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Co-immunoprecipitation of AtNAA50 binding partners

ABSTRACT: In humans and plants, 40 % of the proteome is co-translationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. The regulatory subunit HYPK and the acetyltransferase NAA50 join this complex in humans. Even though both are conserved in Arabidopsis thaliana, only AtHYPK is known to interact with AtNatA. In order to verify the interaction between AtNAA50 and the core NatA subunits and to identify novel AtNAA50 interactors, wildtype leaf protein extracts were mixed with a specific antiserum against AtNAA50 (Armbruster et al., 2020). Subsequently, protein A/G beads were used to precipitate antibody-bound AtNAA50 and its interaction partners. As a negative control, protein A/G beads were incubated with leaf protein extracts lacking the specific antibody against AtNAA50.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Marcin Luzarowski

LAB HEAD: Thomas Ruppert

PROVIDER: PXD043423 | Pride | 2024-03-05

REPOSITORIES: Pride

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

Project description:The ribosome-tethered N-terminal acetyltransferase A (NatA) acetylates 52% of soluble proteins in Arabidopsis thaliana. This co-translational shielding of the N-terminus stabilizes diverse cytosolic plant proteins. The Huntingtin yeast partner K (HYPK) facilitates NatA activity in planta, but in vitro, the N-terminal a-helix1 of HYPK inhibits NatA in the ternary NatA/HYPK complex. Consequently, the mechanism of HYPK function in plants was unknown. To understand the regulatory function of HYPK at the molecular level, we genetically engineered CRISPR/Cas9 mutants expressing either a non-functional HYPK (hypk-cr1) or an N-terminally deleted HYPK version (hypk-cr2). Like NatA-activity-depleted mutants, the hypk-cr1 and –cr2 mutants were more tolerant to drought stress and suffered from global proteome destabilization. Surprisingly, we found that absence of HYPK led to destabilization of the NatA subunits in leaves but not in roots of hypk-cr1. Further genetic and molecular evidence demonstrated that the stability of NatA subunits in leaves is mainly controlled by the C-terminal UBA domain of HYPK. While retaining the UBA domain, NatA activity was still impaired in hypk-cr2 leaves, implying that the N terminus of HYPK also contributes to the regulation of NatA activity. Furthermore, crossing of hypk-cr1 with a NatA-depleted mutant uncovered that HYPK promotes NatA activity not only by stabilizing NatA. Taken together, the activity-promoting function of the HYPK N-terminus depends on the binding of HYPK to NatA via the UBA domain, and is critical for NatA substrates starting with various amino acids. Our findings improve our understanding of the NatA regulatory subunit HYPK.

2024-01-22 | PXD047700 | Pride

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

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Mass-spectrometry based quantification of HYPK, NAA10 and NAA15 protein steady-state levels in Arabidopsis thaliana mutants carrying a T-DNA insertion in the HYPK gene.

Project description:N-alpha terminal acetylation is one of the major protein modifications in eukaryotes carried out by distinct Nats (N-alpha acetyltransferases). The core NatA complex is composed of the catalytic NAA10 and the auxiliary NAA15 subunit and co-translationally acetylates majority of the cytosolic proteins. HYPK interacts with NatA core complex in human and fungus in vivo and in vitro, regulating its activity. Here, a mass spectrometry approach was applied to quantify the steady-state levels of the core NatA subunits, NAA10 and NAA15 and of HYPK in the roots of Arabidopsis mutants carrying a T-DNA insertion in the HYPK gene (AT3G06610).

2022-08-12 | PXD023599 | Pride

AtNAA50 behaviour in the core NatA complex

Project description:In eukaryotes, the N-terminal acetylation (NTA) is one of the most frequent protein modifications. In many organisms, and especially in plants, its biological function remains a mystery. In Arabidopsis thaliana, a large part of the proteome acetylation is catalyzed co-translationally by the action of the core NatA complex, which consitst of NAA10 and NAA15, respectively the catalytic and ribosome-anchoring subunits. This complex interacts with the NAA50 protein (NatE), which also has an N-acetyltransferase in organisms such as human and fruit fly. This project focuses on the effect of AtNAA50 knockouts on the activity of the essential NatA complex.

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Impact of NAA50 depletion on the transcriptome of rosette leaves from the reference plant Arabidopsis thaliana (Col-0).

Project description:In humans and plants, 40 % of the proteome is co-translationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. The regulatory subunit HYPK and the acetyltransferase NAA50 join this complex in humans. Even though both are conserved in Arabidopsis thaliana, only AtHYPK is known to interact with AtNatA. Here we aimed to determine the global transcriptome change in the reference plant Arabidopsis thaliana leaves upon depletion of the acetyltransferase NAA50 (AT5G11340). The wild type and the amiNAA50 #13 mutant were grown on soil for seven weeks on soil under short-day conditions (8.5 h of light, 70-100 μE light photon flux density, 24 °C/18 °C day/night temperature, and 50-60 % humidity) and RNA was extracted with the Universal RNA Kit (Roboclon) according to the manufacturer´s instructions.

