Tracing Compartmentalized NADPH Metabolism in the Cytosol and Mitochondria of Mammalian Cells

• Published in: Molecular cell 2014-07, Vol.55 (2), p.253-263
• Main Authors: Lewis, Caroline A., Parker, Seth J., Fiske, Brian P., McCloskey, Douglas, Gui, Dan Y., Green, Courtney R., Vokes, Natalie I., Feist, Adam M., Vander Heiden, Matthew G., Metallo, Christian M.
• Format: Article
• Language: English
• Subjects: Cell Line, Tumor; Cytosol - metabolism; Glucose - metabolism; Glycine - metabolism; Humans; Isocitrate Dehydrogenase - metabolism; Metabolic Flux Analysis; Mitochondria - metabolism; NADP - metabolism; Serine - metabolism
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2014.05.008

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 location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. 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 these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells. [Display omitted] •Use of 2H stable isotopes to trace NAD(P)H metabolism in cells•Quantification of pentose phosphate pathway contribution to cytosolic NADPH•Reporter system to distinguish cytosolic and mitochondrial NADPH•Resolve direction of otherwise identical compartmentalized redox reactions NAD(P)H cofactors are not transported across cell membranes, with metabolic shuttles required for transport of reducing equivalents between the mitochondria and cytosol. Lewis et al. develop an approach to resolve NADPH-dependent pathways in these distinct metabolic compartments and show that directionality of serine/glycine interconversions can be determined within intact cells.