Zhang Mei, Mileykovskaya Eugenia, Dowhan William
Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, 77225, USA.
J Biol Chem. 2005 Aug 19;280(33):29403-8. doi: 10.1074/jbc.M504955200. Epub 2005 Jun 22.
Digitonin extracts of mitochondria from cardiolipin-containing (wild type) and cardiolipin-lacking (crd1Delta mutant) Saccharomyces cerevisiae subjected to colorless native polyacrylamide gel electrophoresis in the presence of 0.003% digitonin displayed a supercomplex composed of homodimers of complexes III and IV in the former case but only the individual homodimers in the latter case. To avoid treatment with any detergent or dye, we compared organization of the respiratory chain in intact mitochondria from wild type and cardiolipin-lacking cells by using a functional analysis developed previously for the study of the organization of the respiratory chain of S. cerevisiae (Boumans, H., Grivell, L. A., and Berden, J. A. (1998) J. Biol. Chem. 273, 4872-4877). Dependence of the kinetics of NADH oxidation via complexes III, IV, and cytochrome c on the concentration of the complex III-specific inhibitor antimycin A was studied. A linear relationship between respiratory activity and saturation of complex III with antimycin A was obtained for wild type mitochondria consistent with single functional unit kinetics of the respiratory chain. Under the same conditions, cardiolipin-lacking mitochondria displayed a hyperbolic relationship indicating cytochrome c pool behavior. No release of cytochrome c from cardiolipin-lacking mitochondria or mitoplasts under our standard experimental conditions was detected. Identical cytochrome c pool behavior was observed for both wild type and cardiolipin-lacking mitochondria in the presence of a chaotropic agent, which disrupts the interaction between respiratory complexes. The results demonstrate that cardiolipin is essential for association of complexes III and IV into a supercomplex in intact yeast mitochondria.
将含心磷脂(野生型)和缺乏心磷脂(crd1Delta突变体)的酿酒酵母线粒体的洋地黄皂苷提取物,在存在0.003%洋地黄皂苷的情况下进行无色天然聚丙烯酰胺凝胶电泳,结果显示,在前一种情况下有一个由复合物III和IV的同型二聚体组成的超复合物,而在后一种情况下只有单个同型二聚体。为避免使用任何去污剂或染料处理,我们通过使用先前开发的用于研究酿酒酵母呼吸链组织的功能分析方法,比较了野生型和缺乏心磷脂细胞的完整线粒体中呼吸链的组织情况。研究了通过复合物III、IV和细胞色素c进行的NADH氧化动力学对复合物III特异性抑制剂抗霉素A浓度的依赖性。对于野生型线粒体,获得了呼吸活性与抗霉素A对复合物III饱和度之间的线性关系,这与呼吸链的单功能单位动力学一致。在相同条件下,缺乏心磷脂的线粒体呈现出双曲线关系,表明细胞色素c池的行为。在我们的标准实验条件下,未检测到缺乏心磷脂的线粒体或线粒体膜释放细胞色素c。在存在一种破坏呼吸复合物之间相互作用的离液剂的情况下,野生型和缺乏心磷脂的线粒体都观察到了相同的细胞色素c池行为。结果表明,心磷脂对于完整酵母线粒体中复合物III和IV缔合成超复合物至关重要。