Voisine C, Schilke B, Ohlson M, Beinert H, Marszalek J, Craig E A
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Mol Cell Biol. 2000 May;20(10):3677-84. doi: 10.1128/MCB.20.10.3677-3684.2000.
The mitochondrial matrix of the yeast Saccharomyces cerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3 mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in Deltassq1 mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389-18393, 1998). However, the intermediate form in both wild-type and Deltassq1 mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in Deltassq1 mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Deltassq1 cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in Deltassq1 mitochondria. Deltassq1 mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.
酿酒酵母的线粒体基质中含有两种Hsp70家族的分子伴侣，即Ssc1和Ssq1。我们报告称，Ssc1和Ssq1在酵母frataxin同源物（Yfh1）的导入和成熟过程中发挥着相继的作用。在体外，放射性标记的Yfh1无法导入ssc1 - 3突变体线粒体，而是以蛋白酶敏感的前体形式存在。如先前报道，Yfh1中间形式在Δssq1线粒体中仅缓慢加工成成熟形式（S. A. B. Knight、N. B. V. Sepuri、D. Pain和A. Dancis，《生物化学杂志》273:18389 - 18393，1998）。然而，野生型和Δssq1线粒体中的中间形式完全位于内膜内，因为在外膜破裂后它对蛋白酶消化具有抗性。因此，我们得出结论，线粒体中Ssc1的含量比Ssq1大约多1000倍，它是Yfh1导入基质所必需的，而Ssq1是将中间形式有效加工成隔离线粒体中成熟形式所必需的。然而，Δssq1线粒体中成熟Yfh1的稳态水平约为野生型线粒体中的75%，这表明这种加工延迟不会显著影响细胞内浓度。因此，Ssq1可能除了促进Yfh1的加工外还具有其他作用。Ssc1的两倍过表达部分抑制了Δssq1细胞的冷敏感生长表型，以及线粒体铁的积累和通常在Δssq1线粒体中发现的铁硫酶活性缺陷。含有两倍Ssc1的Δssq1线粒体有效地将Yfh1的中间形式转化为成熟形式。观察到的加工缺陷与体内表型抑制之间的这种相关性表明，Ssc1能够执行Ssq1的功能，但仅当它的存在量比Ssq1正常水平大约多2000倍时才行。
