Expression profiles and physiological roles of two types of prefoldins from the hyperthermophilic archaeon Thermococcus kodakaraensis
Expression profiles and physiological roles of two types of prefoldins from the hyperthermophilic archaeon Thermococcus kodakaraensis
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DOI：
10.1016/j.jmb.2008.07.032
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发表时间：
2008-10-03
影响因子：
5.6
通讯作者：
Fujiwara, Shinsuke
中科院分区：
文献类型：
--
作者：
Danno, Atsushi;Fukuda, Wakao;Fujiwara, Shinsuke
超嗜热古菌Thermococcus kodakaraensis在基因组上具有四个前折叠蛋白基因，编码前折叠蛋白的两个α亚基（pfdA和pfdC）和两个β亚基（pfdB和pfdD）。pfdC和pfdD是在火球菌属中未发现的直系同源物的独特基因，而pfdA和pfdB通常在热球菌属和火球菌属中发现。pfdA和pfdB位于不同的位点，pfdC和pfdD在基因组上串联排列。使用特异性抗血清，抗PfdB和抗PfdD的免疫沉淀实验表明，PfdB和PfdD使复合物仅与PfdA和PfdC，分别。以重组形式获得的PfdA/PfdB和PfdC/PfdD复合物对柠檬酸合酶的热聚集都显示出抑制活性。免疫印迹实验表明，PfdA/PfdB复合物在所有检测温度下表达;然而，PfdC/PfdD复合物在93 ℃的热应激条件下特异性表达。转录分析表明，pfdA和pfdB在所有检测的温度下转录水平相同，但pfdC和pfdD在93 ℃下转录水平较高。此外，pfdA和pfdB单独转录，但pfdD与pfdC共转录。在pfdC的上游区域鉴定了一个典型的热休克调节因子（Phr）识别序列。在phr破坏物中测量pfdCD的转录水平，表明与野生型相比，phr破坏物中的pfdCD转录物急剧增加。然而，pfdCD水平也在较高的温度下升高，表明PfdC/PfdD的热诱导主要是通过Phr去阻遏实现的，但一定程度的诱导不受Phr控制。然后构建pfdB和pfdD干扰物，并比较生长曲线。在85 ° C培养下，在野生型和pfdD破坏物之间没有观察到显著差异;然而，在pfdB破坏物中观察到较少的生长。在93摄氏度，pfdD干扰物生长小于野生型，并且pfdB干扰物生长最少。结果表明，PfdA/PfdB复合物在所有生长温度下起着至关重要的作用，PfdC/PfdD复合物有助于在高温环境中的生存。(C)2008爱思唯尔有限公司保留所有权利。
The hyperthermophilic archaeon Thermococcus kodakaraensis possesses four prefoldin genes encoding two alpha subunits (pfdA and pfdC) and two beta subunits (pfdB and pfdD) of prefoldins on the genome. pfdC and pfdD are unique genes whose orthologues are not found in Pyrococcus spp., whereas pfdA and pfdB are commonly found in both Thermococcus and Pyrococcus spp. The pfdA and pfdB are located at different loci, and pfdC and pfdD were tandemly arranged on the genome. Immmunoprecipitation experiments using specific antisera, anti-PfdB and anti-PfdD, revealed that PfdB and PfdD make a complex only with PfdA and PfdC, respectively. Both PfdA/PfdB and PfdC/PfdD complexes obtained as recombinant forms showed inhibitory activity against the thermal aggregation of citrate synthase. Immunoblot experiments indicated that the PfdA/PfdB complex was expressed at all examined temperatures; however, the PfdC/PfdD complex was specifically expressed under heat-stress conditions at 93 degrees C. Transcriptional analyses showed that pfdA and pfdB were transcribed at equal levels at all examined temperatures but pfdC and pfdD were transcribed at higher levels at 93 degrees C. Furthermore, pfdA and pfdB were transcribed individually, but pfdD was cotranscribed with pfdC. A typical Pyrococcus heat-shock regulator (Phr) recognition sequence was identified at the upstream region of pfdC. The transcriptional level of pfdCD was measured in a phr disruptant, showing that the pfdCD transcript in the phr disruptant was drastically increased in comparison with that of the wild type. However, the pfdCD level was also elevated at higher temperature, indicating that heat induction of PfdC/PfdD is mainly achieved by Phr derepression but that a certain degree of induction is not under Phr control. The pfdB and pfdD disruptants were then constructed, and the growth profiles were compared. At 85 degrees C cultivation, no significant difference was observed between the wild type and the pfdD disruptant; however, less growth was observed in the pfdB disruptant. At 93 degrees C, the pfdD disruptant grew less than the wild type, and the pfdB disruptant grew the least. The results suggest that the PfdA/PfdB complex plays a crucial role at all growth temperatures and the PfdC/PfdD complex contributes to survival in a high-temperature environment. (C) 2008 Elsevier Ltd. All rights reserved.