Takahashi M, Hood D A
Departments of Biology and Kinesiology, York University, North York, Ontario M3J 1P3, Canada.
J Biol Chem. 1996 Nov 1;271(44):27285-91. doi: 10.1074/jbc.271.44.27285.
To date, no studies have described the import of proteins in mitochondria obtained from skeletal muscle. In this tissue, mitochondria consist of the functionally and biochemically distinct intermyofibrillar (IMF) and subsarcolemmal (SS) subfractions, which are localized in specialized cellular compartments. This mitochondrial heterogeneity in muscle could be due, in part, to differential rates of protein import. To evaluate this possibility, the import of precursor malate dehydrogenase and ornithine carbamyltransferase proteins was investigated in isolated IMF and SS mitochondria in vitro. Import of these was 3-4-fold greater in IMF compared with SS mitochondria as a function of time. This could account for the higher malate dehydrogenase enzyme activity in IMF mitochondria. Divergent import rates in IMF and SS mitochondria likely result from a differential reliance on various components of the import pathway. SS mitochondria possess a greater content of the molecular chaperones hsp60 and Grp75, yet import is lower than in IMF mitochondria. On the other hand, adriamycin inhibition studies illustrated a greater reliance on acidic phospholipids (i.e. cardiolipin) for the import process in SS mitochondria. Matrix ATP levels were 3-fold higher in IMF mitochondria, but experiments in which ATP depletion was performed with atractyloside and oligomycin illustrated a dissociation between import rates and levels of ATP. In contrast, a close relationship was found between the rate of ATP production (i.e. mitochondrial respiration) and protein import. When respiratory rates in IMF and SS mitochondria were equalized, import rates in both subfractions were similar. These data indicate that 1) import rates are more closely related to the rate of ATP production than the steady state ATP level, 2) import into IMF and SS mitochondrial subfractions is regulated differently, and 3) mitochondrial heterogeneity within a cell type can be due to differences in the rates of protein import, suggesting that this step is a potentially regulatable event in determining the final mitochondrial phenotype.
迄今为止，尚无研究描述从骨骼肌中获取的线粒体中蛋白质的导入情况。在该组织中，线粒体由功能和生化特性不同的肌原纤维间（IMF）和肌膜下（SS）亚组分组成，它们位于特殊的细胞区室中。肌肉中的这种线粒体异质性可能部分归因于蛋白质导入速率的差异。为评估这种可能性，在体外分离的IMF和SS线粒体中研究了前体苹果酸脱氢酶和鸟氨酸氨甲酰基转移酶蛋白的导入情况。随着时间的推移，这些蛋白在IMF中的导入量比在SS线粒体中高3至4倍。这可以解释IMF线粒体中较高的苹果酸脱氢酶活性。IMF和SS线粒体中不同的导入速率可能是由于对导入途径中各种组分的依赖程度不同所致。SS线粒体中分子伴侣hsp60和Grp75的含量更高，但导入量却低于IMF线粒体。另一方面，阿霉素抑制研究表明，SS线粒体的导入过程对酸性磷脂（即心磷脂）的依赖性更强。IMF线粒体中的基质ATP水平高3倍，但用苍术苷和寡霉素进行ATP耗竭的实验表明，导入速率与ATP水平之间没有关联。相反，发现ATP产生速率（即线粒体呼吸）与蛋白质导入之间存在密切关系。当IMF和SS线粒体中的呼吸速率相等时，两个亚组分中的导入速率相似。这些数据表明：1）导入速率与ATP产生速率的关系比与稳态ATP水平的关系更为密切；2）导入到IMF和SS线粒体亚组分的过程受到不同的调节；3）同一细胞类型内的线粒体异质性可能是由于蛋白质导入速率的差异所致，这表明该步骤在决定最终线粒体表型方面可能是一个可调节的事件。
