The Saccharomyces cerevisiae TSC10/YBR265W gene encoding 3- ketosphinganine reductase is identified in a screen for temperature-sensitive suppressors of the Ca²⁺-sensitive csg2Δ mutant

Saccharomyces cerevisiae csg2Δ mutants accumulate the sphingolipid inositolphosphorylceramide, which renders the cells Ca²⁺-sensitive. Temperature-sensitive mutations that suppress the Ca²⁺ sensitivity of csg2Δ mutants were isolated and characterized to identify genes that encode sphingolipid synthesis enzymes. These temperature-sensitive csg2Δ suppressors (tsc) fall into 15 complementation groups. The TSC10/YBR265w gene was found to encode 3-ketosphinganine reductase, the enzyme that catalyzes the second step in the synthesis of phytosphingosine, the long chain base found in yeast sphingolipids. 3-Ketosphinganine reductase (Tsc10p) is essential for growth in the absence of exogenous dihydrosphingosine or phytosphingosine. Tsc10p is a member of the short chain dehydrogenase/reductase protein family. The tsc10 mutants accumulate 3- ketosphinganine and microsomal membranes isolated from tsc10 mutants have low 3-ketosphinganine reductase activity. His₆-tagged Tsc10p was expressed in Escherichia coli and isolated by nickel-nitrilotriacetic acid column chromatography. The recombinant protein catalyzes the NADPH-dependent reduction of 3-ketosphinganine. These data indicate that Tsc10p is necessary and sufficient for catalyzing the NADPH-dependent reduction of 3- ketosphinganine to dihydrosphingosine.