Jenkins G M, Hannun Y A
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29482, USA.
J Biol Chem. 2001 Mar 16;276(11):8574-81. doi: 10.1074/jbc.M007425200. Epub 2000 Oct 30.
The recent findings of sphingolipids as potential mediators of yeast heat stress responses led us to investigate their possible role in the heat-induced cell cycle arrest and subsequent recovery. The sphingolipid-deficient yeast strain 7R4 was found to lack the cell cycle arrest seen in the isogenic wild type. Furthermore, strain lcb1-100, which harbors a temperature-sensitive serine palmitoyltransferase, lacked increased de novo generated sphingoid bases upon heat stress. Importantly, this strain was found to lack the transient heat-induced G0/G1 arrest. These results indicate a role for sphingolipids and specifically those generated in the de novo pathway in the cell cycle arrest response to heat. To determine the bioactive sphingolipid regulating this response, an analysis of key mutants in the sphingolipid biosynthetic and degradation pathways was performed. Strains deleted in sphingoid base kinases, sphingoid phosphate phosphatase, lyase, or dihydrosphingosine hydroxylase were found to display the cell cycle arrest. Also, the knockout of a fatty acyl elongation enzyme, which severely attenuates ceramide production, displayed the arrest. These experiments suggested that the active species for cell cycle arrest were the sphingoid bases. In further support of these findings, exogenous phytosphingosine (10 microM) was found to induce transient arrest. Stearylamine did not induce an arrest, demonstrating chemical specificity, and L-erythro- was not as potent as D-erythro-dihydrosphingosine showing stereospecificity. To investigate a possible arrest mechanism, we studied the hyperstable Cln3 (Cln3-1) strain LDW6A that has been previously shown to be resistant to heat stress-induced cell cycle arrest. The strain containing Cln3-1 was found to be resistant to cell cycle arrest induced by exogenous phytosphingosine, indicating that Cln3 acts downstream of the sphingoid bases in this response. Interestingly, cell cycle recovery from the transient arrest was found to be dependent upon the sphingoid base kinases (LCB4, LCB5). Overall, this combination of genetic and pharmacologic results demonstrates a role for de novo sphingoid base biosynthesis by serine palmitoyltransferase in the transient G0/G1 arrest mediated through Cln3 via a novel mechanism.
近期有关鞘脂类作为酵母热应激反应潜在介质的研究结果，促使我们去探究其在热诱导细胞周期停滞及后续恢复过程中可能发挥的作用。我们发现鞘脂缺陷型酵母菌株7R4缺乏同基因野生型中出现的细胞周期停滞现象。此外，携带温度敏感型丝氨酸棕榈酰转移酶的菌株lcb1 - 100在热应激时从头生成的鞘氨醇碱未增加。重要的是，该菌株缺乏短暂的热诱导G0/G1期停滞。这些结果表明鞘脂类，特别是那些在从头合成途径中产生的鞘脂类，在细胞对热的周期停滞反应中发挥作用。为了确定调节这种反应的生物活性鞘脂，我们对鞘脂生物合成和降解途径中的关键突变体进行了分析。发现缺失鞘氨醇碱激酶、鞘氨醇磷酸磷酸酶、裂解酶或二氢鞘氨醇羟化酶的菌株表现出细胞周期停滞。同样，敲除严重减弱神经酰胺产生的脂肪酰基延长酶也表现出停滞。这些实验表明，细胞周期停滞的活性物质是鞘氨醇碱。进一步支持这些发现的是，发现外源植物鞘氨醇（10微摩尔）可诱导短暂停滞。硬脂胺未诱导停滞，证明了化学特异性，而L - 赤藓醇的效力不如D - 赤藓醇 - 二氢鞘氨醇，显示出立体特异性。为了研究可能的停滞机制，我们研究了超稳定的Cln3（Cln3 - 1）菌株LDW6A，该菌株先前已被证明对热应激诱导的细胞周期停滞具有抗性。发现含有Cln3 - 1的菌株对外源植物鞘氨醇诱导的细胞周期停滞具有抗性，这表明在这种反应中Cln3在鞘氨醇碱的下游起作用。有趣的是，发现从短暂停滞中恢复细胞周期依赖于鞘氨醇碱激酶（LCB4、LCB5）。总体而言，这种遗传学和药理学结果的结合证明了丝氨酸棕榈酰转移酶从头合成鞘氨醇碱生物合成在通过Cln3介导的短暂G0/G1期停滞中通过一种新机制所起的作用。
