Effect of cellular location on the function of ferrochelatase.
Effect of cellular location on the function of ferrochelatase.
复制标题
细胞位置对亚铁螯合酶功能的影响。
DOI：
10.1074/jbc.270.31.18198
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Dailey,HA
中科院分区：
文献类型：
--
作者：
Prasad,AR;Dailey,HA
亚铁螯合酶是血红素生物合成途径的终末酶，是一种核编码蛋白，以前体形式在细胞质中合成，然后转运到线粒体膜内的基质侧。由于酶反应的产物，原血红素IX，几乎只在细胞质隔间或线粒体内膜的细胞质一侧被利用，因此确定铁络合酶在细胞内的位置是否对其有效功能至关重要是有意义的。在一株缺乏铁络合酶的酵母菌中，表达了编码全长铁络合酶的表达载体和缺乏线粒体靶向序列的截短形式的酶。通过酶分析和Western印迹分析，这两种转化细胞产生的铁络合酶总量大致相同，但只有全长结构正确地定位了铁络合酶。在含有截短结构的细胞中，铁络合酶活性存在于所有膜组分中，但不位于线粒体内膜的基质侧。含有任何一种结构的细胞都会产生血红素，尽管具有截短结构的细胞合成的血红素数量明显较少。有趣的是，在铁络合酶定位不当的细胞中，B型细胞色素的数量减少了80%，而C型细胞色素的减少分别只有60%和40%。
Ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is a nuclear encoded protein that is synthesized in the cytoplasm in a precursor form and then is translocated to the matrix side of the inner mitochondrial membrane. Since the product of the enzymatic reaction, protoheme IX, is utilized almost exclusively in the cytoplasmic compartment or on the cytoplasmic side of the inner mitochondrial membrane, it was of interest to determine if the intracellular location of ferrochelatase is crucial for its effective functioning. In a ferrochelatase-deficient strain of the yeastSaccharomyces cerevisiaevectors that coded for full-length ferrochelatase and a truncated form of the enzyme that lacked the mitochondrial targeting sequence were expressed. Both of these transformed cells produce approximately equal total amounts of ferrochelatase, as determined by enzyme assays and Western blot analysis, but only with the full-length construct was ferrochelatase properly localized. In cells containing the truncated construct, ferrochelatase activity was found in all membrane fractions but was not located on the matrix side of the inner mitochondrial membrane. Cells containing either construct produced heme, although the amount of heme synthesized by cells with the truncated construct was significantly less. Interestingly in cells with improperly localized ferrochelatase the amount ofb-type cytochrome decreased by 80% as opposed toc- anda-type cytochromes where the decreases were only 60 and 40%, respectively.