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Disruption of the N^α-Acetyltransferase NatB Causes Sensitivity to Reductive Stress in Arabidopsis thaliana.

ABSTRACT: 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 indicate 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, leaving the role of DOA10 in plants ambiguous.

SUBMITTER: Huber M

PROVIDER: S-EPMC8761761 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:The cytosolic level of inorganic pyrophosphate (PPi) is finely regulated, with PPi hydrolyzed primarily by the vacuolar H+-pyrophosphatase (H+-PPase, VHP1/FUGU5/AVP1) and secondarily by five cytosolic soluble pyrophosphatases (sPPases; PPa1-PPa5) in Arabidopsis thaliana. Loss-of-function mutants of H+-PPase (fugu5s) have been reported to show atrophic phenotypes in their rosette leaves when nitrate is the sole nitrogen source in the culture medium. For this phenotype, two questions remain unanswered: why does atrophy depend on physical contact between shoots and the medium, and how does ammonium prevent such atrophy. To understand the mechanism driving this phenotype, we analyzed the growth and phenotypes of mutants on ammonium-free medium in detail. fugu5-1 showed cuticle defects, cell swelling, reduced ?-glucan levels, and vein malformation in the leaves, suggesting cell wall weakening and cell lethality. Based on the observation in the double mutants fugu5-1 ppa1 and fugu5-1 ppa4 of more severe atrophy compared to fugu5-1, the nitrogen-dependent phenotype might be linked to PPi metabolism. To elucidate the role of ammonium in this process, we examined the fluctuations of sPPase mRNA levels and the possibility of alternative PPi-removing factors, such as other types of pyrophosphatase. First, we found that both the protein and mRNA levels of sPPases were unaffected by the nitrogen source. Second, to assess the influence of other PPi-removing factors, we examined the phenotypes of triple knockout mutants of H+-PPase and two sPPases on ammonium-containing medium. Both fugu5 ppa1 ppa2 and fugu5 ppa1 ppa4 had nearly lethal embryonic phenotypes, with the survivors showing striking dwarfism and abnormal morphology. Moreover, fugu5 ppa1+/- ppa4 showed severe atrophy at the leaf margins. The other triple mutants, fugu5 ppa1 ppa5 and fugu5 ppa2 ppa4, exhibited death of root hairs and were nearly sterile due to deformed pistils, respectively, even when grown on standard medium. Together, these results suggest that H+-PPase and sPPases act in concert to maintain PPi homeostasis, that the existence of other PPi removers is unlikely, and that ammonium may suppress the production of PPi during nitrogen metabolism rather than stimulating PPi hydrolysis.

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Disruption of the N^α-Acetyltransferase NatB Causes Sensitivity to Reductive Stress in Arabidopsis thaliana.

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Dataset Information

Disruption of the Nα-Acetyltransferase NatB Causes Sensitivity to Reductive Stress in Arabidopsis thaliana.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Disruption of the N^α-Acetyltransferase NatB Causes Sensitivity to Reductive Stress in Arabidopsis thaliana.