Nα-acetyltransferase NAA50 mediates plant immunity independent of the Nα-acetyltransferase A complex.

In humans and plants, 40% of the proteome is cotranslationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex is comprised of the catalytic subunit Nα-acetyltransferase 10 (NAA10) and the ribosome-anchoring subunit NAA15. The regulatory subunit Huntingtin Yeast Partner K (HYPK) and the acetyltransferase NAA50 join this complex in humans. Even though both are conserved in Arabidopsis (Arabidopsis thaliana), only AtHYPK is known to interact with AtNatA. Here we uncover the AtNAA50 interactome and provide evidence for the association of AtNAA50 with NatA at ribosomes. In agreement with the latter, a split-luciferase approach demonstrated close proximity of AtNAA50 and AtNatA in planta. Despite their interaction, AtNatA/HYPK and AtNAA50 exerted different functions in vivo. Unlike NatA/HYPK, AtNAA50 did not modulate drought tolerance or promote protein stability. Instead, transcriptome and proteome analyses of a novel AtNAA50-depleted mutant (amiNAA50) implied that AtNAA50 negatively regulates plant immunity. Indeed, amiNAA50 plants exhibited enhanced resistance to oomycetes and bacterial pathogens. In contrast to what was observed in NatA-depleted mutants, this resistance was independent of an accumulation of salicylic acid prior to pathogen exposure. Our study dissects the in vivo function of the NatA interactors HYPK and NAA50 and uncovers NatA-independent roles for NAA50 in plants.

Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity.

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

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.

Structural insight into the DNMT1 reaction cycle by cryo-electron microscopy.

DNMT1 is an essential DNA methyltransferase that catalyzes the transfer of methyl groups to CpG islands in DNA and generates a prominent epigenetic mark. The catalytic activity of DNMT1 relies on its conformational plasticity and ability to change conformation from an auto-inhibited to an activated state. Here, we present four cryo-EM reconstructions of apo DNMT1 and DNTM1: non-productive DNA, DNTM1: H3Ub2-peptide, DNTM1: productive DNA complexes. Our structures demonstrate the flexibility of DNMT1's N-terminal regulatory domains during the transition from an apo 'auto-inhibited' to a DNA-bound 'non-productive' and finally a DNA-bound 'productive' state of DNMT1. Furthermore, we address the regulation of DNMT1's methyltransferase activity by a DNMT1-selective small-molecule inhibitor and ubiquitinated histone H3. We observe that DNMT1 binds DNA in a 'non-productive' state despite the presence of the inhibitor and present the cryo-EM reconstruction of full-length DNMT1 in complex with a di-ubiquitinated H3 peptide analogue. Taken together, our results provide structural insights into the reaction cycle of DNMT1.

HYPK controls stability and catalytic activity of the N-terminal acetyltransferase A in Arabidopsis thaliana.

The ribosome-tethered N-terminal acetyltransferase A (NatA) acetylates 52% of soluble proteins in Arabidopsis thaliana. This co-translational modification of the N terminus stabilizes diverse cytosolic plant proteins. The evolutionary conserved Huntingtin yeast partner K (HYPK) facilitates NatA activity in planta, but in vitro, its N-terminal helix α1 inhibits human NatA activity. To dissect the regulatory function of HYPK protein domains in vivo, we genetically engineer CRISPR-Cas9 mutants expressing a HYPK fragment lacking all functional domains (hypk-cr1) or an internally deleted HYPK variant truncating helix α1 but retaining the C-terminal ubiquitin-associated (UBA) domain (hypk-cr2). We find that the UBA domain of HYPK is vital for stabilizing the NatA complex in an organ-specific manner. The N terminus of HYPK, including helix α1, is critical for promoting NatA activity on substrates starting with various amino acids. Consequently, deleting only 42 amino acids inside the HYPK N terminus causes substantial destabilization of the plant proteome and higher tolerance toward drought stress.

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.

TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination by the E2 Ube2W can be inhibited by N-terminal acetylation, but this doesn't prevent substrate ubiquitination nor degradation. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Further, Trim-Away degrades substrates irrespective of whether they contain lysines or are N-terminally acetylated, which may explain the ability of TRIM21 to counteract fast-evolving pathogens and degrade diverse substrates.

HYPK promotes the activity of the Nα-acetyltransferase A complex to determine proteostasis of nonAc-X2/N-degron-containing proteins.

In humans, the Huntingtin yeast partner K (HYPK) binds to the ribosome-associated Nα-acetyltransferase A (NatA) complex that acetylates ~40% of the proteome in humans and Arabidopsis thaliana. However, the relevance of HsHYPK for determining the human N-acetylome is unclear. Here, we identify the AtHYPK protein as the first in vivo regulator of NatA activity in plants. AtHYPK physically interacts with the ribosome-anchoring subunit of NatA and promotes Nα-terminal acetylation of diverse NatA substrates. Loss-of-AtHYPK mutants are remarkably resistant to drought stress and strongly resemble the phenotype of NatA-depleted plants. The ectopic expression of HsHYPK rescues this phenotype. Combined transcriptomics, proteomics, and N-terminomics unravel that HYPK impairs plant metabolism and development, predominantly by regulating NatA activity. We demonstrate that HYPK is a critical regulator of global proteostasis by facilitating masking of the recently identified nonAc-X2/N-degron. This N-degron targets many nonacetylated NatA substrates for degradation by the ubiquitin-proteasome system.

Structural and functional characterization of the N-terminal acetyltransferase Naa50.

The majority of eukaryotic proteins is modified by N-terminal acetylation, which plays a fundamental role in protein homeostasis, localization, and complex formation. N-terminal acetyltransferases (NATs) mainly act co-translationally on newly synthesized proteins at the ribosomal tunnel exit. NatA is the major NAT consisting of Naa10 catalytic and Naa15 auxiliary subunits, and with Naa50 forms the NatE complex. Naa50 has recently been identified in Arabidopsis thaliana and is important for plant development and stress response regulation. Here, we determined high-resolution X-ray crystal structures of AtNaa50 in complex with AcCoA and a bisubstrate analog. We characterized its substrate specificity, determined its enzymatic parameters, and identified functionally important residues. Even though Naa50 is conserved among species, we highlight differences between Arabidopsis and yeast, where Naa50 is catalytically inactive but binds CoA conjugates. Our study provides insights into Naa50 conservation, species-specific adaptations, and serves as a basis for further studies of NATs in plants.