Cardiolipin-dependent Reconstitution of Respiratory Supercomplexes from Purified Saccharomyces cerevisiae Complexes III and IV
Cardiolipin-dependent Reconstitution of Respiratory Supercomplexes from Purified Saccharomyces cerevisiae Complexes III and IV
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DOI：
10.1074/jbc.m112.425876
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发表时间：
2013-01-04
影响因子：
4.8
通讯作者：
Dowhan, William
中科院分区：
文献类型：
--
作者：
Bazan, Soledad;Mileykovskaya, Eugenia;Dowhan, William
在这里，我们首次报道了从单个复合物III和IV中体外重建呼吸超复合物。复合物III和IV是从酿酒酵母线粒体中纯化出来的。通过电喷雾电离-质谱法测定，配合物III含有8分子心磷脂，配合物IV含有2分子心磷脂。复合物IV也含有Rcf1p。纯化后的配合物混合后未形成超配合物，重组成仅含磷脂酰胆碱和磷脂酰乙醇胺的蛋白脂质体后形成少量的超配合物三聚体III2IV1。在蛋白脂质体重组混合物中进一步加入心磷脂，可形成III2IV1超复合物三聚体和III2IV2超复合物四聚体。在支持四聚体形成方面，没有其他阴离子磷脂像心磷脂那样有效。磷脂酶处理复合物IV阻止三聚体形成在缺乏心脏脂素。心磷脂恢复了三聚体和四聚体的形成。用单粒子电子显微镜对重组的四聚体进行分析，证实了超配合物中单个配合物的天然组织。总之，虽然一些三聚体的形成只依赖于紧密结合的心脂素，但四聚体的形成需要额外的心脂素。这与天然四聚体中高心磷脂含量一致。超复合体形成对心磷脂的依赖表明，生理条件变化导致的心磷脂水平的变化可能控制体内个体呼吸复合体和超复合体之间的平衡。
Here, we report for the first time in vitro reconstitution of the respiratory supercomplexes from individual complexes III and IV. Complexes III and IV were purified from Saccharomyces cerevisiae mitochondria. Complex III contained eight molecules of cardiolipin, and complex IV contained two molecules of cardiolipin, as determined by electrospray ionization-mass spectrometry. Complex IV also contained Rcf1p. No supercomplexes were formed upon mixing of the purified complexes, and low amounts of the supercomplex trimer III2IV1 were formed after reconstitution into proteoliposomes containing only phosphatidylcholine and phosphatidylethanolamine. Further addition of cardiolipin to the proteoliposome reconstitution mixture resulted in distinct formation of both the III2IV1 supercomplex trimer and III2IV2 supercomplex tetramer. No other anionic phospholipid was as effective as cardiolipin in supporting tetramer formation. Phospholipase treatment of complex IV prevented trimer formation in the absence of cardio-lipin. Both trimer and tetramer formations were restored by cardiolipin. Analysis of the reconstituted tetramer by single particle electron microscopy confirmed native organization of individual complexes within the supercomplex. In conclusion, although some trimerformation ocurred dependent only on tightly bound cardio-lipin, tetramer formation required additional cardiolipin. This is consistent with the high cardiolipin content in the native tetramer. The dependence on cardiolipin for supercomplex formation suggests that changes in cardiolipin levels resulting from changes in physiological conditions may control the equilibrium between individual respiratory complexes and supercomplexes in vivo.