Identifying Novel Regulators of Mitochondrial Complex I Biogenesis

项目总结 线粒体复合体I(CI)由44个不同的亚基组成，这些亚基与8个铁-S组装在一起 簇和单个黄素单核苷酸，形成起作用的酶。辅助蛋白被称为 组装因素有助于组装过程；十几个真正的CI组装因素(CIAF)具有 被刻画出来了。然而，大约一半的CI障碍不能追溯到44个CI亚基中的任何一个的突变 或已知的组装因子，这表明CI生物发生的其他调节因子仍有待表征。 发现CI组装的新调节子的理想模型系统至少必须满足4个标准：(I) CI的组装机制应该与人酶的机制非常相似，(Ii)它应该高度富含 线粒体能够相当容易地检测1000个候选基因对CI组装的影响，(Iii) 这样的有机体中的遗传工具箱应该显著地进步到这样的程度，即数千秒的影响 可以快速检测CI组件上的候选基因，最后(Iv)应该可以分析CI组件 在体内，它同时受到发育和环境信号的影响，不容易出现细胞培养假象。 目前还没有研究CI组装的模型系统(在粗糙脉孢菌和各种哺乳动物细胞中 行)满足所有4个标准。为了促进发现CI组装的新调节子，我们正在使用 果蝇线粒体丰富的飞行肌肉作为研究CI组装的新系统 标准。我们发现果蝇骨骼肌CI的生物发生是通过形成~315-，~370-， 已在哺乳动物系统中描述的~550-和~815 kDa CI组装中间体；以及果蝇CI 其亚基的数量与人类的酶相当。重要的是，果蝇同源基因的突变 在人类中描述的CIAF也损害了果蝇的CI组装，进一步表明CI的机制 装配在人类和果蝇之间是保守的。因为NDUFS3(44个CI亚基之一)具有 CI生物发生中的核心作用我们假设CI组装的一些调节因子可能直接与 NDUFS3.在这里，我们建议使用遗传学和蛋白质组学的方法来确定CI组装的新的调节因子 与NDUFS3相互作用；并在果蝇和人类细胞中测试我们的候选调节因子。轻松自如 从飞行肌肉中分离出大量的线粒体，大量的遗传分析工具， 果蝇相对较短的世代时间和有限的基因冗余应该有助于 发现CI组装的新调控因子。

PROJECT SUMMARY Mitochondrial Complex I (CI) is composed of 44 distinct subunits, that are assembled together with eight Fe-S clusters and a single flavin mononucleotide, to form a functioning enzyme. Ancillary proteins referred to as assembly factors assist with the assembly process; and a dozen or so bona fide CI assembly factors (CIAFs) have been characterized. However, about half of CI disorders cannot be traced to mutations in any of the 44 CI subunits or known assembly factors, which suggests that additional regulators of CI biogenesis remain to be characterized. The ideal model system for discovering new regulators of CI assembly will have to satisfy at least 4 criteria: (i) the mechanism of CI assembly should closely mimic that of the human enzyme, (ii) it should be highly enriched with mitochondria to enable the examination of the effects of 1000s of candidate genes on CI assembly rather easily, (iii) the genetic tool kit in such an organism should be significantly advanced to the point where the effects of 1000s of candidate genes on CI assembly can rapidly be tested, and finally (iv) it should be possible to analyze CI assembly in vivo where it is subject to both developmental and environmental signals, and not prone to cell culture artifacts. None of the current model systems for studying CI assembly (in Neurospora crassa and various mammalian cell lines) satisfy all 4 criteria. To facilitate the discovery of novel regulators of CI assembly, we are using the mitochondria-enriched flight muscles in Drosophila as a novel system to study CI assembly as it satisfies all four criteria. We find that CI biogenesis in Drosophila skeletal muscles proceeds via the formation of ~315-, ~370-, ~550-, and ~815 kDa CI assembly intermediates as has been described in mammalian systems; and Drosophila CI has a comparable number of subunits as the human enzyme. Importantly, mutations in Drosophila orthologs of CIAFs described in humans, also impair CI assembly in Drosophila, further showing that the mechanism of CI assembly is conserved between humans and Drosophila. Because NDUFS3 (one of the 44 CI subunits) has a central role in CI biogenesis we hypothesize that some regulators of CI assembly may directly interact with NDUFS3. Here, we propose to use a genetic and proteomic approach to identify novel regulators of CI assembly that interact with NDUFS3; and test our candidate regulators in both Drosophila and human cells. The ease of isolating copious amounts of mitochondria from flight muscles, extensive arsenal of tools for genetic analyses, relatively short generation time, and limited gene redundancy in Drosophila are assets that should facilitate the discovery of new regulators of CI assembly.