Hines V, Schatz G
Biocenter, University of Basel, Switzerland.
J Biol Chem. 1993 Jan 5;268(1):449-54.
Binding of precursors to import receptors on the mitochondrial surface is one of the earliest steps of protein import into mitochondria. In yeast, one of these receptors is a 70-kDa outer membrane protein termed Mas70p. Pulse-chase studies with intact yeast cells had indicated that Mas70p accelerates the import of all mitochondrial precursors tested. In contrast, import experiments with isolated mitochondria suggested that Mas70p accelerated import of only a subset of precursors (Hines, V., Brandt, A., Griffiths, G., Horstmann, H., Brütsch, H., and Schatz, G. (1990) EMBO J. 9, 3191-3200). To resolve this discrepancy, we have now studied the interaction of Mas70p-deficient and wild-type yeast mitochondria with a precursor (pre-alcohol dehydrogenase III) whose import into isolated mitochondria is not accelerated by Mas70p under the usual assay conditions. Mas70p enhanced binding of pre-alcohol dehydrogenase III to the surface of mitochondria in which the electrochemical potential across the inner membrane had been dissipated by an uncoupler; the bound precursor could be efficiently chased into the mitochondria if the potential was restored. The precursor to cytochrome c1 was also bound to mitochondria in a Mas70p-dependent manner. Mas70p also enhanced the direct import of pre-alcohol dehydrogenase III into isolated mitochondria, provided the precursor was first denatured with urea. Under these conditions, the import rate in vitro was more similar to that in intact cells. Mas70p had no effect on the binding or the import of artificial precursors containing mouse dihydrofolate as the "mature" domain. We conclude that Mas70p is an import receptor for most, if not all authentic mitochondrial precursor proteins, but that its function is not always rate-limiting in import experiments with isolated mitochondria.
前体与线粒体外膜表面的输入受体结合是蛋白质输入线粒体的最早步骤之一。在酵母中，其中一种受体是一种70 kDa的外膜蛋白，称为Mas70p。对完整酵母细胞进行的脉冲追踪研究表明，Mas70p可加速所有测试的线粒体前体的输入。相比之下，对分离线粒体进行的输入实验表明，Mas70p仅加速了一部分前体的输入（Hines, V., Brandt, A., Griffiths, G., Horstmann, H., Brütsch, H., and Schatz, G. (1990) EMBO J. 9, 3191 - 3200）。为了解决这一差异，我们现在研究了缺乏Mas70p的酵母线粒体和野生型酵母线粒体与一种前体（前乙醇脱氢酶III）的相互作用，在通常的测定条件下，该前体输入分离的线粒体的过程不会被Mas70p加速。Mas70p增强了前乙醇脱氢酶III与线粒体表面的结合，在内膜上的电化学势已被解偶联剂消散的线粒体中；如果电势恢复，结合的前体可以有效地进入线粒体。细胞色素c1的前体也以依赖Mas70p的方式与线粒体结合。Mas70p还增强了前乙醇脱氢酶III直接输入分离线粒体的过程，前提是该前体首先用尿素变性。在这些条件下，体外输入速率与完整细胞中的更相似。Mas70p对含有小鼠二氢叶酸作为“成熟”结构域的人工前体的结合或输入没有影响。我们得出结论，Mas70p是大多数（如果不是全部）真正的线粒体前体蛋白的输入受体，但在对分离线粒体进行的输入实验中，其功能并不总是限速的。
