Protein N-terminal acetylation is a very common event in eukaryotic cells, occurring on a majority of proteins (9
). However, compared to the yeast homologues, the human protein N-terminal acetyltransferases have not been fully identified or extensively characterized. In this study, we describe the human homologues of the yeast NatC complex. Our candidate subunits, hMak3, hMak10, and hMak31, stably interact with each other, localize to the cytoplasm, and associate with ribosomes. Furthermore, our semipurified hMak3 displays in vitro N-terminal acetyltransferase activity toward peptide substrates matching the observed substrate specificity of the yeast NatC complex, displaying a high degree of substrate specificity. Interestingly, Munro and colleagues showed in a previous study that hNat12/hMak3 functionally complemented yMak3p in Δmak3
yeast strains (12
). Taken together, these findings strongly argue that hMak3 (hNat12) is the human homologue of yeast Mak3p. Based on these observations, we suggest that hMak3, hMak10, and hMak31 constitute the human NatC complex and that hMak3 is the catalytic subunit of the complex. Interestingly, the hMak3 protein (362 amino acids) is 176 amino acids larger than the yeast Mak3p. This is mainly due to an N-terminal region not shared by the yeast homologue. Similarly, the Arabidopsis thaliana
Mak3 also contains additional residues compared to yeast Mak3, and AtMak3 displays enzymatic activity independent of AtMak10 (26
). The function of the N-terminal hMak3 domain is not known. Using the ELM functional site prediction resource (http://elm.eu.org/
), the N-terminal region was found to contain several potential phosphorylation sites, making it a possible region for posttranslational regulation of hMak3 activity. Similarly, the catalytic subunit of hNatA, hArd1, has a C-terminal domain that is phosphorylated to different extents depending on cell culture conditions (24
All the hNatC subunits localize to the cytoplasm and bind ribosomes. However, hMak31 also significantly localizes to the nucleus (Fig. ). The nuclear localization of hMak31 would be expected, as its small size allows it to pass through the nuclear pore complexes. hMak31 (Lsmd1) belongs to the Sm and Sm-like proteins, which associate with RNA and are often involved in RNA processing events. Thus, hMak31 may have a nuclear role linked to RNA processing independent of the hNatC complex. hMak3, hMak10, and hMak31 are all present, both in a ribosome-bound and an unbound state. This pattern is also observed for subunits in the hNatA (T. Arnesen et al., submitted for pubication) and the hNatB complexes (34
). In addition, there seems to be some difference between hMak3, hMak10, and hMak31 with respect to the ratio of ribosome-bound versus unbound protein. In particular, a larger fraction of hMak10 seems to be ribosome bound compared to hMak3 and hMak31. One could hypothesize that hMak10 may function as an anchor for ribosomal binding of the complex, similar to what is observed for hNat1 in the hNatA complex.
In a previous study, Nat13/Nat5 was suggested to be the vertebrate homologue of yMak3p (38
). However, Nat13/Nat5 is the vertebrate protein most similar to yeast Nat5p. Both in humans and in yeast, Nat5 is described to be a subunit of the NatA complex (3
). To our knowledge, Nat5p has not been physically linked to the yeast NatC complex or functionally associated with NatC activity. In our present study we were not able to demonstrate an interaction between hNat13/hNat5 and hMak10. Whether hNat13/hNat5 is capable of binding to the human NatC is therefore doubtful, but more thorough experimentation is needed to settle this issue.
Knockdown of hNatC subunits in HeLa cells leads to cell death and growth arrest. Knockdown cells displayed reduced metabolic activity and DNA synthesis, an increase in the sub-G0/G1 fraction representing nuclear fragmentation, an increase in double-stranded DNA breaks, and PARP cleavage indicating caspase-dependent cell death. All three subunits are important for normal growth, supporting a model where the observed phenotypes are caused by lack of hNatC-mediated N-acetylation. Upon knockdown of hMAK3, hMAK10, or hMAK31 in the colon carcinoma cell lines HCT116 (p53−/−), no apoptosis was induced, in contrast to HCT116 (p53+/+) cells (Fig. ). This difference in response between the cell variants strongly indicates that p53 is essential for induction of apoptosis in hNatC-depleted cells. Our findings suggest that this effect is mediated mainly through the transcriptional activity of p53, since hMAK3 knockdown increased the protein levels of p53, increased p53 Ser37 phosphorylation, and induced the transcription of p53 downstream death effector genes (Fig. ).
