Roboinject

All of the DNA constructs were built using the USER fusion technique. In vitro transcribed cRNAs were injected into the X. laevis oocytes by RoboInject (Multi Channel Systems, Germany). Candidates of membrane transporters were expressed in the S. cerevisiae cell factory designed to produce malic acid. A Leica TCS SP5-II confocal microscope was used for localization studies. HMMER version 3.1b2 and Phyre2 were used for protein motif identification and structure predictions, respectively. Chemicals were quantified by HPLC and LC-MS. Detailed experimental procedures can be found in SI Appendix, Supplementary Materials and Methods and Tables S1–S5.

Oocytes were prepared and injected at stage V-VI with 20 ng of cRNA encoding SLC2A9b. In brief, animals were anesthetized by cooling at 4°C with tricaine mesylate (3-aminobenzoic acid ethyl ester, methane sulfonate salt, 150 mg/l). Small pieces of ovary were isolated in sterile Barth solution (10 mM HEPES pH 7.4, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂, and 0.41 mM CaCl₂, supplemented with 50 µg/ml gentamycin). Oocyte defolliculation occurred in calcium-free modified Barth's solution with 3 mg/ml collagenase NB4 (SERVA Electrophoresis, Heidelberg, Germany). Isolated oocytes were then incubated overnight in standard Barth's solution. After 24 hours, injection of cRNA was performed in at least 1,500 oocytes using an automated injection device (Roboinject, Multi Channel Systems, Reutlingen, Germany). Oocytes were maintained in Barth's Solution for 3 days at 18°C to maximize expression for functional and biochemical studies.
Oocytes were solubilized and crudely homogenized using a 100 µl pipette tip with ice-cold RIPA lysis buffer as described previously [17] (link). After 45 min on ice, samples were centrifuged at 15,000 g (15 min at 4°C). Only the supernatant below the yolk was taken for SDS-PAGE and Western blot analysis.