Human 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. Some regulators of CI assembly may not directly interact with any of the 44 CI subunits, but rather interact with CIAFs to regulate CI assembly indirectly. For instance, they may regulate the stability, subcellular localization, degree of post-translational modification, extent of activation, etc. of a CIAF. We refer to this class of regulators as remote regulators (RRs) of CI assembly. We hypothesize that at least some CI disorders may be attributed to mutations in RRs, many of which have not yet been discovered. The ideal model system for discovering RRs 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 RRs 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. Here, we propose to use a genetic and proteomic approach to identify novel RRs of CI assembly in this system; 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 RRs of CI assembly.
Impaired complex I function has been associated with a host of chronic metabolic and degenerative disorders such as cardiovascular disease and Sarcopenia. Therefore identifying novel regulators of CI assembly has the potential to open up therapeutic opportunities for combating the numerous diseases linked to destabilization of CI.
