FIGURE 5.

Substrate preference of Bar1p matches the properties of the Cer pool with dihydroxy LCB and C₁₆/C₁₈ fatty acids. A, the substrate preference of Bar1p for different LCBs and acyl-CoAs was tested by a ceramide synthase assay with microsomes prepared from a lag1Δlac1Δ S. cerevisiae strain expressing FLAG-tagged P. pastoris Bar1p as its only Cer synthase (incubation time, 5 min). Non-hydroxylated acyl-CoAs with different chain lengths are compared on the left, whereas the preference for α-hydroxylated versus non-hydroxylated C₁₈ acyl-CoA is shown in the middle. Sphing-4-enine was used as the LCB. On the right, the preference for the LCBs sphinganine, sphing-4-enine, and 4-hydroxysphinganine is shown in combination with non-hydroxylated C₁₈ acyl-CoA. In the upper row, the substrate specificity was tested using both a single LCB and a single acyl-CoA per reaction (one reaction per combination), whereas in the lower row, substrate selectivity was tested by offering mixtures of the LCBs or acyl-CoAs to be compared against each other (one reaction per panel). Shown are averages and S.D. from three independent reactions. B, comparison of Cer species from two different S. cerevisiae strains: left, a control strain expressing c-Myc-tagged S. cerevisiae Lag1p; right, a strain expressing FLAG-tagged P. pastoris Bar1p as the only ceramide synthase. The Cer species are grouped by the number of acyl carbons, assuming a C₁₈ (upper row of carbon numbers) or a C₂₀ LCB (lower row; see supplemental Fig. S2), and by the hydroxylation of the LCB (dihydroxy or trihydroxy). Hydroxylation of the fatty acid is not taken into account because there were no significant differences between the strains. Shown are averages and S.D. of three independent experiments. The mol % of the sum of all Cer species was calculated separately for each strain so that the chain length distribution and the hydroxylation pattern, but not the absolute amounts, can be compared between the strains. 100% corresponds to 15 ± 2 nmol/g fresh weight for the Lag1p-expressing strain and 68 ± 8 nmol/g fresh weight for the Bar1p-expressing strain.