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

• Main Authors: Lewis, Caroline A (Author), Parker, Seth J (Author), Fiske, Brian Prescott (Contributor), McCloskey, Douglas (Author), Gui, Dan Yi (Contributor), Green, Courtney R. (Author), Feist, Adam M. (Author), Vander Heiden, Matthew G. (Contributor), Metallo, Christian M. (Author), Vokes, Natalie I. (Author), Lewis, Caroline (Author)
• Other Authors: Massachusetts Institute of Technology. Department of Biology (Contributor), Koch Institute for Integrative Cancer Research at MIT (Contributor), Lewis, Caroline A. (Contributor), Vokes, Natalie (Contributor)
• Format: Article
• Language: English
• Published: Elsevier, 2016-08-15T17:24:40Z.
• Subjects: Article
• Online Access: Get fulltext

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.
National Institutes of Health (U.S.) (NIH grant P30CA147882)
National Institutes of Health (U.S.) (NIH grant U54- CA121852-09)
National Institutes of Health (U.S.) (NIH grant R01CA168653)
David H. Koch Institute for Integrative Cancer Research at MIT (Koch Institute/DFHCC Bridge Project)
David H. Koch Institute for Integrative Cancer Research at MIT (Koch Institute Frontier Research)
Burroughs Wellcome Fund
Damon Runyon Cancer Research Foundation
Kathy and Curt Marble Cancer Research Fund
American Cancer Society (grant IRG #70-002)
United States. Department of Defense (DOD grant W81XWH-13-1-0105)
University of California, San Diego (University of California Cancer Research Coordinating Committee grant)
Searle Scholars Program (Award)