Phosphomimetic Experiments Do Not Support a Causal Role for TFAM Phosphorylation in mtDNA Elimination in Sperm

In sexually reproducing eukaryotes-particularly mammals-mitochondrial DNA (mtDNA) is typically inherited from a single parent, making uniparental mtDNA inheritance a fundamental feature of eukaryotic biology. Recently, it has been suggested that spermatozoa contain no mtDNA because the matrix targeting sequence (MTS) of the mitochondrial transcription factor A (TFAM) becomes phosphorylated, which prevents the mitochondrial import of this protein essential for mtDNA replication. In this study, we used a combination of the GeneSwap technique and phosphomimetic mutations to investigate the impact of TFAM MTS phosphorylation on mtDNA maintenance in cultured cells. TFAM variants carrying phosphomimetic substitutions-S31D, S34D (TFAM-DD), and the double mutants S31D, P32D/S34D, F35D (TFAM-4D)-supported mtDNA maintenance in 143B cells, with their MTSs at least partially processed. This occurred despite the overall negative charge of the MTS in the TFAM-4D variant. Moreover, blocking the MTS processing by a combination of an overall negative charge and a mutation in the arginine residue critical for MTS cleavage did not prevent mtDNA maintenance. These observations led us to conclude that TFAM MTS phosphorylation alone is unlikely to explain mtDNA loss in human sperm during maturation.