NAA50 Is an Enzymatically Active N α-Acetyltransferase That Is Crucial for Development and Regulation of Stress Responses.

Nα-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. In plants, the biological function of NTA remains enigmatic. The dominant N-acetyltransferase (Nat) in Arabidopsis (Arabidopsis thaliana) is NatA, which cotranslationally catalyzes acetylation of ∼40% of the proteome. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. In human (Homo sapiens), fruit fly (Drosophila melanogaster), and yeast (Saccharomyces cerevisiae), this core NatA complex interacts with NAA50 to form the NatE complex. While in metazoa, NAA50 has N-acetyltransferase activity, yeast NAA50 is catalytically inactive and positions NatA at the ribosome tunnel exit. Here, we report the identification and characterization of Arabidopsis NAA50 (AT5G11340). Consistent with its putative function as a cotranslationally acting Nat, AtNAA50-EYFP localized to the cytosol and the endoplasmic reticulum but also to the nuclei. We demonstrate that purified AtNAA50 displays Nα-terminal acetyltransferase and lysine-ε-autoacetyltransferase activity in vitro. Global N-acetylome profiling of Escherichia coli cells expressing AtNAA50 revealed conservation of NatE substrate specificity between plants and humans. Unlike the embryo-lethal phenotype caused by the absence of AtNAA10 and AtNAA15, loss of NAA50 expression resulted in severe growth retardation and infertility in two Arabidopsis transfer DNA insertion lines (naa50-1 and naa50-2). The phenotype of naa50-2 was rescued by the expression of HsNAA50 or AtNAA50. In contrast, the inactive ScNAA50 failed to complement naa50-2 Remarkably, loss of NAA50 expression did not affect NTA of known NatA substrates and caused the accumulation of proteins involved in stress responses. Overall, our results emphasize a relevant role of AtNAA50 in plant defense and development, which is independent of the essential NatA activity.

NatB-Mediated N-Terminal Acetylation Affects Growth and Biotic Stress Responses.

N∝-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes. In humans, NTA is catalyzed by seven Nα-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 N-terminomics, transcriptomics, and physiological assays to characterize the Arabidopsis (Arabidopsis thaliana) 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 (naa20-1, naa20-2, and naa25-1) 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 naa20-1 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.

Downregulation of N-terminal acetylation triggers ABA-mediated drought responses in Arabidopsis.

N-terminal acetylation (NTA) catalysed by N-terminal acetyltransferases (Nats) is among the most common protein modifications in eukaryotes, but its significance is still enigmatic. Here we characterize the plant NatA complex and reveal evolutionary conservation of NatA biochemical properties in higher eukaryotes and uncover specific and essential functions of NatA for development, biosynthetic pathways and stress responses in plants. We show that NTA decreases significantly after drought stress, and NatA abundance is rapidly downregulated by the phytohormone abscisic acid. Accordingly, transgenic downregulation of NatA induces the drought stress response and results in strikingly drought resistant plants. Thus, we propose that NTA by the NatA complex acts as a cellular surveillance mechanism during stress and that imprinting of the proteome by NatA is an important switch for the control of metabolism, development and cellular stress responses downstream of abscisic acid.