![]() Having identified robust protein–protein associations between phosphoglycerate mutase (PGAM), enolase, and pyruvate kinase, we demonstrated that 2-phosphoglyceric acid (2PGA), but not phosphoenolpyruvate (PEP), was fully channeled in the subset of the glycolytic enzymes, which were bound to the mitochondria. In the current study, we used contemporary molecular and cell biological techniques to study the interaction partners of the cytosolically localized glycolytic enzymes. However, it remains unclear how spatial colocalization of plant organelles is achieved or regulated. Following this theory, the exchange of metabolites between respiration and photosynthesis would be more efficient. It has also become apparent that membrane microdomains may be important for organelle trafficking, cellular signaling, and organelle tethering 16, 19, and it has been speculated that metabolite exchange between organelles could be regulated by these microdomains, which could involve direct channeling of molecules and synchronization without the necessity for cytosolic involvement 15, 18, 19. The direct interaction between mitochondrial and chloroplast outer membranes has been demonstrated using transmission electron micrographs (TEMs) of parenchyma cells from Citharexylum myrianthum 21. In addition to the clear metabolic intimacy of the organelles of the plant cell, mitochondria and chloroplasts have frequently been observed to spatially colocalize-a feature that has, among other considerations, been hypothesized to be important to ensure energy-use efficiency 16, 19, 20. The association of organelles has previously been observed and is often postulated to aid metabolic pathways that span organelles, such as photorespiration and nitrogen assimilation, and may also be beneficial for biochemical efficiency 15, 16, 17, 18. Serendipitously, we discovered evidence for a moonlighting role of these enzymes in bringing chloroplasts and mitochondria together. ![]() Here, we set out to perform a comprehensive characterization of these interactions. Despite the initial work being some 15 years old 7, 12, and in contrast to the well-characterized interactome of the plant TCA cycle (tricarboxylic acid cycle) 13, 14, glycolytic assemblies have not been well characterized in plants. Further important experiments revealed that the mitochondrially associated enzymes were capable of sustaining respiratory flux when supplied with isotopically labeled glucose 12 and that the association of the enzymes with mitochondria was dynamic and dependent on the respiratory demand of the cell 4, 7. In plants, as in microbes and mammals, glycolytic enzymes have been demonstrated to be physically associated with mitochondria 12. However, it is important to note that these studies remain somewhat controversial, with the argument being raised that the observations may be an artifact of the measurement method 11. Moreover, using fluorophore-tagged enzymes to follow movement in microfluidic devices, enzymes have been suggested to show chemotactic movement along their substrate gradient 11. The dynamics of glycolytic enzyme assemblies were recently followed using fluorescence resonance energy transfer (FRET) 8 and fluorescence recovery after photobleaching in living human cells 9, demonstrating that the first four enzymes of the glycolytic pathway each independently follows its own specific substrate gradient-providing an important insight into the potential mechanism of assembly of enzyme complexes 4, 9, 10. Recent studies in yeast 5, blood cells 6, and plants 7 demonstrate that glycolytic enzymes colocalize to areas on membranes where ATP is rapidly consumed, suggesting a regulatory role of such enzyme assembles 3, 4. ![]() ![]() Glycolytic enzymes are known to form multienzyme complexes in a broad range of species, yet the function of these assemblies remains unclear 3, 4. Glycolysis represents one of the hallmark pathways of respiration, providing carbon skeletons for the biosynthesis of a wide range of metabolites as well as being at the heart of energy transformations 1, 2.
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