Analysis of lacking Nα-terminal acetylation of proteins by NatA and NatB in Saccharomyces cerevisiae on mRNA levels and protein synthesis
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE140255 | GEO | 2020/07/27
REPOSITORIES: GEO
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Project description:N-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes and is catalyzed in humans by seven Nα-acetyltransferases (NatA-F and NatH). Remarkably, the characterization of the plant Nat machinery and its biological relevance is still in its infancy, although NTA has gained recognition as key regulator of crucial processes like protein turnover, protein-protein interaction and protein targeting. In this study we combined in vitro assays, reverse genetics, quantitative N-terminomics, transcriptomics and physiological assays to characterize the Arabidopsis NatB complex. We show that the plant NatB catalytic (NAA20) and auxiliary subunit (NAA25) form a stable heterodimeric complex that accepts canonical NatB-type substrates in vitro. In planta, NatB complex formation was essential for enzymatic activity. Depletion of NatB subunits to 30% of wild-type level in three Arabidopsis T-DNA insertion mutants (naa20-1, naa20-2, naa25-1) decreased growth to 50% of wild-type level. A complementation approach revealed functional conservation between plant and human catalytic NatB subunits, while yeast NAA20 failed to complement naa20-1. Quantitative N-terminomics of approximately 2000 peptides identified 29 bona fide substrates of the plant NatB. In vivo, NatB preferentially acetylated N-termini starting with the initiator methionine followed by acidic amino acids and contributed 20% of the acetylation marks in the detected plant proteome. The global transcriptome and proteome analyses of NatB-depleted mutants suggested a function of NatB in multiple stress responses. In agreement, we revealed the specific impact of NatB on the resistance of plants to osmotic or high-salt stress. Remarkably, depletion of NatA did not affect these resistances.
2020-04-14 | GSE132978 | GEO
Project description:In Arabidopsis thaliana the evolutionary conserved N-terminal acetyltransferase (Nat) complexes NatA and NatB co-translationally acetylate 60% of the proteome. Both have recently been implicated in the regulation of plant stress responses. While NatA mediates drought tolerance, NatB is required for pathogen resistance and the adaptation to high salinity and high osmolarity. Salt and osmotic stress impair protein folding and result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER-membrane resident E3 ubiquitin ligase DOA10 targets misfolded proteins for degradation during ER stress and is conserved among eukaryotes. In yeast, DOA10 recognizes conditional degradation signals (Ac/N-degrons) created by NatA and NatB. Assuming that this mechanism is preserved in plants, the lack of Ac/N-degrons required for efficient removal of misfolded proteins might explain the sensitivity of NatB mutants to protein harming conditions. In this study, we investigate the response of NatB mutants to dithiothreitol (DTT) and tunicamycin (TM) induced ER stress. We report that NatB mutants are hypersensitive to DTT but not TM, suggesting that the DTT hypersensitivity is caused by an over-reduction of the cytosol rather than an accumulation of unfolded proteins in the ER. In line with this hypothesis, the cytosol of NatB depleted plants is constitutively over-reduced and a global transcriptome analysis reveals that their reductive stress response is permanently activated. Moreover, we demonstrate that doa10 mutants are susceptible to neither DTT nor TM, ruling out a substantial role of DOA10 in ER-associated protein degradation (ERAD) in plants. Contrary to previous findings in yeast, our data indicates that N-terminal acetylation (NTA) does not inhibit ER targeting of a substantial amount of proteins in plants. In summary, we provide further evidence that NatB-mediated imprinting of the proteome is vital for the response to protein-harming stress and rule out DOA10 as the sole recognin for substrates in the plant ERAD pathway.
2021-11-29 | GSE186324 | GEO
Project description:N-terminal protein acetylation is one of the most common protein modifications in eucaryotes and is catalyzed co-translationally by N-terminal aceytltransferases (Nats). Regarding the number of predictaed substrates, NatA is the main Nat complex in human and yeast and is composed of the catalytic subunit NAA10 and the auxilliary subunit NAA15. The down-regulation of NAA10 and NAA15 results in Arabidopsis in drought resistant plants. This study focus on the identification of altered gene expression leading to the drought resistant phenotype. Microarray analysis was used to identify misregulated genes in the NatA depleted mutants responsible for the drought resistant phenotype
2015-07-23 | GSE65414 | GEO
Project description:N-terminal acetylation is a conserved protein modification among eukaryotes, and the yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The enzymes responsible for the bulk of protein N-terminal acetylation in S. cerevisiae are the N-terminal acetyltransferases NatA, NatB and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography (COFRADIC) analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF and MW, yeast NatC substrates also included MY, MK, MM, MA, MV and MS. However, for some of these substrate types, such as MY, MK, MV and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30∆ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast
2023-01-26 | PXD039544 | Pride
Project description:N-terminal protein acetylation is one of the most common protein modifications in eucaryotes and is catalyzed co-translationally by N-terminal aceytltransferases (Nats). Regarding the number of predictaed substrates, NatA is the main Nat complex in human and yeast and is composed of the catalytic subunit NAA10 and the auxilliary subunit NAA15. The down-regulation of NAA10 and NAA15 results in Arabidopsis in drought resistant plants. This study focus on the identification of altered gene expression leading to the drought resistant phenotype. Microarray analysis was used to identify misregulated genes in the NatA depleted mutants responsible for the drought resistant phenotype Arabidopsis thaliana plants were grown on soil for 6 weeks under short-day conditions (8h light / 16h dark cycles) with normal watering and subsequently challenged with drought for 10 days. After 10 days of drought Arabidopsis leaf material was harvested and used for RNA extraction and hybridization on Affymetrix microarrays. Four biological replicates from each genotype and condition were analyzed.
