Cloning and characterization of hNAT5/hSAN: An evolutionarily conserved component of the NatA protein 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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2021, StructureCitation Excerpt :NatA acetylates proteins after removal of the initiator methionine followed by small residues, such as Ala, Cys, Gly, Ser, Thr, or Val by the methionine aminopeptidase (Arnesen et al., 2009; Bienvenut et al., 2012; Mullen et al., 1989; Polevoda et al., 1999; Van Damme et al., 2011b). NatA can associate with an additional subunit, Naa50, forming the NatE complex (Arnesen et al., 2006; Gautschi et al., 2003; Williams et al., 2003). The NatB complex is composed of the catalytic subunit Naa20, the auxiliary subunit Naa25, and acetylates the initiator methionine followed by acidic/hydrophilic residues, such as Asp, Glu, Asn, or Gln (Polevoda et al., 1999; Van Damme et al., 2012).
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2020, International Journal of Biological MacromoleculesCitation Excerpt :The next class is NatD, which only has one catalytic subunit Naa40p (Nat4) and is different from other NATs as only two substrates have been identified for it in yeasts i.e. H2A and H4 [28,30]. The subunits of NatE are Naa50p (Nat5p/San) and two other subunits Naa10p and Naa15p (same as NatA) [31]. Another NAT protein structurally well described is Mpr1 of yeast which composed of six α helices and eight β sheets showing mixed α/β structure.