Chung N, Mao C, Heitman J, Hannun Y A, Obeid L M
Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2001 Sep 21;276(38):35614-21. doi: 10.1074/jbc.M105653200. Epub 2001 Jul 23.
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 phytosphingosine (PHS, 20 microm) or structurally similar or metabolically related molecules, including 3-ketodihydrosphingosine, 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 elo2Delta 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 microm), suggesting that PHS does not need to be converted to PHC. The lcb4Delta lcb5Delta 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 syr2Delta mutant strain was defective in the conversion of DHS to PHS. Interestingly, this strain was resistant to high concentrations of DHS (40 microm) 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.
在酿酒酵母中，我们已经证明了鞘脂类物质通过抑制营养物质的摄取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. Biol. Chem. 275, 17229 - 17232）。在本研究中，我们使用药理学和遗传学方法相结合来确定哪种内源性鞘脂类物质可能是生长抑制的介质。当用外源性植物鞘氨醇（PHS，20微摩尔）或结构相似或代谢相关的分子处理细胞时，包括3 - 酮二氢鞘氨醇、二氢鞘氨醇、C(2) - 植物神经酰胺（PHC）和硬脂胺，只有PHS抑制生长。此外，PHS被证明能抑制尿嘧啶、色氨酸、亮氨酸和组氨酸的摄取。同样，这种作用对PHS具有特异性。然而，由于鞘脂类代谢的动态性质，很难得出生长抑制是由PHS本身引起的结论。通过使用在鞘脂类代谢的各个步骤中存在缺陷的突变酵母菌株，我们进一步确定了PHS的特异性。在PHS转化为PHC过程中存在缺陷的elo2Delta菌株，其神经酰胺的生物合成较慢，并且对PHS（5微摩尔）高度敏感，这表明PHS不需要转化为PHC。lcb4Delta lcb5Delta菌株在PHS转化为PHS 1 - 磷酸的过程中存在缺陷，并且它对PHS的敏感性与野生型菌株相同。syr2Delta突变菌株在二氢鞘氨醇（DHS）转化为PHS的过程中存在缺陷。有趣的是，该菌株对抑制同基因野生型菌株生长的高浓度DHS（40微摩尔）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类物质是PHS或一种密切相关但尚未确定的代谢产物。
