Kasashima Katsumi, Endo Hitoshi
Department of Biochemistry, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan.
Genes Cells. 2015 Dec;20(12):1017-27. doi: 10.1111/gtc.12306. Epub 2015 Oct 7.
Mitochondrial transcription factor A (TFAM) is a key regulator of mitochondrial DNA (mtDNA). TFAM interacts with itself and forms dimers; however, the precise interaction domain in vivo has not yet been determined. We herein showed that human TFAM formed oligomers in mitochondria by in situ chemical cross-linking. We used the separated fluorescent protein, monomeric Kusabira-Green, as a reporter to monitor their self-association in mitochondria. This reporter successfully detected the TFAM-TFAM interaction in cells as fluorescent signals on mitochondria. We also found that the N-terminal high-mobility group box domain was sufficient for this interaction. The expression of the dimer-defective mutant induced enlarged mtDNA nucleoids, suggesting the importance of dimerization in the distribution of mtDNA. The reporter system also supported the association and mixture between independent nucleoids through TFAM by a cell fusion assay using hemagglutinating virus of Japan. We here, for the first time, visualized the interaction of TFAM molecules in mitochondria and proposed its implications for the dynamics of mtDNA nucleoids.
线粒体转录因子A（TFAM）是线粒体DNA（mtDNA）的关键调节因子。TFAM可与自身相互作用并形成二聚体；然而，其在体内的确切相互作用结构域尚未确定。我们在此表明，人类TFAM通过原位化学交联在线粒体中形成寡聚体。我们使用分离的荧光蛋白单体库萨比拉绿作为报告分子，以监测其在线粒体中的自我缔合。该报告分子成功地将细胞中TFAM与TFAM的相互作用检测为线粒体上的荧光信号。我们还发现，N端高迁移率族框结构域足以实现这种相互作用。二聚体缺陷型突变体的表达导致mtDNA类核增大，这表明二聚化在mtDNA分布中具有重要作用。该报告系统还通过使用日本血凝病毒的细胞融合试验，支持了独立类核通过TFAM的缔合和混合。我们首次可视化了线粒体中TFAM分子的相互作用，并提出了其对mtDNA类核动态变化的影响。