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Purification of AtNAA10 and AtHYPK from leaves of transgenic Arabidopsis plants expressing Strep II –tagged NAA10 or HYPK

Project description:In humans and plants, 40 % of the proteome is co-translationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. In humans, the regulatory subunit HYPK and the acetyltransferase NAA50 join this complex. Even though both are conserved in Arabidopsis thaliana, only AtHYPK is known to interact with AtNatA. Here we fused AtNAA10 and AtHYPK with the Strep II®-tag and expressed them separately in stably transformed Arabidopsis thaliana ecotype Col-0 (for AtNAA10) or the hypk-3 (for AtHYPK) mutant (Miklánková et al., 2022). Stably transformed plants of the T2 generation were grown on soil under short-day conditions. Six weeks after germination, the Strep-tagged proteins were purified from leaf material and subjected to mass-spectrometry to identify potential interaction partners.

2024-03-05 | PXD043208 | Pride

Expression data from Arabidopsis thaliana depleted of HYPK or NAA10

Project description:N-alpha terminal acetylation is an abundant protein modification in eukaryotes. The NatA (N-alpha acetyltransferase A) complex is responsible for N-terminal acetylation of majority of the cytosolic proteins and its core is composed of NAA10 and NAA15 subunit. HYPK has been shown to interact with NatA core complex in human and fungus, modulating its activity. Here we address the in vivo function of the plant HYPK protein. A transcriptomics approach was applied to compare transcriptional reprogramming induced by depletion of NAA10 or HYPK mutants in plants.

2022-05-20 | GSE158586 | GEO

AtNAA50 N-terminal acetylation profiling using A. thaliana naa50 KO mutants

Project description:N-terminal acetylation is one of the most abundant protein modifications in eukaryotes and is catalysed in humans by seven N-terminal acetyltransferases. AtNAA50 interreact with the NatA complex (NAA10 and NAA15) to form the NatE complex. In this study, we investigated the Arabidopsis thaliana N-terminal acetylome in leaf cells with KO naa50 gene to identify in vivo specific substrates using the SILProNAQ approach.

2020-07-15 | PXD017770 | Pride

The Nα-acetyltransferase NAA50 mediates plant immunity independent of the NatA complex

Project description:Nα-terminal acetylation (NTA) ranges among the most abundant protein modifications in higher eukaryotes. Over 40 % of the human and plant proteome are co-translationally acetylated by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-binding subunit NAA15. In humans, the regulatory subunit HYPK and the acetyltransferase NAA50 join the complex. Even though both are conserved in Arabidopsis thaliana, only AtHYPK is known to interact with AtNatA. Here we analyse the interactome of AtNAA50 and provide evidence for the association of the enzyme with AtNatA. Moreover we demonstrate that AtNAA50 interacts with ribosome-associated proteins involved in protein translation, folding and translocation. A recent study shows that acetylation protects NatA substrates from proteasomal degradation. In consequence, NatA depletion results in an accelerated protein turnover. We report that this is not true for the depletion of AtNAA50, suggesting that the enzyme is not required for NatA activity. In line with this, novel amiNAA50 knockdown lines do not display the characteristic drought resistance of NatA mutants. Instead, complementary transcriptome and proteome analyses suggest that AtNAA50 is a negative regulator of plant immunity. Pathogen challenges confirm that amiNAA50 plants are resistant to oomycetes and bacteria.

2024-03-01 | PXD040755 | JPOST Repository

Differential N-terminal COFRADIc analyses of Ogden fibroblasts

Project description:The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase gene. The affected males harbour a Ser37Pro mutation in the gene encoding hNaa10, the catalytic subunit of NatA, themajor human NAT. In order to understand the detrimental impact of hNaa10 Ser37Pro, we performed structural, molecular and cellular investigations. Structural models and molecular dynamics simulations of the human NatA and its Ser37Pro mutant suggest differences in regions involved in catalysis and at the interface between hNaa10 and the auxiliary subunit hNaa15. In agreement, biochemical data demonstrate a reduced catalytic capacity and an impaired NatA complex formation with hNaa10 Ser37Pro. Patient derived Naa10 mutant cells show reduced Nt-acetylation for a subset of NatA/NatE-type substrates compared to wild type Naa10 cells. Ogden syndrome fibroblasts further display abnormal cell proliferation and migration capacity, possibly linked to perturbed Retinoblastoma- and MYLK-pathways, respectively. Differential N-terminal combined fractional diagonal chromatography (COFRADIC) was used to quantify the degree and define the patterns of in vivo protein Nt-acetylation and here used to investigate whether patient cells derived from an affected Ogden male (carrying the Naa10 S37P mutation) displayed altered levels of protein Nt-acetylation at the proteome-wide level.

2016-04-12 | PXD001282 | Pride

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