Coustou Virginie, Besteiro Sébastien, Rivière Loïc, Biran Marc, Biteau Nicolas, Franconi Jean-Michel, Boshart Michael, Baltz Théo, Bringaud Frédéric
Laboratoire de Génomique Fonctionnelle des Trypanosomatides, UMR-5162 CNRS, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France.
J Biol Chem. 2005 Apr 29;280(17):16559-70. doi: 10.1074/jbc.M500343200. Epub 2005 Feb 17.
Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic stage of T. brucei expresses a soluble NADH-dependent fumarate reductase (FRDg) in the peroxisome-like organelles called glycosomes. This enzyme is responsible for the production of about 70% of the excreted succinate, the major end product of glucose metabolism in this form of the parasite. Here we functionally characterize a new gene encoding FRD (FRDm1) expressed in the procyclic stage. FRDm1 is a mitochondrial protein, as evidenced by immunolocalization, fractionation of digitonin-permeabilized cells, and expression of EGFP-tagged FRDm1 in the parasite. RNA interference was used to deplete FRDm1, FRDg, or both together. The analysis of the resulting mutant cell lines showed that FRDm1 is responsible for 30% of the cellular NADH-FRD activity, which solves a long standing debate regarding the existence of a mitochondrial FRD in trypanosomatids. FRDg and FRDm1 together account for the total NADH-FRD activity in procyclics, because no activity was measured in the double mutant lacking expression of both proteins. Analysis of the end products of 13C-enriched glucose excreted by these mutant cell lines showed that FRDm1 contributes to the production of between 14 and 44% of the succinate excreted by the wild type cells. In addition, depletion of one or both FRD enzymes results in up to 2-fold reduction of the rate of glucose consumption. We propose that FRDm1 is involved in the maintenance of the redox balance in the mitochondrion, as proposed for the ancestral soluble FRD presumably present in primitive anaerobic cells.
布氏锥虫是一种寄生原生生物，可导致人类昏睡病。布氏锥虫的前循环期在称为糖体的过氧化物酶体样细胞器中表达一种可溶性NADH依赖性延胡索酸还原酶（FRDg）。这种酶负责产生约70%的分泌琥珀酸，琥珀酸是这种寄生虫葡萄糖代谢的主要终产物。在这里，我们对前循环期表达的一种编码FRD的新基因（FRDm1）进行了功能表征。FRDm1是一种线粒体蛋白，免疫定位、洋地黄皂苷透化细胞分级分离以及寄生虫中EGFP标记的FRDm1的表达都证明了这一点。RNA干扰被用于耗尽FRDm1、FRDg或两者。对所得突变细胞系的分析表明，FRDm1负责细胞30%的NADH-FRD活性，这解决了关于锥虫线粒体中是否存在FRD的长期争论。FRDg和FRDm1共同构成了前循环期的总NADH-FRD活性，因为在缺乏两种蛋白质表达的双突变体中未检测到活性。对这些突变细胞系分泌的13C标记葡萄糖终产物的分析表明，FRDm1对野生型细胞分泌的琥珀酸的贡献在14%至44%之间。此外，一种或两种FRD酶的耗尽会导致葡萄糖消耗率降低多达2倍。我们提出，FRDm1参与维持线粒体中的氧化还原平衡，就像原始厌氧细胞中可能存在的祖先可溶性FRD一样。
