PDF(Pubmed)
Abstract：
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 substrates of yeast NatA and NatB has been performed by N-terminomics. 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 between 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 naa30Δ 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.
摘要：
N端乙酰化是真核生物中保守的蛋白质修饰，酿酒酵母是研究这种修饰的有价值的模型系统。负责酿酒酵母中大部分蛋白质N末端乙酰化的酶是N末端乙酰转移酶NatA，NatB和NatC.到目前为止,已通过N-末端组学对酵母NatA和NatB的体内蛋白质底物进行了全蛋白质组鉴定。这里,我们使用酿酒酵母删除了NatC催化亚基Naa30，并通过N末端组合分数对角色谱（COFRADIC）分析鉴定了57种酵母NatC底物。有趣的是,除了以ML开始的规范N-termini之外，MI,MF和MW，酵母NatC底物还包括MY，MK,MM,MA,MV和MS然而,对于其中一些基底类型，比如我的,MK,MV和MS，我们还发现了(残留的)非NatCNAT活动，很可能是由于酵母NatC和NatE/Naa50之间先前建立的冗余。因此，我们已经揭示了不同NAT之间在靶向酵母中蛋氨酸起始N末端的复杂相互作用。此外，我们的结果表明，人NAA30的异位表达拯救了naa30Δ酵母中已知的NatC表型，以及部分恢复的酵母NatCNt-乙酰基体。因此，我们证明了NatC从酵母到人类的进化保守性，从而为研究致病性NAA30变体的未来疾病模型奠定了基础。总的来说,这项工作为NatC介导的N端乙酰化提供了更多的生化和功能见解，并为未来的工作提供了基础，以查明将NatC介导的N端乙酰化缺失与NatC缺失酵母表型联系起来的特定分子机制。
