Figure 8.

Pry function is essential in cells that export fatty acids. A, wild-type, quadruple (pry1Δ pry2Δ faa1Δ faa4Δ), and quintuple (pry1Δ pry2Δ pry3Δ faa1Δ faa4Δ) mutant cells bearing a plasmid-borne copy of FAA1 (pFaa1) were serially diluted 10-fold and stamped on YPD, SC-URA, or 5-fluoroorotic acid (5-FOA) containing solid media. Plates were incubated at 30 °C for 3 days. B, transcriptional shut off of PRY1 results in a time-dependent increase of intracellular fatty acids. Quintuple mutant cells (pry1Δ pry2Δ pry3Δ faa1Δ faa4Δ) bearing a plasmid-borne copy of PRY1 under the transcriptional control of a galactose-inducible promoter (pGal-PRY1) were cultivated in galactose-containing media and diluted into fresh media containing either glucose (Glu) or galactose (Gal). Cells were cultivated for the indicated period of time and intracellular fatty acid levels were quantified by GC-MS. C, export of fatty acids is arrested upon shut off of PRY1 transcription. Cells were cultivated as described for panel B and the ratio of extracellular fatty acids to that of the sum of both intracellular and extracellular fatty acid is shown as an export index. D, blocking fatty acid synthesis rescues growth of cells lacking Pry function. Quintuple mutant cells (pry1Δ pry2Δ pry3Δ faa1Δ faa4Δ) bearing the glucose repressible pGal-PRY1 were cultivated in media containing galactose (Gal), glucose (Glu), or glucose and the fatty acid synthase inhibitor cerulenin (Cer) (0.1 μg/ml). Cell density over time was recorded using a bioimager. The growth curves shown are representative of three independent experiments. Values represent means ± S.D. of three independent determinations.