Abstract
All forms of mammalian actin comprise at their N-terminus a negatively charged region consisting of an N-acetylated aspartate or glutamate followed by two or three acidic residues. This structural feature is unique to actins and important for their interaction with other proteins. The enzyme catalyzing the acetylation of the N-terminal acidic residue is thought to be NAA10, an enzyme that acetylates multiple intracellular proteins. We report here that this acetylation is essentially carried out by NAT6 (Fus2), a protein of unknown function. Tests of the activity of human recombinant NAT6 on a series of purified proteins showed that the best substrate had several acidic residues near its N-terminus. Accordingly NAT6 was particularly active on highly acidic peptides with sequences corresponding to the N-terminus of different forms of mammalian actins. Knocking out of NAT6 in two human cell lines led to absence of acetylation of the first residue of mature beta-actin (Asp2) and gamma-actin-1 (Glu2). Complete acetylation of these two actins was restored by re-expression of NAT6, or by incubation of extracts of NAT6-deficient cells with low concentrations of recombinant NAT6, while NAA10 showed much less or no activity in such assays. Alpha-actin-1 expressed in NAT6-knockout cells was not acetylated at its N-terminus, indicating that the requirement of NAT6 for acetylation of actin N-termini also applies to the skeletal muscle actin isoform. Taken together, our findings reveal that NAT6 plays a critical role in the maturation of actins by carrying out the acetylation of their N-terminal acidic residue.