Eukaryotic initiation factor 2B (eIF2B) initiates and regulates translation initiation in eukaryotes. eIF2B gene mutations cause leukoencephalopathy called vanishing white matter disease (VWM) in humans and slow growth (Slg−) and general control derepression (Gcd−) phenotypes in Saccharomyces cerevisiae. To suppress eIF2B mutations, S. cerevisiae genomic DNA library was constructed in high-copy vector (YEp24) and transformed into eIF2B mutant S. cerevisiae strains. The library was screened for wild-type genes rescuing S. cerevisiae (Slg−) and (Gcd−) phenotypes. A genomic clone, Suppressor-I (Sup-I), rescued S. cerevisiae Slg− and Gcd− phenotypes (gcd7-201 gcn2∆). The YEp24/Sup-I construct contained truncated TAN1, full length EMC4, full length YGL230C, and truncated SAP4 genes. Full length EMC4 (chaperone protein) gene was sub-cloned into pEG (KG) yeast expression vector and overexpressed in gcd7-201 gcn2∆ strain which suppressed the Slg− and Gcd− phenotype. A GST-Emc4 fusion protein of 47 kDa was detected by western blotting using α-GST antibodies. Suppression was specific to gcd7-201 gcn2∆ mutation in eIF2Bβ and Gcd1-502 gcn2∆ in eIF2Bγ subunit. Emc4p overexpression also protected the wild type and mutant (gcd7-201 gcn2∆, GCD7 gcn2∆, and GCD7 GCN2∆) strains from H2O2, ethanol, and caffeine stress. Our results suggest that Emc4p is involved in eIF2B-mediated translational regulation under stress and could provide an amenable tool to understand the eIF2B-mediated defects.

中文翻译：

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酿酒酵母ER膜蛋白复合物亚基4（EMC4）在eIF2B介导的翻译调控和应激条件下的存活中起关键作用

真核生物起始因子2B（eIF2B）在真核生物中起始并调节翻译起始。eIF2B基因突变在人类中引起白脑病，称为消失的白质病（VWM），在酿酒酵母中表现出缓慢的生长（Slg-）和一般控制性抑制（Gcd-）表型。为了抑制eIF2B突变，在高拷贝载体（YEp24）中构建了酿酒酵母基因组DNA文库，并将其转化为eIF2B突变酿酒酵母菌株。筛选该文库中拯救啤酒酵母（Slg-）和（Gcd-）表型的野生型基因。基因组克隆Suppressor-I（Sup-I）拯救了酿酒酵母Slg-和Gcd-表型（gcd7-201 gcn2∆）。YEp24 / Sup-I构建体包含截短的TAN1，全长EMC4，全长YGL230C和截短的SAP4基因。将全长EMC4（伴侣蛋白）基因亚克隆到pEG（KG）酵母表达载体中，并在抑制Slg-和Gcd-表型的gcd7-201gcn2Δ菌株中过表达。使用α-GST抗体通过蛋白质印迹法检测到47 kDa的GST-Emc4融合蛋白。抑制特异于eIF2Bβ中的gcd7-201 gcn2∆突变和eIF2Bγ亚基中的Gcd1-502 gcn2∆。Emc4p过表达还可以保护野生型和突变株（gcd7-201 gcn2∆，GCD7 gcn2∆和GCD7 GCN2∆）免受H2O2，乙醇和咖啡因的胁迫。我们的结果表明Emc4p在压力下参与eIF2B介导的翻译调控，并可能提供一个了解eIF2B介导的缺陷的便利工具。使用α-GST抗体通过蛋白质印迹法检测到47 kDa的GST-Emc4融合蛋白。抑制特异于eIF2Bβ中的gcd7-201 gcn2∆突变和eIF2Bγ亚基中的Gcd1-502 gcn2∆。Emc4p过表达还可以保护野生型和突变株（gcd7-201 gcn2∆，GCD7 gcn2∆和GCD7 GCN2∆）免受H2O2，乙醇和咖啡因的胁迫。我们的结果表明Emc4p在压力下参与eIF2B介导的翻译调控，并可能提供一个了解eIF2B介导的缺陷的便利工具。使用α-GST抗体通过蛋白质印迹法检测到47 kDa的GST-Emc4融合蛋白。抑制特异于eIF2Bβ中的gcd7-201 gcn2∆突变和eIF2Bγ亚基中的Gcd1-502 gcn2∆。Emc4p过表达还可以保护野生型和突变株（gcd7-201 gcn2∆，GCD7 gcn2∆和GCD7 GCN2∆）免受H2O2，乙醇和咖啡因的胁迫。我们的结果表明Emc4p在压力下参与eIF2B介导的翻译调控，并可能提供一个了解eIF2B介导的缺陷的便利工具。