Role for de novo sphingoid base biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae
Role for de novo sphingoid base biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m007425200
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发表时间：
2001-03-16
影响因子：
4.8
通讯作者：
Hannun, YA
中科院分区：
文献类型：
--
作者：
Jenkins, GM;Hannun, YA
神经鞘脂脂作为酵母热应激反应的潜在介体的最新发现使我们研究了它们在热诱导的细胞周期停滞和随后的恢复中的可能作用。鞘磷脂缺陷型酵母菌株7R4缺乏同基因野生型的细胞周期停滞现象，此外，含有温度敏感的丝氨酸棕榈酰基转移酶的菌株Lcb1-100缺乏热应激时新生成的狮身人面像碱基的增加，更重要的是，该菌株缺乏热诱导的瞬时G(0)/G(1)停滞。这些结果表明神经鞘脂脂，特别是在从头开始途径中产生的那些在细胞周期停止反应中对热的作用。为了确定调节这一反应的生物活性鞘脂，对鞘脂生物合成和降解途径中的关键突变进行了分析。缺失狮身人面像碱性激酶、狮身人面像磷酸酶、裂解酶或二氢鞘氨醇羟基酶的菌株表现出细胞周期停滞。此外，一种脂肪酰基延长酶的敲除也显示出抑制作用，这种酶严重削弱了神经酰胺的产生。这些实验表明，细胞周期停滞的活性物种是狮身人面像碱基。进一步支持这些发现的是，外源性植酸鞘氨醇(10微米)可引起一过性停搏。硬脂胺没有诱导细胞停滞，表现出化学特异性，而L-红血球-没有D-红血球-二氢鞘氨醇表现出立体特异性，为了研究可能的停滞机制，我们研究了先前被证明抵抗热应激诱导的细胞周期停滞的超稳Cln3(Cln3-1)菌株LDW6GA。含有Cln3-1的菌株对外源植酸鞘糖苷诱导的细胞周期停滞具有抗性，这表明Cln3在这种反应中作用于狮身人面像碱基的下游。有趣的是，细胞周期的恢复被发现依赖于狮身人面像碱性蛋白激酶(LCB4，LCB5)，总的来说，这种遗传学和药理学结果的结合表明，丝氨酸棕榈酰基转移酶通过一种新的机制在通过Cln3介导的短暂G(0)/G(1)阻滞中发挥作用。
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 G(0)/G(1) 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 muM) 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 LDW6GA 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 G(0)/G(1) arrest mediated through Cln3 via a novel mechanism.