Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells.
Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells.
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DOI：
10.1038/srep46055
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发表时间：
2017-04-06
影响因子：
4.6
通讯作者：
Busch KB
中科院分区：
文献类型：
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作者：
Rieger B;Shalaeva DN;Söhnel AC;Kohl W;Duwe P;Mulkidjanian AY;Busch KB
将呼吸复合物组装成大分子超复合物是目前的一个热门话题，特别是在新获得结构细节的背景下。然而，迄今为止，大多数工作都是用纯化的洗涤剂溶解材料完成的，并且没有现场证实。我们在这里着手记录活细胞中呼吸超复合体的形成。荧光传感器蛋白被放置在细胞色素c氧化酶的特定位置，怀疑是在CI1CIII2CIV1超复合体的表面或埋在该超复合体中。与其他基因座相比，亚基coxviia和CoxVIIc上的传感器报告了密集的蛋白质环境，通过显着缩短的荧光寿命检测到。根据三维建模，coxviia和CoxVIIc埋在CI1CIII2CIV1超复合体中。抑制超复合体支架蛋白HIGD2A和CoxVIIa2l伴随着coxviia -传感器寿命的增加，这与从超复合体中释放CIV一致。引人注目的是，我们的数据为原位定义的稳定超复杂结构提供了强有力的证据。
The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1 supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1 supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.