Figure 2.

Functional characterization of Arabidopsis gene At4g04930 as a sphingolipid Δ4-desaturase. A, Expression of Arabidopsis At4g04930 ORF in a P. pastoris mutant lacking sphingolipid Δ4-desaturase activity restores the synthesis of GlcCer. The P. pastoris mutant disrupted in the endogenous sphingolipid Δ4-desaturase AY700778 was transformed with either empty expression vector or vector containing the Arabidopsis ORF. Sphingolipids were extracted, separated by thin-layer chromatography, and stained by spraying with α-naphthol/sulfuric acid and subsequent heating to 160°C. The presence of GlcCer is clearly visible in P. pastoris mutants complemented with the Arabidopsis ORF (PpΔ4KO + At4g04930) but absent in mutants transformed with the empty vector (PpΔ4KO). For comparison, wild-type P. pastoris cells transformed with the empty vector are also accumulating GlcCer (PpWT). B, GlcCer from P. pastoris cells was isolated and subjected to sphingolipid LCB analysis via deacylation and derivatization with 1-fluoro-2,4-dinitrobenzene. LCBs were fractionated by HPLC and detected by A₃₅₀. GlcCers from wild-type P. pastoris transformed with the empty vector (top trace) and from the P. pastoris sphingolipid Δ4-desaturase mutant transformed with Arabidopsis ORF At4g04930 (bottom trace) both contain Δ4-unsaturated LCBs (predominantly in the form of the C9-methyl-sphinga-4,8-dienine). The P. pastoris mutant transformed with the empty vector does not contain any GlcCers (middle trace).