Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays.
The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum of ABC transporters: We incubate extracts of mouse urine with membrane vesicles prepared from Spodoptera frugiperda Sf9 insect cells overproducing an ABC transporter and determine the compounds transported into the vesicles by LC/MS-based metabolomics. We illustrate the power of this simple "transportomics" approach using ABCC2, a protein present at sites of uptake and elimination. We identified many new substrates of ABCC2 in urine. These included glucuronides of plant-derived xenobiotics, a class of compounds to which humans are exposed on a daily basis. Moreover, we show that the excretion of these compounds in vivo depends on ABCC2: compared to wild-type mice, the urinary excretion of several glucuronides was increased up to 20-fold in Abcc2(-/-) mice. Transportomics has broad applicability, as it is not restricted to urine and can be applied to other ATP-dependent transport proteins as well.
Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Petra
P
Krumpochova
Sunny
S
Sapthu
Jos F
JF
Brouwers
Marcel
M
de Haas
Ric
R
de Vos
Piet
P
Borst
Koen
K
van de Wetering
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D001826:Body Fluids / Q000378:metabolism; D020719:Glucuronides / Q000378:metabolism / Q000652:urine; D006801:Humans; D007700:Kinetics; D055442:Metabolome; D051379:Mice; D018345:Mice, Knockout; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D048789:Phytoestrogens / Q000378:metabolism / Q000652:urine; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015262:Xenobiotics / Q000378:metabolism / Q000652:urine
D018528:ATP-Binding Cassette Transporters; D020719:Glucuronides; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D048789:Phytoestrogens; D011994:Recombinant Proteins; D015262:Xenobiotics; C000715087:ABCC2 protein, human