Figure 4. The phs1 mutation in Sporobolomyces causes reduction in 3-hydroxyacyl-CoA dehydratase activity.

(A) Sporobolomyces (Sp. sp.) wild type (IAM 13481), mutant phs1 (GI209), ura5 auxotroph (AIS2), uracil prototroph phs1+ URA5 (AIS13), and complementing strain phs1 + PHS1 (AIS15) cells were grown in YPD medium at 25°C. As controls, S. cerevisiae (Sa. ce.) wild type (R1158) and pTetO₇-PHS1 (TH-3237) were grown in YPD medium containing 10 µg/ml doxycyclin for 6.5 h at 30°C. Total membranes (4 µg) prepared from the strains were incubated for 15 min at 37°C [¹⁴C]3-hydroxypalmitoyl-CoA (3.6 µM; 10 nCi/µl). After termination of the reactions, lipids were saponified, acidified, extracted, and separated by normal phase TLC. (B) The radioactivities associated with the reaction product 2,3-trans hexadecenoic acid were quantified using a bioimaging analyzer BAS-2500. Values represent the mean ± S.D. from three independent experiments. Statistically significant differences are indicated (**p<0.01; t-test).