Goggin Danica Erin, Lipscombe Richard, Fedorova Elena, Millar A Harvey, Mann Anthea, Atkins Craig Anthony, Smith Penelope Mary Collina
Department of Botany, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia.
Plant Physiol. 2003 Mar;131(3):1033-41. doi: 10.1104/pp.102.015081.
De novo purine biosynthesis is localized to both mitochondria and plastids isolated from Bradyrhizobium sp.-infected cells of cowpea (Vigna unguiculata L. Walp) nodules, but several of the pathway enzymes, including aminoimidazole ribonucleotide synthetase (AIRS [EC 6.3.3.1], encoded by Vupur5), are encoded by single genes. Immunolocalization confirmed the presence of AIRS protein in both organelles. Enzymatically active AIRS was purified separately from nodule mitochondria and plastids. N-terminal sequencing showed that these two isoforms matched the Vupur5 cDNA sequence but were processed at different sites following import; the mitochondrial isoform was five amino acids longer than the plastid isoform. Electrospray tandem mass spectrometry of a trypsin digest of mitochondrial AIRS identified two internal peptides identical with the amino acid sequence deduced from Vupur5 cDNA. Western blots of proteins from mitochondria and plastids isolated from root tips showed a single AIRS protein present at low levels in both organelles. (35)S-AIRS protein translated from a Vupur5 cDNA was imported into isolated pea (Pisum sativum) leaf chloroplasts in vitro by an ATP-dependent process but not into import-competent mitochondria from several plant and non-plant sources. Components of the mature protein are likely to be important for import because the N-terminal targeting sequence was unable to target green fluorescent protein to either chloroplasts or mitochondria in Arabidopsis leaves. The data confirm localization of the protein translated from the AIRS gene in cowpea to both plastids and mitochondria and that it is cotargeted to both organelles, but the mechanism underlying import into mitochondria has features that are yet to be identified.
从头嘌呤生物合成定位于从豇豆（Vigna unguiculata L. Walp）根瘤中感染慢生根瘤菌属细菌的细胞分离得到的线粒体和质体中，但该途径的几种酶，包括氨基咪唑核糖核苷酸合成酶（AIRS [EC 6.3.3.1]，由Vupur5编码），由单基因编码。免疫定位证实两种细胞器中均存在AIRS蛋白。分别从根瘤线粒体和质体中纯化出具有酶活性的AIRS。N端测序表明，这两种同工型与Vupur5 cDNA序列匹配，但在导入后在不同位点进行了加工；线粒体同工型比质体同工型长五个氨基酸。线粒体AIRS胰蛋白酶消化产物的电喷雾串联质谱鉴定出两个内部肽段，与从Vupur5 cDNA推导的氨基酸序列相同。从根尖分离的线粒体和质体蛋白质的蛋白质免疫印迹显示，两种细胞器中均存在低水平的单一AIRS蛋白。从Vupur5 cDNA翻译的(35)S-AIRS蛋白在体外通过ATP依赖的过程导入分离的豌豆（Pisum sativum）叶叶绿体，但不能导入来自几种植物和非植物来源的有导入能力的线粒体。成熟蛋白的组分可能对导入很重要，因为N端靶向序列无法将绿色荧光蛋白靶向拟南芥叶中的叶绿体或线粒体。数据证实了豇豆中从AIRS基因翻译的蛋白定位于质体和线粒体，并且它共靶向这两种细胞器，但导入线粒体的潜在机制具有尚未确定的特征。