2015-07-23 | E-GEOD-65414 | biostudies-arrayexpress
Project description:Co-translational N-terminal (Nt-) acetylation of nascent polypeptides is catalyzed by N-terminal acetyltransferases (NATs). The very N-terminal amino acid sequence is the major factor determining whether or not a given protein is Nt-acetylated. In humans, six different NATs, denoted NatA-NatF, are identified. In the current study we used N-terminal COFRADIC analysis to define the in vivo substrate specificity of Naa50 (Nat5)/NatE as this has long remained elusive. Three yeast strains were generated; a control strain endogenously expressing yNaa50, a deletion strain depleted of yNaa50 and a strain deleted of yNaa50 but ectopically expressing human Naa50. When comparing the Nt-acetylation status of different N-termini in the control strain with the deletion strain, a reduction in Nt-acetylation for several yeast proteins was observed. To our surprise, these substrates were not of the predicted NatE-type substrates, but rather canonical NatA substrates. Further, ectopic expression of hNaa50 mainly resulted in the Nt-acetylation of a selected class of otherwise Nt-free yeast N-termini besides increasing the degree of Nt-acetylation of several other yeast proteins, and as such (partially) complementing those N-termini displaying reduced Nt-acetylation upon yNAA50-deletion. The preferred substrates of hNaa50 were predominantly Met-starting N-termini including Met-Lys-, Met-Val-, Met-Ala-, Met-Tyr-, Met-Phe-, Met-Leu-, Met-Ser- and Met-Thr, and highly overlapped with the previously identified human Naa60/NatF substrate specificity profile. Identification of several hNaa50 substrates with a small amino acid in the second position also revealed a potential interplay between the NATs and methionine aminopeptidases (MetAPs). The initiator Methionine (iMet) is normally cleaved off by MetAPs when the second amino acid is small, but our in vitro data suggest that in contrast to a free iMet, an Nt-acetylated iMet is not hydrolyzed by MetAPs. Thus, Naa50-mediated Nt-acetylation may potentially act as a mechanism to retain the iMet of proteins with a small amino acid at the second position that normally would be hydrolyzed.
2015-05-29 | PXD001437 | Pride
Project description:N-terminal acetylation is a major and vital protein modification catalysed by N-terminal acetyltransferases (NATs). NatF or Nα-acetyltransferase 60 (Naa60) was recently identified as a NAT in higher eukaryotes. Here, we found that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established Naa60 to face the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60 knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins, and that is has a preference for N-termini facing the cytosol. Moreover, NAA60 knockdown caused Golgi fragmentation, indicating an important role in maintenance of Golgi structural integrity. This work uncovers the first NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus far poorly characterized part of the N-terminal acetylome.
2019-06-04 | PXD001155 | Pride
Project description:Protein N-terminal (Nt) acetylation is one of the most abundant modifications in eukaryotes, covering ~50-80 % of the proteome, depending on species. Cells with defective Nt-acetylation display a wide array of phenotypes such as impaired growth, mating defects and increased stress sensitivity. However, the pleiotropic nature of these effects has hampered our understanding of the functional impact of protein Nt-acetylation. The main enzyme responsible for Nt-acetylation throughout the eukaryotic kingdom is the N-terminal acetyltransferase NatA. Here we employed a multi-dimensional proteomics approach to analyze Saccharomyces cerevisiae lacking NatA activity, which caused global proteome remodeling. Pulsed-SILAC experiments revealed that NatA-deficient strains consistently increased degradation of ribosomal proteins compared to wild type. Explaining this phenomenon, thermal proteome profiling uncovered decreased thermostability of ribosomes in NatA-knockouts. Our data are in agreement with a role for Nt-acetylation in promoting stability for parts of the proteome by enhancing the avidity of protein-protein interactions and folding.
2023-10-24 | PXD037510 | Pride
Project description:N-terminal (Nt) acetylation, catalyzed by N-terminal acetyltransferases (NATs), has emerged as an important co-translational modification in eukaryotes, and involves the transfer of the acetyl moiety from acetyl-CoA (Ac-CoA) to the α-amino group of a nascent polypeptide. Here, we report the first global study of Nt-acetylation in response to changes in nutrient availability in S. cerevisiae. Despite major fluctuations in Ac-CoA and histone acetylation levels, our study reveals that the steady-state levels of protein Nt-acetylation remains largely unaffected by changes in cellular metabolism. Accordingly, Ac-CoA does not appear to act as a master switch for protein Nt-acetylation. Interestingly however, we identified two distinct sets of N-termini that are differentially Nt-acetylated following nutrient starvation. The first set of proteins, enriched for annotated N-termini, generally displayed an increased Nt-acetylation in stationary phase compared to exponential growth phase, despite a significant reduction in Ac-CoA levels. In contrast, the second set of proteins, enriched for alternative non-annotated N-termini (i.e. N-terminal proteoforms), generally became less acetylated in stationary phase. In particular, the degree of Nt-acetylation of Pcl8, a negative regulator of glycogen biosynthesis and two components of the pre-ribosome complex (Rsa3 and Rpl7a) increased during starvation. Moreover, the levels of these proteins were regulated by both starvation and the presence of NatA activity. Overall, our data provide the first proteome-wide survey on metabolic regulation of Nt-acetylation, and propose Nt-acetylation-mediated steering of metabolism and ribosome biogenesis.
2018-09-07 | PXD004326 | Pride
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