nat5p have been identified. Strains with deletions in the nat5 gene did not display the same phenotypes as the nat1-Δ and ard1-Δ strains (Gautschi et al., 2003). This could suggest that Nat5p acetylates a limited number of substrates distinct from those of the Nat1p–Ard1p complex. Nat5p has been demonstrated to be dispensable for the acetylation of Nat1p and Ard1p substrates (Polevoda and Sherman, 2003b). San, the D. melanogaster homologue of Nat5p, was demonstrated to be a part of the fly Nat1–Ard1 complex, suggesting functional conservation (Williams et al., 2003). Mutation of San disrupted centromeric sister chromatid cohesion in fruitfly (Williams et al., 2003). In human, only the hARD1–NATH N-α-acetyltransferase complex has been described (Arnesen et al., 2005a). In the present study, we identify hNAT5, the human homologue of Nat5p/San, as a component of the hARD1–NATH complex. Thus, the function of Nat5p and San is probably conserved from yeast and fruitfly to human.

Section snippets

Cloning and expression of the hNat5 gene

A BLASTP 2.2.12 search (Altschul et al., 1997) in the all non-redundant GenBank CDS database using the complete protein sequences of S. cerevisiae Nat5p and D. melanogaster San indicated the putative human homologue (BAB14397.1 derived from AK023090.1). Isolation of RNA and synthesis of cDNA was performed as previously described (Arnesen et al., 2005b). For detection of hNat5 expression, nested primer sets were used to ensure specificity. First reaction (numbers in brackets indicate primer

Identification of hNat5

Previously, phylogenetic analysis of different protein N-terminal α-amino-acetyltransferases from several species revealed that the putative human Nat5p homologue (BAB14397.1) grouped with yeast Nat5p indicating that they belong to the same group (Polevoda and Sherman, 2003b). Using the complete protein sequences of yeast Nat5p and fruitfly San in a BLASTP search, we verified that the putative human hNAT5, BAB14397.1, was indeed the human protein with highest sequence similarity to the yeast

Discussion

We have studied the human homologue of the S. cerevisiae Nat5p and D. melanogaster San and named it hNAT5. hNAT5 belongs to the GNAT family of Acetyltransferases. The NatA complex formation seen in yeast (Gautschi et al., 2003) and fruitfly (Williams et al., 2003) is found to be conserved in human by the present study. The substrates of hNAT5 are unknown, but given its association with NATH it is likely that specific protein N-termini are cotranslationally acetylated by hNAT5. The subcellular

Acknowledgements

We thank C. Hoff and K. Starheim for technical assistance. This work was supported by The Norwegian Cancer Society (Grants to TA, JEV, JRL), The Locus of Experimental Cancer Research (University of Bergen) and The Meltzer Foundation (Grant to TA).

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