HYPK affects proteostasis by facilitating co-translational imprinting of the proteome with acetylation marks
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)
TISSUE(S): Leaf
SUBMITTER: 
Mario Oroshi
LAB HEAD: Prof. Dr. Matthias Mann
PROVIDER: PXD022120 | Pride | 2022-04-13
REPOSITORIES: Pride
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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.
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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).
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Project description:The human N-terminal acetyltransferase E (NatE) including its associated NatA co-translationally acetylates the N-terminus of about 40-60% of the proteome to mediate diverse biological processes including protein half-life, localization and protein interaction. In eukaryotes, the NatE complex contains the NAA50 catalytic subunit with substrate specificity for N-terminal methionine acetylation and NatA, which facilitates ribosomal targeting of the complex for co-translational activity. NatA, contains NAA10 catalytic and NAA15 auxiliary subunits, and forms a complex with a protein with intrinsic NAA10 inhibitory activity, HYPK. The molecular basis for how the human NAA10 and NAA50 catalytic subunits within NatE complex coordinate function and how HYPK regulates NatE activity is unknown. Here, we characterize the biochemical interplay between the human NAA10 and NAA50 catalytic subunits of NatE and its regulation by HYPK and correlate this to the cryo-EM structures of the human NatE and NatE/HYPK complexes. We show that NAA50 and HYPK exhibit negative cooperative binding to NatA in vitro and in human cells, by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contributes to NAA10 activity inhibition through structural alteration of the NAA10 substrate binding site. NatE is about 8-fold more active than NAA50, likely due to a reduced entropic cost for substrate binding through NatA tethering, but is inhibited by HYPK through structural alteration of the NatE substrate binding site. Taken together, these studies reveal the molecular basis for coordinated N-terminal acetylation by the NAA10 and NAA50 catalytic subunits of NatE and its modulation by HYPK.
2020-03-13 | PXD017031 | Pride
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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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.
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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
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 | PXD043600 | Pride
Project description:N-terminal acetylation is one of the most common protein modifications in eukaryotes. The NatA complex is the dominant regulator of the N-acetylome in all eukaryotes. Here, we show that the imprinting of the proteome with acetylation marks by the essential NatA complex is a critical trigger for stabilizing plant proteins. Depleting 70% of the NatA activity by downregulation of the catalytic or the ribosome anchoring subunit causes 4-fold faster degradation of total proteins in Arabidopsis. This proteome dataset compares the NatA depleted Arabidopsis lines amiNAA10 and amiNAA15 to wild type Arabidopsis.
2022-04-13 | PXD024122 | Pride
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. 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.
2024-03-05 | PXD043423 | Pride
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