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

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.

组蛋白赖氨酸去甲基化酶（histone lysine demethylases）的磷酸化是细胞调控染色质动态以调节应激响应的重要机制。已有研究证实，酿酒酵母（Saccharomyces cerevisiae）H3K36me2/3去甲基化酶Rph1p是多种信号事件的整合枢纽。然而，当前学界对于多数Rph1p磷酸化位点如何影响或反映其在应激响应通路中的功能，以及该过程如何调控细胞应激响应的认知仍较为匮乏。本研究聚焦Rph1p磷酸化在盐胁迫响应中的调控作用。实验结果显示，在急性高盐胁迫刺激下，Rph1p会发生广泛磷酸化，尤其富集于脯氨酸相邻的残基位点。通过基因组水平的磷酸化缺失突变筛选，我们鉴定出一组盐胁迫响应相关的核心磷酸化位点——S410、T411、S412及S689，这些位点对于酵母细胞在该胁迫条件下的存活与生长至关重要。但上述位点并未伴随H3K36甲基化的整体水平变化，这提示在盐胁迫响应过程中，特定位点的磷酸化可能并非通过调控H3K36甲基化水平发挥功能，而是将Rph1p靶向招募至染色质的特定区域。对生长迟缓的Rph1p-S412A与Rph1p-S689A突变体开展蛋白质组学分析后发现，其蛋白组呈现细胞结构与形态相关蛋白、以及线粒体关联蛋白的显著功能富集。上述研究结果表明，Rph1p在S412与S689位点的磷酸化对于调控酵母细胞的盐胁迫响应具有关键意义，为阐明所有真核细胞的应激响应调控机制提供了全新的研究视角与理论参考。