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
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
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DOI：
10.1074/jbc.273.46.30688
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发表时间：
1998-11-13
影响因子：
4.8
通讯作者：
Dunn, T
中科院分区：
文献类型：
--
作者：
Beeler, T;Bacikova, D;Dunn, T
酿酒酵母csg 2 δ突变体积累鞘脂肌醇磷酸神经酰胺，这使得细胞Ca 2+敏感。分离抑制csg 2 Delta突变体的Ca 2+敏感性的温度敏感性突变，并鉴定编码鞘脂合成酶的基因。这些温度敏感的csg 2 Delta抑制子（tsc）分为15个互补组。发现TSC 10/YBR 265 w基因编码3-酮鞘氨醇还原酶，该酶催化植物鞘氨醇合成的第二步，植物鞘氨醇是酵母鞘脂中发现的长链碱基。3-酮鞘氨醇还原酶（Tsc 10 p）是在缺乏外源性二氢鞘氨醇或植物鞘氨醇的情况下生长所必需的，Tsc 10 p是短链脱氢酶/还原酶蛋白家族的成员。tsc 10突变体积累3-酮基鞘氨醇，从tsc 10突变体分离的微粒体膜具有低的3-酮基鞘氨醇还原酶活性。His（6）标记的Tsc 10 p在大肠杆菌中表达，并通过镍-次氮基三乙酸柱层析分离。重组蛋白催化NADPH依赖性还原3-酮鞘氨醇。这些数据表明，Tsc 10 p是必要的，足以催化NADPH依赖性还原3-酮鞘氨醇二氢鞘氨醇。
Saccharomyces cerevisiae csg2 Delta mutants accumulate the sphingolipid 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 that 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 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. His(6)-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.