In organello formaldehyde crosslinking of proteins to mtDNA:: Identification of bifunctional proteins
In organello formaldehyde crosslinking of proteins to mtDNA:: Identification of bifunctional proteins
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DOI：
10.1073/pnas.140063197
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发表时间：
2000-07-05
影响因子：
11.1
通讯作者：
Butow, RA
中科院分区：
文献类型：
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作者：
Kaufman, BA;Newman, SM;Butow, RA
线粒体DNA的分离单位是蛋白质-DNA复合物，称为类核。为了了解类核蛋白如何促进mtDNA的组织和遗传，我们开发了一种细胞器内甲醛交联方法来鉴定与mtDNA相关的蛋白质。使用高度纯化的线粒体，我们观察到一个时间依赖性的蛋白质与mtDNA的交联，通过等密度氯化铯梯度沉降确定。我们检测到大约20种与mtDNA交联的蛋白质，并确定了11种，主要是通过质谱法。其中之一是Abf 2 p，这是一种丰富的高迁移率族蛋白，已知在类核形态和mtDNA交易中发挥作用。除了其他几种具有已知DNA结合特性或在mtDNA维持中起作用的蛋白质外，我们还鉴定了其他未预料到的mtDNA相关蛋白质，如分子伴侣Hsp 60 p和克雷布斯循环蛋白Kgd 2 p。遗传实验表明，hsp 60-ts突变体在允许温度下具有小诱导表型，kgd 2 δ突变增加了abf 2 δ突变的小诱导表型。交联和DNA凝胶转移实验表明，热休克蛋白60 p结合到单链DNA与一个假定的mtDNA复制起点的模板链的高度特异性。这些数据确定了参与rho（+）mtDNA稳定性的双功能蛋白质。
The segregating unit of mtDNA is a protein-DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNA organization and inheritance, we have developed an in organello formaldehyde crosslinking procedure to identify proteins associated with mtDNA. Using highly purified mitochondria, we observed a time-dependent crosslinking of protein to mtDNA as determined by sedimentation through isopycnic cesium chloride gradients. We detected approximate to 20 proteins crosslinked to mtDNA and identified 11, mostly by mass spectrometry. Among them is Abf2p, an abundant, high-mobility group protein that is known to function in nucleoid morphology, and in mtDNA transactions. In addition to several other proteins with known DNA binding properties or that function in mtDNA maintenance, we identified other mtDNA-associated proteins that were not anticipated, such as the molecular chaperone Hsp60p and a Krebs cycle protein, Kgd2p. Genetic experiments indicate that hsp60-ts mutants have a petite-inducing phenotype at the permissive temperature and that a kgd2 Delta mutation increases the petite-inducing phenotype of an abf2 Delta mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with high specificity for the template strand of a putative origin of mtDNA replication. These data identify bifunctional proteins that participate in the stability of rho(+) mtDNA.