Tools to Resolve Compartmentalized Metabolism in Mammalian Cells

Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective subcellular location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods.  To address this need we have developed an approach to resolve NADP(H)-dependent pathways in subcellular compartments within intact mammalian cells. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in distinct cellular locations. Using this system we determine the direction of several metabolic cycles that are channelled between the mitochondria and cytosol, highlighting the ability of this approach to resolve compartment-specific redox reactions in intact cells.