NatC is believed to acetylate relatively few substrates compared to NatA in a manner of high amino acid sequence specificity at the N-terminal region. This could make hMak3 a potential target for drug-mediated cancer treatment, as previously discussed for hNatA (6
). An important goal of future studies will be to further elaborate the pathways through which hMAK3
knockdown mediates p53-dependent apoptosis.
In combination with the previous studies of the hNatA and hNatB complexes (1
) our present results emphasize the biological importance of protein N-terminal acetylation in human cells. It should be noted that hMAK3
knockdown cells in general demonstrated a stronger phenotype than hMAK10
knockdown cells. These effects may indicate that hMak3 has an additional function independent of hMak10 and hMak31. Leister and coworkers demonstrated that A. thaliana
Mak3 alone is able to functionally replace the yeast NatC complex (26
). Also, in contrast to AtMak3, knockout of AtMak10
did not result in any obvious defects. This indicates that Mak3 can have functions independent of the NatC complex in organisms higher than yeast.
The oligopeptides MLALI and MLGTG are identical to the N termini of the predicted human NatC substrates hArl8b and mTOR, respectively (38
). hArl8b was recently described to be N-terminally acetylated, and an intact N terminus is essential for its association with lysosomes (20
). mTOR was suggested to be a direct downstream target for NatC acetylation in zebrafish. Our in vitro acetyltransferase assays support our hypothesis that both hArl8b and mTOR are substrates for hNatC in vivo (Fig. ). Knockdown of hMAK3
in HeLa cells induces a change in subcellular distribution of hArl8b-GFP. The observed change was statistically significant, but only a fraction of the cells (~15%) display altered hArl8b-GFP localization as a consequence of hMAK3 knockdown (Fig. ). The observed phenotype changes will to some extent be dependent on the transfection efficiency of sihMAK3
: only a fraction of the hArl8b-GFP-expressing cells will have efficient knockdown of hMAK3
. Among this subpopulation, one would expect a larger fraction of cells to display aberrant hArl8b-GFP localization. Furthermore, large-scale proteomics analyses of NAT knockdown in human cell lines have demonstrated only partial downstream N-terminal acetylation effects in agreement with our observations (9
). Combined, these data strongly support that hArl8b indeed is an hNatC substrate in vivo. This is to our knowledge the only human NatC substrate where a clear consequence is observed when the N-terminal acetylation is lost. Also, it is the only known case with a link between a specific human NAT and a substrate of which N-terminal acetylation affects the substrate function. Further characterization of functionally important human NatC substrates will be of great importance in order to understand the overall impact of N-terminal acetylation, and in particular, N-terminal acetylation mediated by the hNatC complex.
With the present study, the human homologues of all three major yeast NAT complexes, NatA, NatB, and NatC, have been experimentally determined. In Table we present an overview of the currently known human protein N-terminal acetyltransferases. The nomenclature of this enzyme class is under revision (B. Polevoda, T. Arnesen, and F. Sherman, unpublished data), and the NatC components will be denoted as follows: Naa30 (Mak3), Naa35 (Mak10), and Naa38 (Mak31).
In summary, we here identify hMak3, hMak10, and hMak31 as the subunits of the human NatC complex. Knockdown of these subunits induces p53-dependent apoptosis. hMak3-mediated acetylation is necessary for the lysosomal localization and function of hArl8b (Fig. ).
FIG. 11. Schematic representation of functions associated with the human NatC complex. (A) The hNatC complex, composed of the subunits hMak3, hMak10, and hMak31, associates with ribosomes and acetylates nascent Met-Leu and similar polypeptides. One protein of (more ...)