Dumont M E, Cardillo T S, Hayes M K, Sherman F
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Mol Cell Biol. 1991 Nov;11(11):5487-96. doi: 10.1128/mcb.11.11.5487-5496.1991.
Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.
血红素通过细胞色素c血红素裂合酶共价连接到细胞色素c上。为了测试在体内细胞色素c导入线粒体过程中血红素附着是否必需，已使用细胞色素c抗体来分析酿酒酵母各种菌株的细胞质和线粒体中脱辅基细胞色素c和全细胞色素c的分布。缺乏血红素裂合酶的菌株在细胞质中积累脱辅基细胞色素c。即使在血红素裂合酶水平正常的情况下，对于一种改变的脱辅基细胞色素c也观察到类似的细胞质积累，其中丝氨酸残基取代了通常作为血红素附着位点的两个半胱氨酸残基。然而，在线粒体内也发现了可检测量的这种改变的脱辅基细胞色素c。在完全缺乏血红素裂合酶的菌株中，内化的改变的脱辅基细胞色素c水平降低，而在过表达血红素裂合酶的菌株中则大大增加。识别血红素裂合酶的抗体被用于证明该酶存在于线粒体内膜的外表面，并且在线粒体内膜和外膜的接触位点没有富集。这些结果表明，脱辅基细胞色素c通过一个自由可逆的过程穿过线粒体外膜，在内膜间隙中与血红素裂合酶结合，然后通过不可逆地转化为全细胞色素c并伴随折叠成天然构象而被困在线粒体内。缺乏共价连接血红素能力的改变的脱辅基细胞色素c仅在其仍然与血红素裂合酶结合的程度上在线粒体中积累。
