Functional Studies Of Mitofilin And CHCHD6 In Regulating Mitochondrial Structure And Function

Mitochondria possess an outer membrane (OMM) and an inner membrane (IMM), which invaginates to form cristae membrane (CM). The maintenance of CM mainly depends on the mitochondrial contact site (MICOS) complex, which also has a function in mitochondrial protein import.Mitofilin and CHCHD6, which physically interact, are core and novel components of the MICOS respectively. In this study, we generated knockdown/knockout HeLa clones of Mitofilin and CHCHD6 by using transcription activator-like effector nucleases (TALENs). Transmission electron microscopy (TEM) revealed that vesicle-like cristae morphology appeared in cell lines lacking Mitofilin, and mitochondria exhibited lower cristae density in CHCHD6-knockout cells. Immunoblot analysis showed that knockdown of Mitofilin, but not knockout of CHCHD6, affected their binding partners in controlling cristae morphology. We next performed immunoprecipitation and mass spectra (IP-MS) experiments with Mitofilin and CHCHD6 antibodies respectively. Although we did not find out new MICOS components, we directly identified a complex containing Mitofilin, Sam50, and CHCHD 3 and 6. We also demonstrated that Mitofilin and CHCHD6 directly interact with Sam50 and CHCHD6 can interact with Mitofilin and OPA1 in cristae junctions (CJs). In addition, we observed that Mitofilin-knockdown cells showed decreased mitochondrial membrane potential (??m) and intracellular ATP content, which were minimally affected in CHCHD6-knockout cells. Taken together, we conclude that the integrity of MICOS and its efficient interaction with Sam50 are indispensable for cristae organization, which is linked to mitochondrial function.As a core component of MICOS complex, Mitofilin has been reported to interact with the TOM and SAM complex in yeast. In this study, we found that Mitofilin can interact with core components of the SAM and TIM23 complex in HeLa cells whereas Mitofilin can not interact with core components of the TOM and TIM22 complex. We also demonstrated that expressions of some IMM proteins, such as OPA1, CHCHD3 and CHCHD6,were decreased in Mitofilin-knockdown cells. These results indicate that Mitofilin may promote mitochondrial protein import by interacting with Sam50 and Tim23 in mammalian cells.Additionally, we focused on Lamin A/C and Vimentin, which were indicated as new Mitofilin binding partners in the IP-MS result of Mitofilin. Lamin A/C are components of the nuclear lamina which functions in governing the separation of the nuclear content from the rest of the cell. Mutations in the gene encoding Lamin A/C cause a group of rare genetic diseases named laminopathies. Vimentin is one of the intermediate filaments that functions in structural support, signal transduction and organelle positioning of a cell. Vimentin has been reported to support mitochondrial morphology and organization. In this study, we found that the expression of Lamin A/C are decreased in Mitofilin knockdown cells whereas they do not interact with Mitofilin. Additionally, we demonstrated that Vimentin is a binding partner of Mitofilin whereas its expression level and distribution is unchanged in Mitofilin knockdown cells.