Journal of Biological Chemistry ( IF 5.5 ) Pub Date : 2022-06-16 , DOI: 10.1016/j.jbc.2022.102148 Maria Laura Gaspar 1 , Manuel A Aregullin 1 , Yu-Fang Chang 1 , Stephen A Jesch 1 , Susan A Henry 1
Depletion of exogenous inositol in yeast results in rising levels of phosphatidic acid (PA) and is correlated with increased expression of genes containing the inositol-dependent upstream activating sequence (UASINO) promoter element. INO1, encoding myo-inositol 3-phosphate synthase, is the most highly regulated of the UASINO-containing genes, but its mechanism of regulation is not clear. In the current study we determined the relative timing and kinetics of appearance of individual molecular species of PA following removal of exogenous inositol in actively growing wild type, pah1Δ, and ole1ts strains. We report that the pah1Δ strain, lacking the PA phosphatase, exhibits a delay of about 60 min in comparison to wild type before initiating derepression of INO1 expression. The ole1ts mutant on the other hand, defective in fatty acid desaturation, when grown at a semi-restrictive temperature, exhibited reduced synthesis of PA 34:1 species and elevated synthesis of PA 32:1 species. Importantly, we found these changes in the fatty acid composition in the PA pool of the ole1ts strain were associated with reduced expression of INO1, indicating that synthesis of PA 34:1 is involved in optimal expression of INO1 in the absence of inositol. Using deuterium-labeled glycerol in short-duration labeling assays, we found that changes associated with PA 34:1 species 34:1 were uniquely correlated with increased expression of INO1 in all three strains. These data indicate that the signal for activation of INO1 transcription is not necessarily the overall level of PA, but rather levels of a specific species of newly synthesized PA 34:1.
中文翻译:
磷脂酸种类 34:1 介导用于酵母脂质合成的肌醇 3-磷酸合酶基因 INO1 的表达
酵母中外源肌醇的消耗导致磷脂酸(PA)水平升高,并且与含有肌醇依赖性上游激活序列(UAS INO)启动子元件的基因表达增加相关。INO1编码肌醇 3-磷酸合酶,是 UAS INO含基因中受调节最高的基因,但其调节机制尚不清楚。在目前的研究中,我们确定了在积极生长的野生型、pah1 Δ 和ole1 ts菌株中去除外源肌醇后,单个 PA 分子种类出现的相对时间和动力学。我们报告说pah1与野生型相比,缺乏 PA 磷酸酶的 Δ 菌株在开始抑制INO1表达之前表现出约 60 分钟的延迟。另一方面,脂肪酸去饱和有缺陷的ole1 ts突变体在半限制性温度下生长时,表现出 PA 34:1 物种的合成减少和 PA 32:1 物种的合成增加。重要的是,我们发现ole1 ts菌株PA 池中脂肪酸组成的这些变化与INO1的表达减少有关,表明 PA 34:1 的合成与INO1的最佳表达有关在没有肌醇的情况下。在短期标记测定中使用氘标记的甘油,我们发现与 PA 34:1 物种 34:1 相关的变化与所有三种菌株中INO1的表达增加唯一相关。这些数据表明,激活INO1转录的信号不一定是 PA 的整体水平,而是新合成的特定种类 PA 34:1 的水平。