Activities, substrate specificity, and genetic interactions of fission yeast Siw14, a cysteinyl-phosphatase-type inositol pyrophosphatase.
Activities, substrate specificity, and genetic interactions of fission yeast Siw14, a cysteinyl-phosphatase-type inositol pyrophosphatase.
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肌醇焦磷酸 1,5-IP8 是一种信号分子，可调节裂殖酵母裂殖酵母中磷酸盐和多磷酸盐的稳态。 1,5-IP8 水平由将 5-IP7 转化为 1,5-IP8 的 Asp1 激酶结构域和水解 1,5-IP8 的 β-磷酸的两种焦磷酸酶（Asp1 焦磷酸酶结构域（组氨酸酸性磷酸酶家族）和 Aps1 焦磷酸酶（Nudix 家族））之间的平衡决定。在这里，我们将裂殖酵母 Siw14 (SpSiw14) 描述为半胱氨酰磷酸酶家族成员和酿酒酵母 Siw14 的同源物，作为参与肌醇焦磷酸分解代谢的第三种裂殖酵母焦磷酸酶。我们发现 SpSiw14 的底物库包括无机焦磷酸盐、无机多磷酸盐和肌醇焦磷酸盐 5-IP7、1-IP7 和 1,5-IP8，以及通用底物对硝基苯磷酸盐。遗传分析表明，(i) 通过 C189S 突变消除 SpSiw14 蛋白或使 SpSiw14 焦磷酸酶失活对粟酒裂殖酵母的生长没有影响，但在没有 Aps1 的情况下是致命的；(ii) siw14Δ aps1Δ 的合成致死率取决于 Asp1 激酶对 1,5-IP8 的合成。我们得出的结论是，SpSiw14 和 Aps1 焦磷酸酶在裂殖酵母中具有必要但多余的功能，并且它们的合成致死性是过多 1,5-IP8 毒性作用的结果。通过裂殖酵母 3' 加工/终止机制的功能丧失突变来抑制 siw14Δ aps1Δ 致死率，强化了过度转录终止作为 1,5-IP8 中毒基础的情况。肌醇焦磷酸信号分子 1,5-IP8 通过其作为 RNA 3' 加工和转录终止激动剂的作用来调节裂殖酵母磷酸盐稳态。细胞 1,5-IP8 水平由肌醇多磷酸激酶 Asp1 和几种肌醇焦磷酸酶的活性之间的平衡决定。在这里，我们将粟酒裂殖酵母 Siw14 (SpSiw14) 描述为一种半胱氨酰磷酸酶家族焦磷酸酶，能够水解磷酸酐底物无机焦磷酸盐、无机多磷酸盐和肌醇焦磷酸盐 5-IP7、1-IP7 和 1,5-IP8。遗传分析表明 SpSiw14 参与体内 1,5-IP8 分解代谢，因为： 在缺乏 Nudix 型肌醇焦磷酸酶 Aps1 的情况下，SpSiw14 活性的丧失是致命的； siw14Δ aps1Δ 致死率取决于 Asp1 激酶合成 1,5-IP8。 3′-加工/终止因子的功能丧失突变对 siw14Δ aps1Δ 致死率的抑制表明转录过早终止是 1,5-IP8 中毒的原因。
Inositol pyrophosphate 1,5-IP8 is a signaling molecule that regulates phosphate and polyphosphate homeostasis in the fission yeast Schizosaccharomyces pombe. 1,5-IP8 levels are dictated by a balance between the Asp1 kinase domain that converts 5-IP7 to 1,5-IP8 and two pyrophosphatases—the Asp1 pyrophosphatase domain (histidine acid phosphatase family) and the Aps1 pyrophosphatase enzyme (Nudix family)—that hydrolyze the β-phosphates of 1,5-IP8. Here, we characterize S. pombe Siw14 (SpSiw14), a cysteinyl-phosphatase family member and a homolog of Saccharomyces cerevisiae Siw14, as a third fission yeast pyrophosphatase implicated in inositol pyrophosphate catabolism. We find that SpSiw14’s substrate repertoire embraces inorganic pyrophosphate, inorganic polyphosphate, and the inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8, in addition to the generic substrate p-nitrophenylphosphate. Genetic analyses revealed that (i) elimination of the SpSiw14 protein or inactivation of the SpSiw14 pyrophosphatase by the C189S mutation had no effect on S. pombe growth but was lethal in the absence of Aps1 and (ii) the synthetic lethality of siw14∆ aps1∆ depended on the synthesis of 1,5-IP8 by the Asp1 kinase. We conclude that SpSiw14 and Aps1 pyrophosphatases have essential but redundant functions in fission yeast, and that their synthetic lethality is a consequence of the toxic effects of too much 1,5-IP8. Suppression of siw14∆ aps1∆ lethality by loss-of-function mutations of components of the fission yeast 3′-processing/termination machinery fortifies the case for overzealous transcription termination as the basis for 1,5-IP8 toxicosis. The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3′-processing and transcription termination. Cellular 1,5-IP8 levels are determined by a balance between the activities of the inositol polyphosphate kinase Asp1 and several inositol pyrophosphatase enzymes. Here, we characterize Schizosaccharomyces pombe Siw14 (SpSiw14) as a cysteinyl-phosphatase-family pyrophosphatase enzyme capable of hydrolyzing the phosphoanhydride substrates inorganic pyrophosphate, inorganic polyphosphate, and inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8. Genetic analyses implicate SpSiw14 in 1,5-IP8 catabolism in vivo, insofar as: loss of SpSiw14 activity is lethal in the absence of the Nudix-type inositol pyrophosphatase enzyme Aps1; and siw14∆ aps1∆ lethality depends on synthesis of 1,5-IP8 by the Asp1 kinase. Suppression of siw14∆ aps1∆ lethality by loss-of-function mutations of 3′-processing/termination factors points to precocious transcription termination as the cause of 1,5-IP8 toxicosis.
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DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
影响因子：
--
作者：
Randall, Thomas A.;Gu, Chunfang;Li, Xingyao;Wang, Huanchen;Shears, Stephen B.
通讯作者：
Shears, Stephen B.
影响因子：
2.9
作者：
Florio, Tyler J.;Lokareddy, Ravi K.;Cingolani, Gino
通讯作者：
Cingolani, Gino
影响因子：
11.4
作者：
Safrany, ST;Caffrey, JJ;Shears, SB
通讯作者：
Shears, SB
影响因子：
4.8
作者：
Gu, Chunfang;Nguyen, Hoai-Nghia;Shears, Stephen B.
通讯作者：
Shears, Stephen B.