Centre for Organismal Studies, Heidelberg University, 69120 Heidelberg, Germany.
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris Saclay, 91198 Gif-sur-Yvette cedex, France.
Plant Physiol. 2020 Feb;182(2):792-806. doi: 10.1104/pp.19.00792. Epub 2019 Nov 19.
N-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes. In humans, NTA is catalyzed by seven N-acetyltransferases (NatA-F and NatH). Remarkably, the plant Nat machinery and its biological relevance remain poorly understood, although NTA has gained recognition as a key regulator of crucial processes such as protein turnover, protein-protein interaction, and protein targeting. In this study, we combined in vitro assays, reverse genetics, quantitative -terminomics, transcriptomics, and physiological assays to characterize the Arabidopsis () NatB complex. We show that the plant NatB catalytic (NAA20) and auxiliary subunit (NAA25) form a stable heterodimeric complex that accepts canonical NatB-type substrates in vitro. In planta, NatB complex formation was essential for enzymatic activity. Depletion of NatB subunits to 30% of the wild-type level in three Arabidopsis T-DNA insertion mutants (, , and ) caused a 50% decrease in plant growth. A complementation approach revealed functional conservation between plant and human catalytic NatB subunits, whereas yeast NAA20 failed to complement Quantitative N-terminomics of approximately 1000 peptides identified 32 bona fide substrates of the plant NatB complex. In vivo, NatB was seen to preferentially acetylate N termini starting with the initiator Met followed by acidic amino acids and contributed 20% of the acetylation marks in the detected plant proteome. Global transcriptome and proteome analyses of NatB-depleted mutants suggested a function of NatB in multiple stress responses. Indeed, loss of NatB function, but not NatA, increased plant sensitivity toward osmotic and high-salt stress, indicating that NatB is required for tolerance of these abiotic stressors.
N-端乙酰化（NTA）是真核生物中最丰富的蛋白质修饰之一。在人类中，NTA 由七种 N-乙酰转移酶（NatA-F 和 NatH）催化。值得注意的是，尽管 NTA 已被认为是调节蛋白质周转、蛋白质-蛋白质相互作用和蛋白质靶向等关键过程的关键调节剂，但植物 Nat 机制及其生物学相关性仍知之甚少。在这项研究中，我们结合了体外测定、反向遗传学、定量 N-端组学、转录组学和生理测定，以表征拟南芥（）NatB 复合物。我们表明，植物 NatB 催化（NAA20）和辅助亚基（NAA25）形成稳定的异二聚体复合物，可在体外接受典型的 NatB 型底物。在植物中，NatB 复合物的形成对于酶活性至关重要。在三个拟南芥 T-DNA 插入突变体（、和）中，NatB 亚基的消耗降至野生型水平的 30%，导致植物生长减少 50%。互补方法表明植物和人类催化 NatB 亚基之间的功能保守性，而酵母 NAA20 未能互补。对大约 1000 个肽的定量 N-端组学分析鉴定了 32 个植物 NatB 复合物的真正底物。在体内，NatB 优先乙酰化以起始 Met 为起始的 N 端，随后是酸性氨基酸，并在检测到的植物蛋白质组中贡献了 20%的乙酰化标记。NatB 缺失突变体的全转录组和蛋白质组分析表明 NatB 在多种应激反应中具有功能。事实上，NatB 功能的丧失，但不是 NatA，增加了植物对渗透和高盐胁迫的敏感性，表明 NatB 是耐受这些非生物胁迫的必需条件。
