Mao C, Wadleigh M, Jenkins G M, Hannun Y A, Obeid L M
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1997 Nov 7;272(45):28690-4. doi: 10.1074/jbc.272.45.28690.
We have identified the yeast sphingosine resistance gene (YSR2) of Saccharomyces cerevisiae as encoding a protein that specifically dephosphorylates dihydrosphingosine 1-phosphate (DHS-1-P), and we refer to this protein as dihydrosphingosine-1-phosphate phosphatase. Overexpression of YSR2 conferred sphingosine resistance to the dihydrosphingosine-1-P lyase-defective mutant (JS16) of S. cerevisiae, which is hypersensitive to sphingosine. The ysr2Delta deletion mutant of S. cerevisiae accumulated DHS-1-P compared with its wild type strain upon labeling with D-erythro-[4, 5-3H]dihydrosphingosine, whereas overexpression of YSR2 increased dephosphorylation of DHS-1-P. An epitope-tagged fusion protein (YSR2-Flag) was partially purified and found to specifically dephosphorylate DHS-1-P to yield dihydrosphingosine. YSR2 failed to dephosphorylate ceramide 1-phosphate or phosphatidic acid. Functionally, the mutant bearing the ysr2Delta deletion decreased labeling of sphingolipids and increased labeling of glycerolipids dramatically following in vivo labeling with D-erythro-[3H]dihydrosphingosine, but it slightly affected labeling of sphingolipids with inositol. Taken together, these results identify YSR2 as dihydrosphingosine-1-phosphate phosphatase. They also raise the intriguing possibility that phosphorylation followed by dephosphorylation is required for incorporation of exogenous long chain sphingoid bases into sphingolipids.
我们已确定酿酒酵母的酵母鞘氨醇抗性基因（YSR2）编码一种能特异性使二氢鞘氨醇1-磷酸（DHS-1-P）去磷酸化的蛋白质，我们将此蛋白质称为二氢鞘氨醇-1-磷酸磷酸酶。YSR2的过表达赋予酿酒酵母对二氢鞘氨醇-1-P裂解酶缺陷型突变体（JS16）鞘氨醇抗性，该突变体对鞘氨醇高度敏感。酿酒酵母的ysr2Delta缺失突变体在用D-赤藓糖-[4,5-3H]二氢鞘氨醇标记后，与其野生型菌株相比积累了DHS-1-P，而YSR2的过表达增加了DHS-1-P的去磷酸化。一种表位标记的融合蛋白（YSR2-Flag）被部分纯化，并发现能特异性使DHS-1-P去磷酸化生成二氢鞘氨醇。YSR2不能使神经酰胺1-磷酸或磷脂酸去磷酸化。在功能上，携带ysr2Delta缺失的突变体在用D-赤藓糖-[3H]二氢鞘氨醇进行体内标记后，显著降低了鞘脂的标记并增加了甘油脂的标记，但对含肌醇鞘脂的标记影响较小。综上所述，这些结果确定YSR2为二氢鞘氨醇-1-磷酸磷酸酶。它们还提出了一个有趣的可能性，即外源长链鞘氨醇碱基掺入鞘脂需要先磷酸化然后去磷酸化。
