Moore A L, Walters A J, Thorpe J, Fricaud A C, Watts F Z
Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton, U.K.
Yeast. 1992 Nov;8(11):923-33. doi: 10.1002/yea.320081103.
A technique is described for the isolation and purification of intact, respiratory-competent mitochondria from Schizosaccharomyces pombe. The purified mitochondria are capable of oxidizing NADH and succinate as respiratory substrates, indicating the presence of succinate dehydrogenase and an NADH dehydrogenase located on the outer surface of the inner membrane. Mitochondria display good respiratory control with an ADP/O ratio of < 2. Respiratory activity is linearly dependent upon the redox poise of the quinone pool, suggesting the presence of an unbranched respiratory pathway to molecular oxygen. Immunogold labelling using antisera raised against mitochondrial HSP70 proteins (SSP1, SSC1 and PHSP1) from three different species, namely S. pombe, Saccharomyces cerevisiae and the plant Pisum sativum respectively, has been used to investigate the presence and ultrastructure of the mitochondria isolated by this procedure. The immunocytochemistry was carried out using cells containing wild-type levels of SSP1 protein and cells over-expressing the protein. These results also demonstrate the capacity of mitochondria to import increased levels of protein in vivo. In vitro import experiments using COXIV-DHFR indicate that purified S. pombe mitochondria can efficiently import this precursor, and that protein translocation is dependent upon an oxidizable substrate and a membrane potential.
本文描述了一种从粟酒裂殖酵母中分离和纯化完整的、具有呼吸功能的线粒体的技术。纯化后的线粒体能够氧化NADH和琥珀酸作为呼吸底物，这表明在内膜外表面存在琥珀酸脱氢酶和NADH脱氢酶。线粒体表现出良好的呼吸控制，ADP/O比值<2。呼吸活性与醌池的氧化还原状态呈线性相关，表明存在一条通向分子氧的无分支呼吸途径。分别使用针对来自粟酒裂殖酵母、酿酒酵母和植物豌豆三种不同物种的线粒体HSP70蛋白（SSP1、SSC1和PHSP1）产生的抗血清进行免疫金标记，以研究通过该程序分离的线粒体的存在和超微结构。免疫细胞化学实验使用了含有野生型水平SSP1蛋白的细胞和过表达该蛋白的细胞。这些结果还证明了线粒体在体内导入增加水平蛋白质的能力。使用COXIV-DHFR进行的体外导入实验表明，纯化的粟酒裂殖酵母线粒体能够有效导入该前体，并且蛋白质转运依赖于可氧化底物和膜电位。
