Fig. 2. Sply encodes the Drosophila sphingosine-1-phosphate lyase. (A) CLUSTALW alignment of Sply. The predicted protein product of Sply is 49% and 43% identical and 68% and 60% similar to human and yeast SPL protein sequences respectively. The broken line indicates the putative transmembrane region. The unbroken line indicates a consensus pyridoxal phosphate-binding motif. (B) Sply gene organization. Open reading frames GH13783 and LP04413 and location of transposon insertion (P{PZ}Sply⁰⁵⁰⁹¹) are indicated. (C) Overexpression of Sply in a Saccharomyces cerevisiae SPL mutant restores sphingosine resistance. The LP04413 and GH13783 cDNAs were cloned into yeast expression vector pYES2 and transformed into a yeast SPL mutant strain (dpl1), as described in the Materials and Methods. The transformed strain (dpl1 + Sply) was compared with wild type (DPL1) and SPL mutant overexpressing endogenous yeast SPL (dpl1 + DPL1) strains in a sphingosine resistance assay. Dilutions of saturated cultures for each strain are indicated above. (D) Expression of Sply in a Saccharomyces cerevisiae SPL mutant restores SPL enzyme activity. Whole cell extracts of Saccharomyces cerevisiae wild type, SPL mutant (dpl1) and SPL mutant overexpressing Sply (dpl1 + Sply) strains were analyzed for SPL activity.