Functional characterization of a gene encoding a fourth ATP sulfurylase isoform from Arabidopsis thaliana
Functional characterization of a gene encoding a fourth ATP sulfurylase isoform from Arabidopsis thaliana
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DOI：
10.1016/s0378-1119(00)00132-3
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发表时间：
2000-05-02
期刊：
影响因子：
3.5
通讯作者：
Saito, K
中科院分区：
文献类型：
--
作者：
Hatzfeld, Y;Lee, S;Saito, K
硫酸盐同化途径的第一种酶是植物叶绿体和细胞质中的三磷酸腺苷转移酶。在拟南芥中，克隆发现了三种三磷酸腺苷硫酰化酶亚型(APS1、-2和-3)，它们似乎都定位于叶绿体中。在本研究中，通过搜索表达序列标签(EST)数据库和筛选拟南芥基因组文库来寻找胞质异构体。第四种异构体APS4被鉴定出来，但它也编码一种定位于叶绿体的异构体。APS基因都含有四个内含子。内含子位于每个APS基因的编码序列中的相同位置。在APS3和APS4基因的启动子中发现了一个可能的TATA盒，但其他启动子之间没有发现序列相似的区域。对APS4基因和基因组克隆的联合分析表明，该蛋白由469个氨基酸组成，与拟南芥APS1亚类的同源性最高。APS4基因能够对酵母ATP硫酰化酶(Met3)突变体进行功能互补，并且重组酶具有ATP硫酰化酶活性。APS4蛋白在其氨基末端显示了一个叶绿体靶向多肽，当它与绿色荧光蛋白融合时，能够将报告分子靶向叶绿体。APS4mRNA在根和叶中的表达水平基本一致，不受硫饥饿和O-乙酰丝氨酸处理的诱导。在鉴定出第四种定位于叶绿体的三磷酸腺苷硫酰化酶后，海桑胞质异构体的来源尚不清楚。根据序列分析，推测APS2可能编码胞质三磷酸腺苷硫酰基酶。(C)2000 Elsevier Science B.V.保留所有权利。
ATP sulfurylase (ATP: sulfate adenylyl transferase, EC 2.7.7.4), the first enzyme of the sulfate assimilation pathway, is present in the chloroplast and cytosol of plants. In Arabidopsis thaliana cDNA cloning revealed the existence of three ATP sulfurylase isoforms (APS1, -2, and -3) all of which appear to be localized in plastids. In the present study the cytosolic isoform was sought by searching the expressed sequence tag (EST) database and by screening A. thaliana genomic libraries. A fourth isoform, APS4, was identified, but it also encodes a plastid-localized isoform. The APS genes all contain four introns. The introns are located at identical positions within the coding sequence of each of the APS genes. A putative TATA box was identified in the promoter of the APS3 and APS4 genes, but no regions of sequence similarity were found among the other promoters. Combined analysis of an APS4 cDNA and genomic clone revealed that the deduced protein is 469 amino acids and is most homologous to the A. thaliana APS1 subclass. The APS4 cDNA was able to functionally complement a yeast ATP sulfurylase (met3) mutant and the recombinant enzyme displayed ATP sulfurylase activity. The APS4 protein exhibits a plastid targeting peptide at its amino terminus that, when fused to green fluorescent protein, was able to target the reporter to chloroplasts. APS4 mRNA was detected at a similar steady-slate level in roots and leaves, and its expression was not induced by sulfur starvation or by O-acetylserine treatment. Having identified a fourth plastid-localized ATP sulfurylase, the origin of cytosolic isoform in A. thaliana remains unclear. Based on sequence analysis, it is hypothesized that APS2 may encode the cytosolic ATP sulfurylase. (C) 2000 Elsevier Science B.V. All rights reserved.