Claros M G, Perea J, Shu Y, Samatey F A, Popot J L, Jacq C
Ecole Normale Supérieure, Laboratoire de Génétique Moléculaire, Unité de Recherche Associée au Centre National de la Recherche Scientifique no. 1302, Paris, France.
Eur J Biochem. 1995 Mar 15;228(3):762-71.
The apocytochrome b gene, exclusively encoded by the mitochondrial genome, was engineered so that it could be expressed in the yeast cytoplasm. Different combinations of the apocytochrome b transmembrane domains were produced in the form of hybrid proteins fused to both the N-terminal mitochondrial targeting sequence of the ATPase subunit 9 from Neurospora crassa and to a cytoplasmic version of the bI4 RNA maturase, localised on the N-terminal and C-terminal sides, respectively, of the hydrophobic stretches. The bI4 RNA maturase, which can complement mitochondrial mutations, was used as an in vivo reporter to assess the mitochondrial import of the different groups of transmembrane helices. This new, reliable and sensitive reporter activity allowed us to experimentally determine the limitations to the mitochondrial import of hydrophobic proteins. All eight transmembrane helices of apocytochrome b could be imported into mitochondria, either alone or in combination, but no more than three to four transmembrane helices could be imported together at one time. This limit is close to that observed in the population of nuclear-encoded mitochondrial proteins. The hydrophobic characteristics of engineered and natural proteins targeted to the mitochondrial inner membrane revealed two factors important in the import process. These were (a) the local hydrophobicity of a transmembrane segment, and (b) the average regional hydrophobicity of the protein over an extended length of 60-80 residues. Such features may have played a major role in the evolution of mitochondrial genomes.
脱辅基细胞色素b基因仅由线粒体基因组编码，经过改造后可在酵母细胞质中表达。以融合蛋白的形式产生了脱辅基细胞色素b跨膜结构域的不同组合，这些融合蛋白分别与来自粗糙脉孢菌的ATP酶亚基9的N端线粒体靶向序列以及bI4 RNA成熟酶的细胞质形式融合，分别位于疏水片段的N端和C端。能够弥补线粒体突变的bI4 RNA成熟酶被用作体内报告基因，以评估不同跨膜螺旋组的线粒体导入情况。这种新的、可靠且灵敏的报告基因活性使我们能够通过实验确定疏水蛋白线粒体导入的限制因素。脱辅基细胞色素b的所有八个跨膜螺旋都可以单独或组合导入线粒体，但一次同时导入的跨膜螺旋不超过三到四个。这个限制与在核编码线粒体蛋白群体中观察到的情况相近。靶向线粒体内膜的工程蛋白和天然蛋白的疏水特性揭示了导入过程中两个重要因素。这两个因素是：（a）跨膜片段的局部疏水性；（b）蛋白质在60 - 80个残基的延伸长度上的平均区域疏水性。这些特征可能在线粒体基因组的进化中发挥了重要作用。
