Miyakawa Isamu, Sato Hiroshi, Maruyama Yukari, Nakaoka Tomoko
Department of Physics, Informatics and Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan.
J Gen Appl Microbiol. 2003 Apr;49(2):85-93. doi: 10.2323/jgam.49.85.
Mitochondrial (mt) nucleoids were isolated from yeast Kluyveromyces lactis with morphological intactness. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed more than 20 proteins that are associated with the mt-nucleoids. However, the protein profile of the mt-nucleoids of K. lactis was significantly different from that of the mt-nucleoid proteins from Saccharomyces cerevisiae. SDS-DNA PAGE, which detected an Abf2p, a major mitochondrial DNA-binding protein, among the mt-nucleoid proteins of S. cerevisiae on a gel, detected only a 17-kDa protein in the K. lactis mt-nucleoid proteins. The 17-kDa protein was purified as homogeneous from the mt-nucleoids by a combination of acid extraction, hydroxyapatite chromatography and DNA-cellulose chromatography. The 17-kDa protein introduced a negative supercoil into circular plasmid DNA in the presence of topoisomerase I, as does S. cerevisiae Abf2p, and it packed K. lactis mtDNA into nucleoid-like particles in vitro. These results, together with the determination of the N-terminal amino acid sequence, suggested that the 17-kDa protein is an Abf2p homologue of K. lactis and plays structural roles in compacting mtDNA in cooperation with other nucleoid proteins.
从乳酸克鲁维酵母中分离出形态完整的线粒体（mt）类核。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示有20多种蛋白质与mt类核相关。然而，乳酸克鲁维酵母mt类核的蛋白质谱与酿酒酵母mt类核蛋白质的谱显著不同。SDS-DNA PAGE在凝胶上检测到酿酒酵母mt类核蛋白质中的一种主要线粒体DNA结合蛋白Abf2p，而在乳酸克鲁维酵母mt类核蛋白质中仅检测到一种17 kDa的蛋白质。通过酸提取、羟基磷灰石色谱和DNA纤维素色谱相结合的方法，从mt类核中纯化出了均一的17 kDa蛋白质。与酿酒酵母Abf2p一样，17 kDa蛋白质在拓扑异构酶I存在的情况下将负超螺旋引入环状质粒DNA，并在体外将乳酸克鲁维酵母mtDNA包装成类核样颗粒。这些结果，连同N端氨基酸序列的测定，表明17 kDa蛋白质是乳酸克鲁维酵母的Abf2p同源物，并与其他类核蛋白协同在压缩mtDNA中发挥结构作用。
