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

Abstract

N^α-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 N^α-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.

Figure 1.

In vitro Nat and N^ε-Lys acetyltransferase (Kat) activity of AtNAA50. A, Trx-His₆-AtNAA50 was purified from E. coli by immobilized metal affinity chromatography. B, Purified Trx-His₆-AtNAA50 was incubated for 1 h at 37°C with 45 µm [³H]acetyl-CoA and 0.2 mm of the synthetic MLGP and SESS peptides. After incubation, the peptide was enriched via specific interaction with SP Sepharose, and the amount of [³H]acetyl incorporated in the peptide was quantified by scintillation counting. As a negative control, AtNAA50 was heat inactivated at 95°C for 10 min. Data are presented as means ± sd (n = 4, P < 0.05 by Student’s t test). The experiment was repeated independently. C, Web logo of 28 N termini found to be specifically acetylated at their iMet after the expression of AtNAA50 in E. coli. D, Alignment of AtNAA50 and HsNAA50 protein sequences with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Lys residues that are acetylated in humans are shaded in blue; Lys residues that are acetylated in Arabidopsis are shaded in red. Lys residues that are predicted to be acetylated by Phosida (www.phosida.com) are marked in red. The conserved catalytic residues are shaded in green. E, Immunodetection of acetylated Lys residues on AtNAA50. Eleven micromolar purified AtNAA50 was incubated with 5 mm acetyl-CoA for 0, 20, 40, or 60 min at 37°C. Subsequently, the reaction was stopped by the addition of SDS loading buffer. The auto-Kat activity was determined via immunological detection of acetylated Lys residues with an α-acetylated Lys antibody. Heat-inactivated AtNAA50 (15 min at 95°C) served as a negative control (60 min). As loading control, AtNAA50 was visualized using Amido Black (n = 3). The experiment was repeated independently. F, Quantification of signals shown in E for the AtNAA50 autoacetylation. The Amido Black loading control was used for normalization. Data are presented as means ± se. Different lowercase letters indicate individual groups identified by pairwise multiple comparisons with a Holm-Sidak one-way ANOVA (P < 0.05, n = 3).

Figure 2.

AtNAA50 is localized in the cytosol, the nucleus, and the ER. AtNAA50-EYFP was transiently expressed in N. benthamiana epidermal cells. A, Counterstaining with DAPI shows that AtNAA50-EYFP localizes to the nucleus (marked with arrows). B, AtNAA50-EYFP colocalizes with the RFP-tagged ER marker VMA12. The experiment was repeated independently. Bars = 50 µm.

Figure 3.

Mutations in NAA50 lead to a severe growth reduction and infertility. A, Gene model for AtNAA50 (AT5G11340). Black boxes represent exons. The two T-DNA insertion lines naa50-1 (SAIL_1210_A02) and naa50-2 (SAIL_1186_A03) are marked with triangles. UTR, Untranslated region. B, Transcript levels of NAA50, NAA10, and NAA15 in naa50-2 mutants and wild-type (WT) plants (Student’s t test, P < 0.05, n = 3). The experiment was repeated independently. C, The NAA50 protein was detected with a specific antiserum in wild-type plants and naa50-2 mutants. Amido Black staining of the transferred proteins on the membrane served as a loading control (LC). D, Representative images of homozygous naa50-1 and naa50-2 mutants compared with wild-type plants of the same age. Plants were grown for 8 weeks under short-day conditions and subsequently transferred to long-day conditions. Although occasionally naa50 mutants were able to flower, none of these plants produced seeds. Images were digitally extracted for comparison.

Figure 4.

NAA50 function is conserved among humans and plants but not yeast. A, C, and E, Representative phenotypes of plants grown for approximately 8 weeks under short-day conditions. naa50-2 mutants were complemented with AtNAA50 (A), HsNAA50 (C), and ScNAA50 (E). Images were digitally extracted for comparison. Bars = 2 cm. B and D, The fresh weight of naa50-2 complemented with AtNAA50 (B) and HsNAA50 (D) was measured. The wild type (WT) served as a control. Data are presented as means ± se. Statistically significant differences are indicated by different letters (Holm-Sidak one-way ANOVA, P < 0.05). This experiment was performed with n > 5 plants and was independently repeated. F, The expression of the ScNAA50 transcript in three individuals of naa50-2:ScNAA50 and the wild type was confirmed via RT-qPCR. A sample without genomic DNA (water) served as a negative control.

Figure 5.

N-terminal protein acetylation in NAA50-depleted mutants. A to C, Distribution of the NTA yields in naa50-2 (gray) and the wild type (black) for all quantified N termini (+/−N-terminal Met excision [NME]; A), N termini after removal of their iMet (+NME; B), and N termini retaining their iMet (−NME; C). D, Volcano plot of acetylated N termini found in the wild type and naa50-2. Proteins with statistically significant alteration of N-terminal acetylation yield are labeled in green (P < 0.05, greater than 2-fold change): 1, AT2G14880; 2, AT5G03370. For these experiments, whole plants were collected and pooled to obtain 100 mg of plant material of wild-type (n = 2) and naa50-2 (n = 3) plants. FDR, False discovery rate.

Figure 6.

The expression of 732 protein groups is deregulated in naa50-2 mutants compared with wild-type plants. A, Volcano plot depicting the significantly regulated (greater than 1.4-fold change, LIMMA P < 0.05, n = 4) protein groups in blue. Proteins with unaltered abundance in naa50-2 are labeled in gray. NAA10 and NAA15 are indicated in green. Proteins were extracted from 4-week-old naa50-2 and wild-type seedlings grown under short-day conditions on one-half strength MS medium. B, The differentially regulated proteins (first pie chart, down-regulated in naa50-2 compared with the wild type; second pie chart, up-regulated in naa50-2 compared with the wild type) are localized in a variety of cellular compartments, according to the SUBAcon localization of the SUBA4 Arabidopsis subcellular localization database (Hooper et al., 2017). For comparison, the distribution of localizations was calculated for all proteins detected in the MS approach (third pie chart, designated background). The full list of quantified protein groups is available in Supplemental Table S2. N.A., Not available.