Skrzypek M S, Nagiec M M, Lester R L, Dickson R C
Department of Biochemistry and Lucille P. Markey Cancer Center, University of Kentucky Medical Center, Lexington, Kentucky 40536-0298, USA.
J Bacteriol. 1999 Feb;181(4):1134-40. doi: 10.1128/JB.181.4.1134-1140.1999.
Sphingolipid long-chain bases and their phosphorylated derivatives, for example, sphingosine-1-phosphate in mammals, have been implicated as signaling molecules. The possibility that Saccharomyces cerevisiae cells also use long-chain-base phosphates to regulate cellular processes has only recently begun to be examined. Here we present a simple and sensitive procedure for analyzing and quantifying long-chain-base phosphates in S. cerevisiae cells. Our data show for the first time that phytosphingosine-1-phosphate (PHS-1-P) is present at a low but detectable level in cells grown on a fermentable carbon source at 25 degreesC, while dihydrosphingosine-1-phosphate (DHS-1-P) is only barely detectable. Shifting cells to 37 degreesC causes transient eight- and fivefold increases in levels of PHS-1-P and DHS-1-P, respectively, which peak after about 10 min. The amounts of both compounds return to the unstressed levels by 20 min after the temperature shift. These data are consistent with PHS-1-P and DHS-1-P being signaling molecules. Cells unable to break down long-chain-base phosphates, due to deletion of DPL1 and LCB3, show a 500-fold increase in PHS-1-P and DHS-1-P levels, grow slowly, and survive a 44 degreesC heat stress 10-fold better than parental cells. These and other data for dpl1 or lcb3 single-mutant strains suggest that DHS-1-P and/or PHS-1-P act as signals for resistance to heat stress. Our procedure should expedite experiments to determine how the synthesis and breakdown of these compounds is regulated and how the compounds mediate resistance to elevated temperature.
鞘脂长链碱及其磷酸化衍生物，比如哺乳动物中的1 - 磷酸鞘氨醇，已被认为是信号分子。酿酒酵母细胞也利用长链碱磷酸盐来调节细胞过程的可能性直到最近才开始被研究。在此，我们提出了一种简单且灵敏的方法，用于分析和定量酿酒酵母细胞中的长链碱磷酸盐。我们的数据首次表明，在25摄氏度以可发酵碳源培养的细胞中，植物鞘氨醇 - 1 - 磷酸（PHS - 1 - P）以低但可检测的水平存在，而二氢鞘氨醇 - 1 - 磷酸（DHS - 1 - P）仅勉强可检测到。将细胞转移至37摄氏度会分别导致PHS - 1 - P和DHS - 1 - P水平瞬时增加8倍和五倍，在约10分钟后达到峰值。温度转移后20分钟，这两种化合物的量均恢复到未受胁迫时的水平。这些数据与PHS - 1 - P和DHS - 1 - P作为信号分子一致。由于DPL1和LCB3缺失而无法分解长链碱磷酸盐的细胞，其PHS - 1 - P和DHS - 1 - P水平增加500倍，生长缓慢，并且在44摄氏度热胁迫下的存活能力比亲代细胞强10倍。dpl1或lcb3单突变菌株的这些及其他数据表明，DHS - 1 - P和/或PHS - 1 - P作为耐热胁迫的信号。我们的方法应能加快实验进程，以确定这些化合物的合成与分解是如何被调控的，以及这些化合物如何介导对高温的抗性。
