Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells
Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells
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DOI：
10.1105/tpc.012500
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发表时间：
2003-09-01
期刊：
影响因子：
11.6
通讯作者：
Sweetlove, LJ
中科院分区：
文献类型：
--
作者：
Giegé, P;Heazlewood, JL;Sweetlove, LJ
线粒体除了合成三磷酸腺苷外，还具有广泛的代谢功能，并含有多种蛋白质来执行这些功能。在这里，我们提出了一个意想不到的发现，在拟南芥细胞的线粒体部分中存在糖酵解酶。对这一线粒体片段的蛋白质组学分析显示，构成糖酵解途径的10种酶中有7种。其中四种酶(甘油醛-3-P脱氢酶、醛缩酶、磷酸甘油酸变位酶和烯醇化酶)也存在于线粒体膜间隙/外膜部分。酶活性分析证实，整个糖酵解途径存在于分离的拟南芥线粒体中，并且这些活性对蛋白酶处理的敏感性表明，糖酵解酶存在于线粒体的外部。通过在拟南芥原生质体中表达烯醇化酶和醛缩酶黄色荧光蛋白的融合，在体内证实了糖酵解酶与线粒体的关联。黄色荧光蛋白荧光信号显示，这两种融合蛋白存在于整个细胞质中，但也集中在与线粒体特异性探针MitoTracker Red共定位的点状区域。此外，当提供适当的辅因子时，分离的完整线粒体能够将C-13-葡萄糖代谢成三氯乙酸循环的C-13标记的中间产物，这表明完整的糖酵解序列存在于这个亚细胞部分中并具有活性。在这些数据的基础上，我们认为整个糖酵解途径是通过附着在植物线粒体外膜的胞液表面而与植物线粒体联系在一起的，这种糖酵解的微区划允许丙酮酸直接提供给线粒体，在线粒体中用作呼吸底物。
Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase- and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of C-13-glucose to C-13-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.