Preparation of S. cerevisiae cells for in vivo transport assays and fluorescence imaging

S. cerevisiae cells were grown in synthetic uracil and lysine dropout medium containing; 2% (w/v) glucose or raffinose, 0.67% (w/v) yeast nitrogen base without amino acids, 2 gr/L drop-out lysine, uracil Kaiser mix. Constitutive expression of Lyp1 from pFB021 and Lyp1(62-590) from pFB022 was done in BY4742 or BY4742 ∆lys1, ∆vba1, ∆vba2 strains, which were cultivated in the presence of glucose and absence of lysine and uracil and supplemented with 800 mg/L lysine-lysine dipeptide. BY4742 or BY4742 ∆lyp1 strains carrying plasmids with a Gal promoter were grown in the presence of raffinose with 800 mg/L lysine-lysine dipeptide or 69 mg/L lysine, were induced with 0.2% (w/v) galactose for 2 hours prior to the transport assay or fluorescence imaging. For the determination of the K_m for Lyp1 in vivo Lyp1-YPet was constitutively expressed from pFB018 in the 22∆6AAL⁵ strain and cultivated in the presence of glucose and 200 mg/L lysine-lysine dipeptide and absence of lysine and uracil. Cultures were grown at 30 °C with 180 RPM shaking. Subcultures were grown for two-to-three consecutive days and never exceeded an OD₆₀₀ of 1. Cells were centrifuged at 3,000 × g for 5 min at 4 °C, supernatant was decanted and cells were suspended in ice-cold 100 mM potassium phosphate, 10 mM glucose, pH 6.0. This step was performed twice before suspension of the cells to an OD₆₀₀ of 5.