肌醇焦磷酸1,5-IP8调节裂变酵母磷酸盐稳态调节子的表达,通过其作为抑制PHOmRNA合成的上游lncRNAs转录早熟终止的激动剂的作用,包含磷酸获得基因pho1,pho84和tgp1。1,5-IP8水平由将5-IP7转化为1,5-IP8的Asp1N末端激酶结构域和三种肌醇焦磷酸酶-Asp1C末端结构域(组氨酸酸性磷酸酶)之间的平衡决定,Siw14(一种半胱氨酸磷酸酶),和Aps1(一种Nudix酶)。在这项研究中,我们报道了Aps1的生化和遗传特征,并分析了Asp1,Siw14和Aps1突变对细胞肌醇焦磷酸水平的影响.我们发现Aps1的底物库包括无机多磷酸盐,5-IP7、1-IP7和1,5-IP8。与5-IP7相比,Aps1对1-IP7的水解表现出〜两倍的偏好,与野生型细胞相比,aps1Δ细胞的1-IP7水平高两倍。虽然Aps1和Siw14都不是增长所必需的,在YES培养基上,aps1Δsiw14Δ双突变是致命的。这种致死性是IP8中毒的表现,由此,过量的1,5-IP8驱动tgp1的去抑制,导致Tgp1介导的甘油磷酸胆碱的摄取。我们能够在缺乏甘油磷酸胆碱的ePMGT培养基上恢复aps1Δsiw14突变体,并通过删除tgp1来抑制aps1Δsiw14在YES上的严重生长缺陷。然而,通过删除tgp1无法缓解aps1Δasp1-H397A菌株的严重生长缺陷,这表明该双焦磷酸酶突变体中的1,5-IP8水平超过了一个阈值,超过该阈值的过度热情终止会影响其他基因,导致细胞毒性。
目的:通过lncRNA介导的干扰抑制裂殖酵母PHO基因tgp1,pho1和pho84对1,5-IP8代谢的变化敏感,1,5-IP8是一种信号分子,可作为早熟lncRNA终止的激动剂。1,5-IP8由5-IP7的磷酸化形成,并由来自三个不同酶家族的肌醇焦磷酸酶分解代谢:Asp1(组氨酸酸性磷酸酶),Siw14(一种半胱氨酸磷酸酶),和Aps1(一种Nudix水解酶)。这项研究需要对Aps1进行生化表征,并分析Asp1,Siw14和Aps1突变如何影响体内生长和肌醇焦磷酸池。Aps1催化无机多磷酸盐的水解,体外5-IP7、1-IP7和1,5-IP8,与5-IP7相比,1-IP7具有〜两倍的偏好。aps1细胞的1-IP7水平比野生型细胞高两倍。aps1Δsiw14Δ双突变是致命的,因为过量的1,5-IP8会触发tgp1的抑制,导致甘油磷酸胆碱的毒性摄取。
Inositol pyrophosphate 1,5-IP8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1, pho84, and tgp1, via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP7 to 1,5-IP8 and three inositol
pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1\'s substrate repertoire embraces inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8. Aps1 displays a ~twofold preference for hydrolysis of 1-IP7 versus 5-IP7 and aps1∆ cells have twofold higher levels of 1-IP7 vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1∆ siw14∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP8 toxicosis, whereby excessive 1,5-IP8 drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1∆ siw14∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1∆ siw14∆ on YES by deleting tgp1. However, the severe growth defect of an aps1∆ asp1-H397A strain could not be alleviated by deleting tgp1, suggesting that 1,5-IP8 levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity.
OBJECTIVE: Repression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP8, a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP8 is formed by phosphorylation of 5-IP7 and catabolized by inositol
pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo. Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8 in vitro, with a ~twofold preference for 1-IP7 over 5-IP7. aps1∆ cells have twofold higher levels of 1-IP7 than wild-type cells. An aps1∆ siw14∆ double mutation is lethal because excessive 1,5-IP8 triggers derepression of tgp1, leading to toxic uptake of glycerophosphocholine.