Identification and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme
Identification and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme
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DOI：
10.1128/jb.180.11.2875-2882.1998
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发表时间：
1998-06-01
影响因子：
3.2
通讯作者：
Pronk, JT
中科院分区：
文献类型：
--
作者：
Boles, E;de Jong-Gubbels, P;Pronk, JT
丙酮酸是几种氨基酸的前体，可以通过丙酮酸激酶由磷酸烯醇丙酮酸合成。然而，缺乏丙酮酸激酶活性的酿酒酵母的pyk 1 pyk 2突变体在限定培养基中的乙醇上正常生长，表明存在丙酮酸合成的替代途径。苹果酸酶是这一作用的候选者，它催化苹果酸氧化脱羧为丙酮酸。开放阅读框架YKL 029 c，这是同源的苹果酸酶基因从其他生物体的破坏，废除了苹果酸酶活性的葡萄糖生长的细胞提取物。相反，从MET 25启动子过表达YXL 029 c/MAE 1导致苹果酸酶活性增加高达33倍。对突变体的生长研究表明，Pyk 1 p或Mae 1 p的存在是在乙醇上生长所必需的。缺乏这两种酶的突变体可以通过向乙醇培养物中加入丙氨酸或丙酮酸来拯救。单独破坏MAE 1不会导致明确的表型。通过测定野生型培养物中的酶活性和MAE 1 mRNA水平以及通过测量携带MAE 1：：lacZ融合物的菌株中的β-半乳糖苷酶活性来研究MAE 1的调节。在摇瓶培养物和碳限制恒化器培养物中，MAE 1均组成型表达。亚细胞分离实验表明，苹果酸酶在葡萄糖厌氧生长过程中有3 ~ 4倍的诱导作用。酿酒酵母是一种线粒体酶。其调节和定位表明在厌氧生长条件下提供线粒体内NADPH或丙酮酸的作用。然而，由于无效突变体仍然可以厌氧生长，这种功能显然不是必需的。
Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase, Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression of YXL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol, Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring beta-galactosidase activities in a strain carrying a MAE1::lacZ fusion, Both in shake flask cultures and in carbon-limited chemostat cultures, MAE1 was constitutively expressed. A three-to fourfold induction was observed during anaerobic growth on glucose, Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.