Subcellular localization of two types of ferrochelatase in cucumber
Subcellular localization of two types of ferrochelatase in cucumber
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DOI：
10.1007/s00425-003-1019-2
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发表时间：
2003-08-01
期刊：
影响因子：
4.3
通讯作者：
Takamiya, K
中科院分区：
文献类型：
--
作者：
Masuda, T;Suzuki, T;Takamiya, K
亚铁螯合酶（protoheme ferrolyase，EC 4.99.1.1）是一种存在于高等植物质体和线粒体中的酶，催化亚铁离子插入原卟啉IX形成血红素。采用离体导入法，对豌豆（PisumsativumL.）在细胞器中，已经提出亚铁螯合酶的两种同种型之一（1型）被双重靶向到质体和线粒体中，并且在两种细胞器中对血红素生物合成起作用。然而，最近，铁螯合酶的线粒体靶向受到争议，因为豌豆线粒体似乎接受多种叶绿体蛋白，包括拟南芥（L.）嘿为了明确高等植物亚铁螯合酶的亚细胞定位，研究了两种亚铁螯合酶（CsFeC 1和CsFeC 2）在黄瓜中的亚细胞定位。在子叶中，一个显着水平的特定铁螯合酶活性检测类囊体膜，但只有微量的活性检测在线粒体。特异性抗体的Western印迹分析表明，抗CsFeC 2抗血清与光合和非光合组织中的质体发生交叉反应。抗CsFeC 1没有交叉反应与线粒体，但CsFeC 1是清楚地检测到非光合组织的质体。使用绿色荧光蛋白的原位瞬时表达试验表明，以及CsFeC 2，CsFeC 1的N-末端转运肽的融合蛋白只针对质体，但不进入线粒体。这些结果表明，在黄瓜中，CsFeC 1和CsFeC 2都只靶向质体，而不是线粒体。黄瓜基因组或cDNA文库的筛选不允许分离任何其他铁螯合酶同源基因。这里提供的数据意味着对高等植物线粒体血红素生物合成的重新考虑。
It is widely believed that ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous ion into protoporphyrin IX to form protoheme, exists in both plastids and mitochondria of higher plants. By in vitro import assay with isolated pea (Pisum sativum L.) organelles, it has been proposed that one of two isoforms of ferrochelatase (type 1) is dual-targeted into both plastids and mitochondria, and functions for heme biosynthesis in the both organelles. Recently, however, mitochondrial targeting of ferrochelatase is being disputed since pea mitochondria appeared to accept a variety of chloroplast proteins including the type-1 ferrochelatase of Arabidopsis thaliana (L.) Heynh. To clarify the precise subcellular localization of ferrochelatase in higher plants, here we investigated the subcellular localization of two types of ferrochelatase (CsFeC1 and CsFeC2) in cucumber (Cucumis sativus L.). In cotyledons, a significant level of specific ferrochelatase activity was detected in thylakoid membranes, but only a trace level of activity was detectable in mitochondria. Western blot analysis with specific antibodies showed that anti-CsFeC2 antiserum cross-reacted with plastids in photosynthetic and non-photosynthetic tissues. Anti-CsFeC1 did not cross-react with mitochondria, but CsFeC1 was clearly detectable in plastids from non-photosynthetic tissues. In situ transient-expression assays using green fluorescent protein demonstrated that, as well as CsFeC2, the N-terminal transit peptide of CsFeC1 targeted the fusion protein solely into plastids, but not into mitochondria. These results demonstrated that in cucumber both CsFeC1 and CsFeC2 are solely targeted into plastids, but not into mitochondria. Screening of a cucumber genomic or cDNA library did not allow any other ferrochelatase homologous gene to be isolated. The data presented here imply the reconsideration of mitochondrial heme biosynthesis in higher plants.