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N-terminal acetylation in Arabidopsis thaliana, and the effects of Naa10/15 or HypK knock-outs

ABSTRACT: This project is about the study of the effects of several proteins on the proteome of Arabidopsis thaliana, and more specifically on the N-terminal acetylome of this plant. To this end were analysed KO samples of either an N-alpha acetyltransferase (Naa10 or Naa15) or the chaperonne protein HypK.

INSTRUMENT(S): LTQ Orbitrap Velos

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

TISSUE(S): Leaf

SUBMITTER: Jean Baptiste BOYER

LAB HEAD: Carmela GIGLIONE

PROVIDER: PXD023195 | Pride | 2022-08-12

REPOSITORIES: Pride

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

Miklánková Pavlína P Linster Eric E Boyer Jean-Baptiste JB Weidenhausen Jonas J Mueller Johannes J Armbruster Laura L Lapouge Karine K De La Torre Carolina C Bienvenut Willy W Sticht Carsten C Mann Matthias M Meinnel Thierry T Sinning Irmgard I Giglione Carmela C Hell Rüdiger R Wirtz Markus M

Science advances 20220615 24

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 promot ...[more]

PMID: 35704578

Similar Datasets

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

2024-01-22 | PXD043600 | 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

Global and protein-specific analysis reveals Nt-acetylation-independent turnover of SQE1 by Arabidopsis DOA10-like E3 ligases

Project description:The Acetylation-dependent (Ac/) N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by specific E3-ligases (Ac/N-recognins). To date, no Ac/N-recognins have been defined in plants. Here we use molecular, genetic, and multi-omics approaches to characterise potential roles for Arabidopsis DOA10-like E3-ligases in the Nt-acetylation-(NTA-) dependent turnover of proteins at global and protein-specific scales. Arabidopsis has two ER-localised DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no significant differences in the global NTA profile compared to wildtype, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we show that turnover of ER-localised squalene epoxidase 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta does not depend on NTA, but Nt-acetyltransferases indirectly impact its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis, and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.

2024-01-26 | PXD043217 | Pride

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

Expression data from Arabidopsis thaliana depleted of HYPK or NAA10

Project description:Expression data from Arabidopsis thaliana depleted of HYPK or NAA10

| PRJNA665720 | ENA

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

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.

2020-07-15 | PXD017430 | Pride

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

LC-MS/MS analysis of immunoprecipitated NAA15-V5 variants

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