NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulphur proteins in Arabidopsis thaliana.
NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulphur proteins in Arabidopsis thaliana.
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DOI：
10.1111/tpj.13409
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发表时间：
2017-02
期刊：
影响因子：
--
通讯作者：
Balk J
中科院分区：
文献类型：
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作者：
Bastow EL;Bych K;Crack JC;Le Brun NE;Balk J
铁硫（FeS）簇组装的胞质途径的蛋白质是保守的，除了植物缺乏CFD 1（胞质FeS簇缺陷1）基因。这就提出了一个问题，即NBP 35（核苷酸结合蛋白35 kDa）是后生动物中CFD 1的异聚体伴侣，它如何在植物中发挥作用。 首先，我们在拟南芥中创建了NBP 35的可行突变等位基因，以克服先前报道的敲除突变的胚胎致死性。具有小于30%NBP35蛋白的RNAi敲低系令人惊讶地显示与野生型没有发育或生物化学差异。将Cys 14取代为Ala，使N-末端Fe 4S 4簇在体外不稳定，导致轻度生长缺陷和细胞溶质FeS酶（如乌头酸酶和醛氧化酶）活性的显著降低。DNA糖基化酶ROS 1活性仅部分降低，而黄嘌呤脱氢酶活性完全没有降低。强烈耗尽NBP 35蛋白与Cys 14到Ala取代组合的植物具有扭曲的叶片发育和降低的FeS酶活性。为了找到NBP 35的蛋白质相互作用伴侣，进行了酵母双杂交筛选，鉴定了NBP 35和DRE 2（核糖体蛋白S14表达的去阻遏）。已知NBP 35形成二聚体，并且DRE 2在胞质FeS蛋白组装途径的上游起作用。NBP 35-DRE 2相互作用不被Cys 14替换为Ala所破坏。我们的研究结果表明，NBP 35在植物中保守的FeS蛋白的成熟中具有功能，并且与DRE 2的功能密切相关。NBP 35（核苷酸结合蛋白35 kDa）的敲除突变体是胚胎致死的，这已经排除了功能研究。在这里，我们产生了在N末端铁硫（FeS）簇结合基序中具有Cys 14至Ala取代的细胞系，其表现出几种细胞溶质FeS酶的活性降低。此外，发现NBP 35与DRE 2相互作用，DRE 2是该途径的上游蛋白质。因此，我们证明NBP 35在FeS蛋白的成熟中具有功能。
Proteins of the cytosolic pathway for iron‐sulphur (FeS) cluster assembly are conserved, except that plants lack a gene for CFD1 (Cytosolic FeS cluster Deficient 1). This poses the question of how NBP35 (Nucleotide‐Binding Protein 35 kDa), the heteromeric partner of CFD1 in metazoa, functions on its own in plants. Firstly, we created viable mutant alleles of NBP35 in Arabidopsis to overcome embryo lethality of previously reported knockout mutations. RNAi knockdown lines with less than 30% NBP35 protein surprisingly showed no developmental or biochemical differences to wild‐type. Substitution of Cys14 to Ala, which destabilized the N‐terminal Fe4S4 cluster in vitro, caused mild growth defects and a significant decrease in the activity of cytosolic FeS enzymes such as aconitase and aldehyde oxidases. The DNA glycosylase ROS1 was only partially decreased in activity and xanthine dehydrogenase not at all. Plants with strongly depleted NBP35 protein in combination with Cys14 to Ala substitution had distorted leaf development and decreased FeS enzyme activities. To find protein interaction partners of NBP35, a yeast‐two‐hybrid screen was carried out that identified NBP35 and DRE2 (Derepressed for Ribosomal protein S14 Expression). NBP35 is known to form a dimer, and DRE2 acts upstream in the cytosolic FeS protein assembly pathway. The NBP35–DRE2 interaction was not disrupted by Cys14 to Ala substitution. Our results show that NBP35 has a function in the maturation of FeS proteins that is conserved in plants, and is closely allied to the function of DRE2. Knockout mutants of NBP35 (Nucleotide‐Binding Protein 35 kDa) are embryo lethal, which has precluded functional studies. Here we generated a line with a Cys14 to Ala substitution in the N‐terminal iron‐sulfur (FeS) cluster binding motif, which exhibited a decrease in activities of several cytosolic FeS enzymes. In addition, NBP35 was found to interact with DRE2, a protein upstream in the pathway. Thus we demonstrate that NBP35 has a function in the maturation of FeS proteins.