Glucose metabolism is biochemically intertwined between energy metabolism and building block biosynthesis in living cells. However, it has not been investigated yet how its metabolic network is orchestrated to govern glucose flux in space and time. We demonstrate that glucosome assemblies behave like liquid droplets in human cells and thus reversibly respond to environmental changes. In addition, we characterize a molecular architecture of the glucosome, which appears to be constructed from higher-ordered oligomeric structures of its scaffolder enzyme along with transient enzyme-enzyme interactions. Importantly, we reveal that enzymatic compositions of glucosomes are altered when they are spatially in proximity to mitochondria to functionally couple glycolysis with mitochondrial metabolism in human cells. Collectively, we propose that the subcellular localization-function relationship between glucosomes and mitochondria represents one of fundamental principles by which 4-dimensional metabolic networks are not only dynamically but also efficiently regulated in living human cells.
