United States Department of Defense
Uniformed Services University of the Health Sciences
Date of this Version
1998
Abstract
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 nickelnitrilotriacetic 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.
Comments
Published in Journal of Biological Chemistry (1998) 273(46): pp. 30688-30694