GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production
GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production
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DOI：
10.1002/yea.2948
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发表时间：
2013-04-01
期刊：
影响因子：
2.6
通讯作者：
Locy, Robert D.
中科院分区：
文献类型：
--
作者：
Cao, Juxiang;Barbosa, Jose M.;Locy, Robert D.
GABA分流通路涉及谷氨酸脱羧酶（GAD）、GABA氨基转移酶（GAT）和琥珀酸半醛脱氢酶（SSADH）三种酶。这些酶协同作用将谷氨酸（-酮戊二酸）转化为琥珀酸盐。这些基因的缺失突变导致酿酒酵母在45℃下生长缺陷，并与野生型和单突变菌株进行了双突变和三突变构建的耐热性比较。尽管野生型和所有突变株对50℃的短暂热胁迫高度敏感，但在40℃下进行30 min的非致死预处理诱导了野生型和所有突变株对50℃的耐受性。突变株在45℃下的敏感性与诱导的50℃处理相似。测定了野生型和各突变株细胞内活性氧中间体（ROI）的积累。各突变体在不同胁迫条件下的ROI积累与突变株的热敏感性相关。添加ROI清除剂n -叔丁基-苯基硝基酮（N-tert-butyl- phenylnitrone， PBN）能提高突变体的存活率，并能强烈抑制ROI的积累，但对野生型的影响不显著。在45℃的致死热暴露过程中，测量细胞内GABA、谷氨酸和-酮戊二酸显示，在uga1和uga2突变体中，GABA和-酮戊二酸的积累水平更高，而在gad1突变体中，谷氨酸的积累水平更高。这些结果表明，GABA分流途径在保护酵母细胞免受热损伤中起着至关重要的作用，通过限制热应激时-酮戊二酸向琥珀酸的碳通量来产生ROI。版权所有：John Wiley & Sons， Ltd。
The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (-ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45 degrees C. Double and triple mutation constructs were compared for thermotolerance with the wild-type and single mutant strains. Although wild-type and all mutant strains were highly susceptible to brief heat stress at 50 degrees C, a non-lethal 30 min at 40 degrees C temperature pretreatment induced tolerance of the wild-type and all of the mutants to 50 degrees C. The mutant strains collectively exhibited similar susceptibility at 45 degrees C to the induced 50 degrees C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild-type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N-tert-butyl--phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild-type. Measurement of intracellular GABA, glutamate and -ketoglutarate during lethal heat exposure at 45 degrees C showed higher levels of accumulation of GABA and -ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from -ketoglutarate to succinate during heat stress. Copyright (c) 2013 John Wiley & Sons, Ltd.