Choline Metabolism Profiling in Yeast

Strains were grown to log phase (A_600nm ≈ 0.8) in YNB medium, at which point ¹⁴C-choline-GPC (≅100,000 cpm/ml) (American Radiolabeled Chemicals 3880) was added. After 1 h of growth in the presence of label, cultures were harvested and the cell pellets were treated with 5% TCA for 20 min on ice. The suspension was pelleted, and aliquots of both the pellet (containing membrane) and the water-soluble TCA extract were subjected to LSC using a Tri-Carb 4910 TR liquid scintillation analyzer (PerkinElmer) as described (13 (link)). Lipids were extracted from the membrane fraction and subject to TLC followed by phosphoimager to confirm PC as the sole labeled lipid as described (15 (link)). Choline-containing metabolites were separated using anion exchange chromatography as described (62 (link)).