Abstract
Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by spectrofluorometric quantification. These techniques are, however, limited to substrates that are available as radiolabeled isotopes or to when the substrate is fluorescent. This prompted us to develop a transport assay where we could in principle detect transport activity for any organic metabolite regardless of its availability as radiolabeled isotope or fluorescence properties. In this protocol we describe the use of X. laevis oocytes as a heterologous host for expression of secondary active transporters and how to perform uptake assays followed by detection and quantification of transported metabolites by liquid chromatography-mass spectrometry (LC-MS). We have successfully used this method for identification and characterization of transporters of the plant defense metabolites called glucosinolates and cyanogenic glucosides ( Jørgensen et al., 2017 ), however the method is usable for the characterization of any transporter whose substrate can be detected by LC-MS.
Highlights
In this protocol we describe the use of X. laevis oocytes as a heterologous host for expression of secondary active transporters and how to perform uptake assays followed by detection and quantification of transported metabolites by liquid chromatography-mass spectrometry (LC-MS)
For identification and characterization of plant specialized metabolite transporters, it can be very challenging to generate radiolabeled isotopes of a target substrate. To overcome this challenge we developed a protocol for detecting and quantifying transport of specialized metabolites into X. laevis oocytes by use of LC-MS
We plot the analyte concentration as a function of the signal intensity from the LC-MS and calculate the linear equation to describe the relationship between these two values (Crocoll et al, 2016)
Summary
1. Pipette filter tips (e.g., Biotix, catalog numbers: M-0012-9FC, M0020-9FC, M-0300-9FC, M1250-9FC96). 2. Petri dishes for oocyte washing (e.g., 90 mm diameter, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 263991). 3. 24-well NuncTM cell-culture dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 142475). 1.5 ml tubes (e.g., 1.5 ml microfuge tubes, ‘Easy Fit’, Almeco, catalog number: 02.023.01001) 6. THALIANA GLUCOSINOLATE TRANSPORTER-1 (AtGTR1) (UniProt, catalog number: Q944G5), is used as an example here. Methanol for HPLC ≥99.9% (Sigma-Aldrich, catalog number: 34860) 11. Acetonitrile for HPLC (Sigma-Aldrich, catalog number: 34851) 13. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670) 16. Calcium chloride dihydrate (CaCl2·2H2O) (EMD Millipore, catalog number: 1.02382) 17. Tris-HCl solutions (Trisma hydrochloride) (Sigma-Aldrich, catalog number: T3253) 19.
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