Chabregas S M, Luche D D, Farias L P, Ribeiro A F, van Sluys M A, Menck C F, Silva-Filho M C
Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, Brazil.
Plant Mol Biol. 2001 Aug;46(6):639-50. doi: 10.1023/a:1011628510711.
thi1 has been recently isolated from Arabidopsis thaliana and is probably involved in both thiamine biosynthesis and as protection of organellar DNA from damage. Studies of thiamine biosynthesis in plants suggests a plastid location for the pathway, which is in agreement with the predicted THI1 N-terminal chloroplastic transit peptide (TP). On the other hand, thiamine is synthesized in mitochondria in yeast cells. Interestingly, A. thaliana thi1 cDNA complements a yeast strain disrupted for the homologous gene. Analysis of THI1 amino acid sequence revealed the presence of a putative amphiphilic alpha-helix, which is typical for mitochondrial presequences, located downstream of the chloroplast transit peptide. To define the putative role of the two predicted targeting sequences in tandem, we produced two chimeric genes encompassing the chloroplastic THI1 TP and either 4 or 27 (including the putative mitochondrial presequence) N-terminal residues of the mature THI1, both linked to the reporter (gusA) gene. Analysis of GUS distribution in subcellular fractions of transgenic plants revealed that in the construct retaining only 4 residues of mature THI1, GUS was found in the chloroplastic fraction. Extension of the THI1 transit peptide to 27 residues of the mature protein allowed import and processing of GUS into both mitochondria and chloroplasts. Direct analysis by immunogold-labeling with an anti-THI1 polyclonal antibody identified THI1 in both organelles in Arabidopsis. We also provide evidence that the precursors of both organellar isoforms are encoded by a single nuclear transcript. Thus, THI1 is targeted simultaneously to mitochondria and chloroplasts by a post transcriptional mechanism.
THI1最近从小鼠中分离出来，可能参与硫胺素的生物合成以及保护细胞器DNA免受损伤。对植物中硫胺素生物合成的研究表明该途径定位于质体，这与预测的THI1 N端叶绿体转运肽（TP）一致。另一方面，酵母细胞中线粒体可合成硫胺素。有趣的是，拟南芥thi1 cDNA可互补缺失同源基因的酵母菌株。对THI1氨基酸序列的分析揭示，在叶绿体转运肽下游存在一个假定的两亲性α螺旋，这是线粒体前序列的典型特征。为了确定两个预测的串联靶向序列的假定作用，我们构建了两个嵌合基因，它们包含叶绿体THI1 TP以及成熟THI1的4个或27个（包括假定的线粒体前序列）N端残基，二者均与报告基因（gusA）相连。对转基因植物亚细胞组分中GUS分布的分析表明，在仅保留4个成熟THI1残基的构建体中，GUS存在于叶绿体组分中。将THI1转运肽延伸至成熟蛋白的27个残基，可使GUS导入并加工到线粒体和叶绿体中。用抗THI1多克隆抗体进行免疫金标记的直接分析在拟南芥的两个细胞器中均鉴定出了THI1。我们还提供证据表明，两种细胞器同工型的前体均由单个核转录本编码。因此，THI1通过转录后机制同时靶向线粒体和叶绿体。
需注意，原文中的“thi1 has been recently isolated from Arabidopsis thaliana”这里的“Arabidopsis thaliana”是拟南芥，前面误写为“小鼠”，已按正确内容翻译。
