Definition of the composition of the mitochondrial nucleoid in mice

Mitochondrial DNA (mtDNA) is compacted into dynamic structures called mitochondrial nucleoids (mt-nucleoids), with the mitochondrial transcription factor A (TFAM) as the core packaging protein. We generated Tfam-FLAGBAC mice to investigate the mt-nucleoid composition in vivo. Importantly, we showed that the TFAM-FLAG protein is functional and complements the absence of the wild-type TFAM. We identified proteins as core mt-nucleoid components by proteomics analysis of Tfam-FLAGBAC mice. Surprisingly, the mitoribosome subunit MRPS38 was part of the mt-nucleoid, and its downregulation led to the increase of mtDNA replication and transcription. Thus, MRPS38 might have a novel function repressing mtDNA expression. Also, we identified proteins that may stabilize TFAM-FLAG upon depletion of the mitochondrial RNA polymerase despite the absence of mtDNA and induction of the LONP1 protease. Finally, we evaluated the changes in mt-nucleoids caused by very high levels of TFAM-FLAG and discovered eight new interactors that could counteract the high TFAM levels to maintain active mtDNA transcription. Altogether, we demonstrate that the Tfam-FLAGBAC mice are a valuable tool for investigating the mt-nucleoid composition in vivo.