THE SACCHAROMYCES-CEREVISIAE TSC10 YBR265W GENE ENCODING 3-KETOSPHINGANINE REDUCTASE IS IDENTIFIED IN A SCREEN FOR TEMPERATURE-SENSITIVE SUPPRESSORS OF THE CA2+-SENSITIVE CSG2-DELTA MUTANT/

Saccharomyces cerevisiae csg2 Delta mutants accumulate the sphingolipi d inositolphosphorylceramide, which renders the cells Ca2+-sensitive. Temperature-sensitive mutations that suppress the Ca2+ sensitivity of csg2 Delta mutants were isolated and characterized to identify genes t hat encode sphingolipid synthesis enzymes. These temperature-sensitive csg2 Delta 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 phytosph ingosine, the long chain base found in yeast sphingolipids. 3-Ketosphi nganine reductase (Tsc10p) is essential for growth in the absence of e xogenous dihydrosphingosine or phytosphingosine, Tsc10p is a member of the short chain dehydrogenase/reductase protein family. The tsc10 mut ants accumulate 3-ketosphinganine and microsomal membranes isolated fr om tsc10 mutants have low 3-ketosphinganine reductase activity. His(6) -tagged Tsc10p was expressed in Escherichia coli and isolated by nicke l-nitrilotriacetic acid column chromatography. The recombinant protein catalyzes the NADPH-dependent reduction of 3-ketosphinganine. These d ata indicate that Tsc10p is necessary and sufficient for catalyzing th e NADPH-dependent reduction of 3-ketosphinganine to dihydrosphingosine .