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Proline-adjacent phosphosites on S. cerevisiae histone demethylase Rph1p are salt stress responsive and important for cell growth in the salt stress response

ABSTRACT: Phosphorylation of histone lysine demethylases is an important mechanism by which the cell modulates chromatin dynamics to regulate its response to stress. There is evidence that the Saccharomyces cerevisiae H3K36me2/3 demethylase, Rph1p, is an integrator of many signalling events. However, there is limited understanding of how most Rph1p phosphosites affect or reflect its role in stress response pathways and how this modulates a cellular response. Here, we investigated the role of Rph1p phosphorylation in the salt stress response. We showed that Rph1p is extensively phosphorylated in response to acute high salt stress, particularly at proline-adjacent residues. Genomic phosphonull mutations identified a subset of salt-stress responsive phosphosites – S410, T411, S412 and S689 – that are important for yeast cell growth in this condition. However, these sites were not associated with gross changes in H3K36 methylation, suggesting that phosphorylation at specific sites may instead direct Rph1p to specific regions of the chromatin in the salt stress response. Proteomic investigation of slow growing Rph1p-S412A and Rph1p-S689A mutants revealed functional enrichment for proteins involved in cell structure and morphology, as well as mitochondria-associated proteins. These findings show that phosphorylation of Rph1p at S412 and S689 is important for modulating the yeast cell’s response to salt stress, offering insights into stress-responsive regulatory mechanisms in all eukaryotic cells.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Nicola Karakatsanis

LAB HEAD: Marc R

PROVIDER: PXD059945 | Pride | 2025-11-03

REPOSITORIES: Pride

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Proline-Adjacent Phosphosites on Saccharomyces cerevisiae Histone Demethylase Rph1p are Salt Stress Responsive and Important for Cell Growth Under Salt Stress.

Karakatsanis Nicola M NM Hamey Joshua J JJ Wilkins Marc R MR

Molecular & cellular proteomics : MCP 20250908 10

Phosphorylation of histone lysine demethylases is an important mechanism by which the cell modulates chromatin dynamics to regulate its response to stress. There is evidence that the Saccharomyces cerevisiae H3K36me2/3 demethylase, Rph1p, is an integrator of many signaling events. However, the regulatory function of most Rph1p phosphosites in stress response pathways remains unknown. Here, we investigated the role of Rph1p phosphorylation in the salt stress response. We showed that Rph1p is phos ...[more]

PMID: 40930238

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Project description:The foodborne pathogen Listeria monocytogenes experiences osmotic stress in many habitats, including foods and the gastrointestinal tract of the host. While osmotic stress induced changes in expression have been investigated for specific genes, identifying and understanding genome-wide temporal changes in the transcriptome due to salt stress will provide insights into how this pathogen adapts to and survives this stress, leading to development of new intervention strategies. To determine the short-term and long-term responses to salt stress, we exposed exponential phase cells of L. monocytogenes H7858 to 6% NaCl in Brain Heart Infusion (BHI) broth at 7°C and 37°C and extracted RNA after 2.5%, 5%, 10%, and 20% of lag phase and during exponential growth in BHI + 6% NaCl, and evaluated temporal changes in transcript levels with microarrays. The temperature dependent short-term response to salt stress included a significant increase in transcript levels of the alternative sigma factor σB, with maximum expression at 2.5-5% of lag phase at 37°C, and at 20% of lag phase at 7°C. Transcript levels of genes known to be regulated by σB, including inlAB, opuCA, glpFK, and gadBC, were significantly upregulated at the same relative time points as σB As indicated by significantly elevated transcript levels during exponential growth in BHI + 6% NaCl, genes encoding proteins involved in purine and pyrimidine synthesis and amino acid biosynthesis are part of the long-term response to salt stress at 37°C. Transcript levels of virulence genes plcA, mpl, actA, and plcB were also significantly upregulated during exponential growth under salt stress at 37°C. Genes encoding proteins involved in protein degradation and stability and cell membrane modifications are part of the long-term response to salt stress at 7°C. Overall, an initial alteration in the transcriptome occurs, decreasing transcript levels of genes encoding proteins involved in energy conversion and metabolism, while increasing transcript levels of those involved in the stress response controlled by σB, followed by a long-term response, including increased expression of genes encoding compatible solute transporters and general stress proteins, facilitating growth under salt stress. A reference design was used to analyze gene expression differences. Exponential phase cultures at 37C and at 7C were used as a reference for salt stressed samples at each temperature. Salt stressed samples from the same relative time point were compared across temperatures. 4 biological replicates were tested for each comparison.

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	Caffeine_rep1_5uL.raw	Raw
	Caffeine_rep3_5uL.mgf	Mgf
	Caffeine_rep3_5uL.raw	Raw
	Caffeine_rep5_5uL.mgf	Mgf