The N-terminal Segment of the Human ANKZF1 negatively regulates its internal mitochondrial targeting signal to prevent its mitochondrial localization

ANKZF1, the human orthologue of yeast Vms1, is a multi-domain cytosolic protein. Occasionally, the protein is also found in mitochondria, although the reason for its mitochondrial localization and the mechanism of its mitochondrial targeting remains unclear. Despite the absence of any predicted Mitochondrial Targeting Sequence or Domain (MTS/MTD) in the protein, ANKZF1 possesses multiple internal Matrix-targeting sequence-like sequences (iMTS-Ls). In this study, we show that ANKZF1’s N-terminal 73 residues negatively regulate its mitochondrial targeting. Interestingly, 1-72-ANKZF1 peptide hinders the mitochondrial targeting of Δ73-ANKZF1 upon co-expression. Using a series of truncation mutants of ANKZF1, we further show that iMTS-Ls constituted by residues 231-240 of ANKZF1 is indispensable for its mitochondrial localization. The sequence of 231-240 residues is extremely conserved across different organisms, indicating the importance of this segment for conditional mitochondrial targeting of the protein. Importantly, 231-324 residues containing two consecutive predicted iMTS-Ls constitute an independent mitochondrial signal sequence and can target Green Fluorescent Protein (GFP) to mitochondria when fused to N-terminus. Further, by Molecular Dynamic simulation, we show that the deletion of N-terminal 74 amino acids of ANKZF1 leads to a massive structural rearrangement within the protein leading to the opening of its C-terminal part and solvent exposure of the 231-240 residues. We posit that these structural rearrangements and exposure of the internal mitochondrial targeting sequence in the absence of N-terminal segment of ANKZF1 leads to its mitochondrial translocation.