Tracing Compartmentalized NADPH Metabolism in the Cytosol and Mitochondria of Mammalian Cells
Author(s)Lewis, Caroline A.; Parker, Seth J.; Fiske, Brian Prescott; McCloskey, Douglas; Gui, Dan Yi; Green, Courtney R.; Vokes, Natalie; Feist, Adam M.; Vander Heiden, Matthew G.; Metallo, Christian M.; Lewis, Caroline A.; ... Show more Show less
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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.
DepartmentDavid H. Koch Institute for Integrative Cancer Research at MIT; Massachusetts Institute of Technology. Department of Biology
Lewis, Caroline A., Seth J. Parker, Brian P. Fiske, Douglas McCloskey, Dan Y. Gui, Courtney R. Green, Natalie I. Vokes, Adam M. Feist, Matthew G. Vander Heiden, and Christian M. Metallo. "Tracing Compartmentalized NADPH Metabolism in the Cytosol and Mitochondria of Mammalian Cells" Molecular Cell 55:2 (July 2014), pp. 253-263.
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