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外文期刊>The biochemical journal
>Disruption and overexpression of the Schizosaccharomyces pombe aps1 gene, and effects on growth rate, morphology and intracellular diadenosine 5?,5'-P1,P5-pentaphosphate and diphosphoinositol polyphosphate concentrations
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Disruption and overexpression of the Schizosaccharomyces pombe aps1 gene, and effects on growth rate, morphology and intracellular diadenosine 5?,5'-P1,P5-pentaphosphate and diphosphoinositol polyphosphate concentrations
piSchizosaccharomyces pombe/i Aps1 is an enzyme that degrades both diadenosine oligophosphates (Apsubin/i/subA, in/i = 5 or 6) and diphosphoinositol polyphosphates {diphosphoinositol pentakisphosphate (iPP/i-InsiP/isub5/sub) and bisdiphosphoinositol tetrakisphosphate ([iPP/i]sub2/sub-InsiP/isub4/sub)} iin vitro/i. The iin vivo/i substrates of Aps1 are unknown. We report here the identification of Apsub5/subA, iPP/i-InsiP/isub5/sub, [iPP/i]sub2/sub-InsiP/isub4/sub and a novel diphosphoinositol polyphosphate ([iPP/i]subix/i/sub-InsiP/isubix/i/sub) in iS. pombe/i using HPLC methods. Apsub5/subA was present at 0.06pmol/mg of protein (approx. 4nM). iPP/i-InsiP/isub5/sub, [iPP/i]subix/i/sub-InsiP/isubix/i/sub and [iPP/i]sub2/sub-InsiP/isub4/sub were present at 15pmol/mg (approx. 1.1iμ/iM), 15pmol/mg (approx. 1.1iμ/iM) and 30pmol/mg (approx. 2.2iμ/iM) respectively, while the intracellular concentration of InsiP/isub6/sub was 0.5nmol/mg of protein (approx. 36iμ/iM). Disruption of iaps1/i resulted in a 52% decrease in Apsub6/subA hydrolase activity iin vitro/i, no detectable change in the intracellular Apsub5/subA concentration, and 3-fold increased intracellular concentrations of iPP/i-InsiP/isub5/sub and [iPP/i]subix/i/sub-InsiP/isubix/i/sub. Disruption of iaps1/i resulted in no detectable change in morphology or growth rate in minimal or rich media at 30°C. Overexpression of iaps1/i via two different plasmids that resulted in 60% and 6-fold increases above wild-type enzymic activity iin vitro/i caused no detectable changes in the intracellular concentrations of [iPP/i]sub2/sub-InsiP/isub4/sub, [iPP/i]subix/i/sub-InsiP/isubix/i/sub or iPP/i-InsiP/isub5/sub, but paradoxical increases of approx. 2.5- and 55-fold respectively in the intracellular Apsub5/subA concentration. Overexpression of iaps1/i also resulted in a reduced growth rate and in morphological changes, including swollen, rounded and multiseptate cells. No phenotypic changes or changes in intracellular Apsub5/subA occurred upon overexpression of iaps1/iE93Q, which encodes a mutated Aps1 lacking significant enzymic activity. We conclude that Aps1 degrades iPP/i-InsiP/isub5/sub and [iPP/i]subix/i/sub-InsiP/isubix/i/subiin vivo/i./p
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机译：> 裂殖酵母（Schizosaccharomyces pombe） Aps1是一种酶，可降解两种低聚磷酸二腺苷（Ap n A， n = 5或6）和二磷酸肌醇多磷酸盐（diphosphoinositol pentakisphosphate（ PP -Ins P 5 ）和双二磷酸肌醇四磷酸（[ PP ] 2 -Ins P 4 ）}体外。 Aps1的体内底物未知。我们在这里报告Ap 5 A， PP -Ins P 5 ，[ PP < / i>] 2 -Ins P 4 和新型二磷酸肌醇多磷酸酯（[ PP ] < i> x -Ins P x ）在 S中。使用HPLC方法测定pombe 。 Ap 5 A的含量为0.06pmol / mg蛋白质（约4nM）。 PP -Ins P 5 ，[ PP ] x -Ins P x 和[ PP ] 2 -Ins P 4 的含量为15 pmol/mg（约1.1 μ M），15pmol / mg（约1.1 μ M）和30 pmol/mg（约2.2 μ M），而Ins P 6 的细胞内浓度为0.5nmol / mg蛋白（约36μM）。 aps1 的破坏导致Ap 6 A水解酶活性体外降低52％，而细胞内Ap 5则没有可检测到的变化 A浓度和 PP -Ins P 5 和[ PP >] x -Ins P x 。 aps1 的破坏导致在30°C的最小或富媒体中形态或生长速率均未检测到变化。 aps1 的过表达是通过两个不同的质粒导致的，它们的野生型酶活性在体外分别比野生型酶高60％和6倍，导致细胞内[[< i> PP ] 2 -Ins P 4 ，[ PP ] x -Ins P x 或 PP -Ins P 5 ，但矛盾的增加了细胞内Ap 5 A浓度分别为2.5倍和55倍。 aps1 的过表达还导致生长速率降低和形态变化，包括肿胀，圆形和多分隔的细胞。 aps1 E93Q的过表达不会引起表型变化或细胞内Ap 5 A的变化，该基因编码的突变型Aps1缺乏明显的酶活性。我们得出结论，Aps1降解了 PP -Ins P 5 和[ PP ] x < / i> -Ins P x 体内。
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