Expanded in vivo substrate profile of the yeast N-terminal acetyltransferase NatC

N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein sub-strates of yeast NatA and NatB has been performed by N-ter-minomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay be-tween different NATs in targeting methionine-starting N -termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30 Delta yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.

Automated summary

N-terminal acetylation of proteins is a conserved modification in eukaryotes, and the yeast Saccharomyces cerevisiae is a valuable model for studying this process. The bulk of N-terminal acetylation in S. cerevisiae is catalyzed by NatA, NatB, and NatC. In this study, the authors identified 57 yeast NatC substrates using a combination of techniques. They also uncovered some non-NatC NAT activity, suggesting redundancy between NatC and NatE/Naa50. Furthermore, the authors demonstrated the evolutionary conservation of NatC from yeast to humans and its potential implications for studying pathogenic variants. Overall, this work provides valuable insights into NatC-mediated N-terminal acetylation.