Glycolytic Enzymes Associate Dynamically with Mitochondria in Response to Respiratory Demand and Support Substrate Channeling

Abstract

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using 13C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from 13C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.

Reactive Oxygen Species Production by Potato Tuber Mitochondria Is Modulated by Mitochondrially Bound Hexokinase Activity

“…In this work we observed mt-HK activity (Table I) in isolated PTM, in agreement with a recent report (Graham et al, 2007). The mt-HK activity found in PTM is highly dependent on oxygen consumption, because the activation of mitochondrial respiration by succinate restores the activity levels of the enzyme (Fig.…”

“…Previous studies have shown that HK is bound to mitochondria in mammalian tissues, in different plant species (Galina et al, 1995;da-Silva et al, 2001;Wilson, 2003;Meyer et al, 2006;Claeyssen and Rivoal, 2007), and even in PTM (Graham et al, 2007). To check whether a particulate HK activity from potato tuber is also associated with this organelle, in Table I we determined the activities of HK, oligomycin-sensitive F 0 F 1 ATPase, a mitochondrial enzyme, and Glc-6-P dehydrogenase (G6PDH), a cytosolic and plastid enzyme (Dennis and Green, 1975;Miernyk and Dennis, 1983) in different fractions of potato tuber homogenates.…”

“…Recently, an authentic HK activity was detected in PTM (Graham et al, 2007) and its involvement in potato tuber glycolysis suggested, but its involvement in PTM ROS generation was not explored. We then raise the hypothesis that HK bound to PTM would contribute to produce a steady-state ADP recycling that regulates ROS formation.…”

Linking Post-Translational Modifications and Variation of Phenotypic Traits

“…Such constraints may be related to the existence of macromolecular complexes associating enzymes belonging to the same metabolic pathway. These so-called metabolons (59) were described for glycolytic enzymes in several organisms ranging from plants (60,61), to human (62,63) and yeast (64). Metabolon allows passing (channeling) the intermediary metabolites from one enzyme to the consecutive one within a given metabolic pathway, forming a metabolite tunnel.…”

Spatial Distribution of Cellular Function: The Partitioning of Proteins between Mitochondria and the Nucleus in MCF7 Breast Cancer Cells

“…While direct investigation of possible nuclear functions for other individual glycolytic enzymes detected in the present experiments is now desirable, we conclude that there is already a substantial body of corroborating evidence showing that a majority of glycolytic enzymes can be present and have important functions in the nucleus (see discussion). Apart from hexose kinases HK1 and HK2, [77][78][79] the presence of glycolytic enzymes in mitochondria has been less researched, but we note evidence that in cancer cells glycolytic enzymes may form large cytosolic complexes 73 and that association of large protein complexes that include glycolytic enzymes with yeast and plant mitochondria have been reported, [80][81][82] that membrane association of glycolytic enzymes has been found in pancreatic cancer cells, 83 and that many glycolytic enzymes have been reported to concentrate in the MAM contacts between mitochondria and the endoplasmic reticulum after viral infection. 84 Overall, there are very substantial indications that even for nominally cytosolic glycolytic proteins the present methods reflect functionally important subcellular protein distributions.…”