Biosensor reveals multiple sources for mitochondrial NAD⁺.
Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations.
Vollum Institute, Oregon Health & Science University, Portland, OR 97239, USA.
Xiaolu A
XA
Cambronne
Vollum Institute, Oregon Health & Science University, Portland, OR 97239, USA.
Melissa L
ML
Stewart
Vollum Institute, Oregon Health & Science University, Portland, OR 97239, USA.
DongHo
D
Kim
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, USA.
Amber M
AM
Jones-Brunette
Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR 97239, USA.
Rory K
RK
Morgan
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, USA.
David L
DL
Farrens
Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR 97239, USA.
Michael S
MS
Cohen
Vollum Institute, Oregon Health & Science University, Portland, OR 97239, USA.
Richard H
RH
Goodman
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D015374:Biosensing Techniques; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D009243:NAD / Q000032:analysis / Q000378:metabolism*; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000037:antagonists & inhibitors; D017354:Point Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D037761:Sirtuins / Q000378:metabolism
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D009243:NAD; D011065:Poly(ADP-ribose) Polymerases; C484008:NMNAT2 protein, human; C484009:NMNAT3 protein, human; D009612:Nicotinamide-Nucleotide Adenylyltransferase; D037761:Sirtuins; D011088:DNA Ligases; C120640:DNA ligase (NAD)