Regev-Rudzki Neta, Karniely Sharon, Ben-Haim Nitzan Natani, Pines Ophry
Department of Molecular Biology, Hebrew University Medical School, Jerusalem 91120, Israel.
Mol Biol Cell. 2005 Sep;16(9):4163-71. doi: 10.1091/mbc.e04-11-1028. Epub 2005 Jun 22.
The distribution of identical enzymatic activities between different subcellular compartments is a fundamental process of living cells. At present, the Saccharomyces cerevisiae aconitase enzyme has been detected only in mitochondria, where it functions in the tricarboxylic acid (TCA) cycle and is considered a mitochondrial matrix marker. We developed two strategies for physical and functional detection of aconitase in the yeast cytosol: 1) we fused the alpha peptide of the beta-galactosidase enzyme to aconitase and observed alpha complementation in the cytosol; and 2) we created an ACO1-URA3 hybrid gene, which allowed isolation of strains in which the hybrid protein is exclusively targeted to mitochondria. These strains display a specific phenotype consistent with glyoxylate shunt elimination. Together, our data indicate that yeast aconitase isoenzymes distribute between two distinct subcellular compartments and participate in two separate metabolic pathways; the glyoxylate shunt in the cytosol and the TCA cycle in mitochondria. We maintain that such dual distribution phenomena have a wider occurrence than recorded currently, the reason being that in certain cases there is a small fraction of one of the isoenzymes, in one of the locations, making its detection very difficult. We term this phenomenon of highly uneven isoenzyme distribution "eclipsed distribution."
相同的酶活性在不同亚细胞区室之间的分布是活细胞的一个基本过程。目前，酿酒酵母乌头酸酶仅在线粒体中被检测到，它在三羧酸（TCA）循环中发挥作用，被认为是线粒体基质标记物。我们开发了两种在酵母细胞质中对乌头酸酶进行物理和功能检测的策略：1）我们将β-半乳糖苷酶的α肽与乌头酸酶融合，并在细胞质中观察到α互补；2）我们创建了一个ACO1-URA3杂交基因，该基因允许分离出杂交蛋白仅靶向线粒体的菌株。这些菌株表现出与乙醛酸循环消除一致的特定表型。我们的数据共同表明，酵母乌头酸酶同工酶分布在两个不同的亚细胞区室之间，并参与两条独立的代谢途径；细胞质中的乙醛酸循环和线粒体中的TCA循环。我们认为这种双重分布现象的发生比目前记录的更为广泛，原因是在某些情况下，其中一种同工酶在其中一个位置的含量很少，使得其检测非常困难。我们将这种同工酶分布高度不均的现象称为“重叠分布”。
