Metabolic studies on Saccharomyces cerevisiae containing fused citrate synthase/malate dehydrogenase.
Metabolic studies on Saccharomyces cerevisiae containing fused citrate synthase/malate dehydrogenase.
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DOI：
10.1021/bi00205a003
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发表时间：
1994-10
期刊：
影响因子：
2.9
通讯作者：
C. Lindbladh;R. Brodeur;W. Small;G. Lilius;L. Bülow;K. Mosbach;P. Srere
中科院分区：
文献类型：
--
作者：
C. Lindbladh;R. Brodeur;W. Small;G. Lilius;L. Bülow;K. Mosbach;P. Srere
我们构建了两种不同的融合蛋白，分别由CS1的C端与MDH1的N端和HSA的N端融合而成。在CS1和MDH1缺失的酿酒酵母突变株中表达融合蛋白，并对转化子的表型进行了鉴定。结果表明，融合蛋白被转运到线粒体中，并恢复了酵母突变株CS1-、MDH1-和CS1-/MDH1-在醋酸盐上生长的能力。对分离的线粒体中CS1活性的测定表明，表达天然CS1的菌株比亲本增加了10倍。在含有CS1/MDH1融合蛋白的转化子中，观察到亲本水平的CS1，而在表达CS1/HSA结合物的菌株中观察到五分之一的水平。对分离的线粒体的耗氧量研究表明，亲本类型的酵母与表达不同融合蛋白或天然CS1的菌株之间没有任何显著差异。[3(-13)C]丙酸用于研究含有CS1/MDH1融合载体的酵母细胞的Krebs TCA循环代谢。13C核磁共振研究在呼吸活性亲本酵母细胞中进行，并使用由表达天然CS1的CS1突变体和分别表达CS1/MDH1和CS1/HSA融合蛋白的基因工程酵母细胞组成。[3(-13)C]丙酸被认为在进入线粒体的TCA循环之前被代谢成[2(-13)C]琥珀酰辅酶A。然后，这种代谢物通过两个对称的中间体琥珀酸和富马酸被氧化，然后转化为苹果酸、草乙酸酯和其他代谢物，如丙氨酸。
We have constructed two different fusion proteins consisting of the C-terminal end of CS1 fused in-frame to the N-terminal end of MDH1 and HSA, respectively. The fusion proteins were expressed in mutants of Saccharomyces cerevisiae in which CS1 and MDH1 had been deleted and the phenotypes of the transformants characterized. The results show that the fusion proteins are transported into the mitochondria and that they restore the ability for the yeast mutants CS1-, MDH1-, and CS1-/MDH1- to grow on acetate. Determination of CS1 activity in isolated mitochondria showed a 10-fold increase for the strain that expressed native CS1, relative to the parental. In the transformant with CS1/MDH1 fusion protein, parental levels of CS1 were observed, while one-fifth this amount was observed for the strain expressing the CS1/HSA conjugate. Oxygen consumption studies on isolated mitochondria did not show any significant differences between parental-type yeast and the strains expressing the different fusion proteins or native CS1. [3(-13)C]Propionate was used to study the Krebs TCA cycle metabolism of yeast cells containing CS1/MDH1 fusion constructs. The 13C NMR study was performed in respiratory-competent parental yeast cells and using the genetically engineered yeast cells consisting of CS1- mutants expressing native CS1 and the fusion proteins CS1/MDH1 and CS1/HSA, respectively. [3(-13)C]Propionate is believed to be metabolized to [2(-13)C]succinyl-CoA before it enters the TCA cycle in the mitochondria. This metabolite is then oxidized through two symmetrical intermediates, succinate and fumarate, followed by conversion to malate, oxalacetate, and other metabolites such as alanine.(ABSTRACT TRUNCATED AT 250 WORDS)