NatA complex

Abstract: Nα-terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys- and Val- N termini. NatA is composed of subunits encoded by yARD1 and yNAT1 in yeast and hARD1 and hNAT1 in humans. A yeast ard1-Δ nat1-Δ strain was phenotypically complemented by hARD1 hNAT1, suggesting that yNatA and hNatA are similar. However, heterologous combinations, hARD1 yNAT1 and yARD1 hNAT1, were not functional in yeast, suggesting significant structural subunit differences between the species. Proteomics of a yeast ard1-Δ nat1-Δ strain expressing hNatA demonstrated that hNatA acts on nearly the same set of yeast proteins as yNatA, further revealing that NatA from humans and yeast have identical or nearly identical specificities. Nevertheless, all NatA substrates in yeast were only partially N-acetylated, whereas the corresponding NatA substrates in HeLa cells were mainly completely N-acetylated. Overall, we observed a higher proportion of N-terminally acetylated proteins in humans (84%) as compared with yeast (57%). N-acetylation occurred on approximately one-half of the human proteins with Met-Lys- termini, but did not occur on yeast proteins with such termini. Thus, although we revealed different N-acetylation patterns in yeast and humans, the major NAT, NatA, acetylates the same substrates in both species.

Abstract: N-terminal acetylation is ubiquitous among eukaryotic proteins and controls a myriad of biological processes. Of the N-terminal acetyltransferases (NATs) that facilitate this cotranslational modification, the heterodimeric NatA complex has the most diversity for substrate selection and modifies the majority of all N-terminally acetylated proteins. Here, we report the X-ray crystal structure of the 100-kDa holo-NatA complex from Schizosaccharomyces pombe, in the absence and presence of a bisubstrate peptide-CoA-conjugate inhibitor, as well as the structure of the uncomplexed Naa10p catalytic subunit. The NatA-Naa15p auxiliary subunit contains 13 tetratricopeptide motifs and adopts a ring-like topology that wraps around the NatA-Naa10p subunit, an interaction that alters the Naa10p active site for substrate-specific acetylation. These studies have implications for understanding the mechanistic details of other NAT complexes and how regulatory subunits modulate the activity of the broader family of protein acetyltransferases.

Abstract: The human NatA protein N-alpha-terminal-acetyltransferase complex is responsible for cotranslational N-terminal acetylation of proteins with Ser, Ala, Thr, Gly, and Val N termini. The NatA complex is composed of the catalytic subunit hNaa10p (hArd1) and the auxiliary subunit hNaa15p (hNat1/NATH). Using immunoprecipitation coupled with mass spectrometry, we identified endogenous HYPK, a Huntingtin (Htt)-interacting protein, as a novel stable interactor of NatA. HYPK has chaperone-like properties preventing Htt aggregation. HYPK, hNaa10p, and hNaa15p were associated with polysome fractions, indicating a function of HYPK associated with the NatA complex during protein translation. Knockdown of both hNAA10 and hNAA15 decreased HYPK protein levels, possibly indicating that NatA is required for the stability of HYPK. The biological importance of HYPK was evident from HYPK-knockdown HeLa cells displaying apoptosis and cell cycle arrest in the G(0)/G(1) phase. Knockdown of HYPK or hNAA10 resulted in increased aggregation of an Htt-enhanced green fluorescent protein (Htt-EGFP) fusion with expanded polyglutamine stretches, suggesting that both HYPK and NatA prevent Htt aggregation. Furthermore, we demonstrated that HYPK is required for N-terminal acetylation of the known in vivo NatA substrate protein PCNP. Taken together, the data indicate that the physical interaction between HYPK and NatA seems to be of functional importance both for Htt aggregation and for N-terminal acetylation.