title: Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells.
abstract: 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.
authors.affiliation: The Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Caroline A
authors.initials: CA
authors.lastname: Lewis
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Seth J
authors.initials: SJ
authors.lastname: Parker
authors.affiliation: The Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Fiske
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Douglas
authors.initials: D
authors.lastname: McCloskey
authors.affiliation: The Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Dan Y
authors.initials: DY
authors.lastname: Gui
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Courtney R
authors.initials: CR
authors.lastname: Green
authors.affiliation: The Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Natalie I
authors.initials: NI
authors.lastname: Vokes
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Feist
authors.affiliation: The Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Dana-Farber Cancer Institute, Boston, MA 02115, USA. Electronic address: mvh@mit.edu.
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Vander Heiden
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA; Institute of Engineering and Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address: cmetallo@ucsd.edu.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Metallo
mesh_terms: D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D064688:Metabolic Flux Analysis; D008928:Mitochondria / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
chemical_list: D012694:Serine; D009249:NADP; C538784:IDH2 protein, human; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human; D005947:Glucose; D005998:Glycine