Phytosphingosine as a specific inhibitor of growth and nutrient import in Saccharomyces cerevisiae
Phytosphingosine as a specific inhibitor of growth and nutrient import in Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m105653200
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发表时间：
2001-09-21
影响因子：
4.8
通讯作者：
Obeid, LM
中科院分区：
文献类型：
--
作者：
Chung, NJ;Mao, CG;Obeid, LM
在酵母中，我们已经证明鞘脂通过抑制营养输入在热应激反应中发挥必要作用（Chung， N., Jenkins， G. M., Hannun， Y. a ., Heitman， J., and Obeid， L. M. (2000) J.生物化学，275,17229-17232）。在这项研究中，我们采用药理学和遗传学相结合的方法来确定哪种内源性鞘脂可能是生长抑制的介质。当细胞被外源性植物鞘氨酸（PHS, 20mum）或结构相似或代谢相关的分子（包括3-酮二氢鞘氨酸、二氢鞘氨酸、c -2-植物神经酰胺（PHC）和硬脂胺）处理时，只有PHS抑制细胞生长。此外，小灵通被证明可以抑制尿嘧啶、色氨酸、亮氨酸和组氨酸的摄取。同样，这种效果是小灵通所特有的。然而，由于鞘脂代谢的动态性，很难断定生长抑制是由小灵通本身引起的。通过使用鞘脂代谢各步骤缺陷的突变酵母菌株，我们进一步确定了PHS的特异性。elo2 δ菌株在将小灵通转化为PHC方面存在缺陷，其神经酰胺的生物合成速度较慢，对小灵通过敏（5mum），表明小灵通不需要转化为PHC。lcb4 δ lcb5 δ菌株在小灵通转化为小灵通1-磷酸方面存在缺陷，对小灵通的敏感性与野生型菌株相当。syr2 δ突变株在将DHS转化为PHS方面存在缺陷。有趣的是，该菌株对抑制等基因野生型菌株生长的高浓度DHS （40 muM）具有抗性，这表明DHS需要转化为PHS才能抑制生长。总之，这些数据表明抑制酵母生长的活性鞘脂是PHS或一种密切相关但尚未确定的代谢物。
In the yeast Saccharomyces cerevisiae, we have demonstrated a necessary role for sphingolipids in the heat stress response through inhibition of nutrient import (Chung, N., Jenkins, G. M., Hannun, Y. A., Heitman, J., and Obeid, L. M. (2000) J. Biol Chem. 275,17229-17232). In this study, we used a combination of pharmacological and genetic approaches to determine which endogenous sphingolipid is the likely mediator of growth inhibition. When cells were treated with exogenous phytosphin-gosine (PHS, 20 muM) or structurally similar or metabolically related molecules, including 3-ketodihydrosphin-gosine, dihydrosphingosine, C-2-phytoceramide (PHC), and stearylamine, only PHS inhibited growth. Also, PHS was shown to inhibit uptake of uracil, tryptophan, leucine, and histidine. Again this effect was specific to PHS. Because of the dynamic nature of sphingolipid metabolism, however, it was difficult to conclude that growth inhibition was caused by PHS itself. By using mutant yeast strains defective in various steps in sphingolipid metabolism, we further determined the specificity of PHS. The elo2 Delta strain, which is defective in the conversion of PHS to PHC, was shown to have slower biosynthesis of ceramides and to be hypersensitive to PHS (5 muM), suggesting that PHS does not need to be converted to PHC. The lcb4 Delta lcb5 Delta strain is defective in the conversion of PHS to PHS 1-phosphate, and it was as sensitive to PHS as the wild-type strain. The syr2 Delta mutant strain was defective in the conversion of DHS to PHS. Interestingly, this strain was resistant to high concentrations of DHS (40 muM) that inhibited the growth of an isogenic wild-type strain, demonstrating that DHS needs to be converted to PHS to inhibit growth. Together, these data demonstrate that the active sphingolipid species that inhibits yeast growth is PHS or a closely related and yet unidentified metabolite.