The N‐terminal segment of the human <scp>ANKZF1</scp> negatively regulates its internal mitochondrial targeting signal to prevent localization to mitochondria

ANKZF1, the mammalian ortholog of yeast Vms1, is a multidomain cytosolic protein. Occasionally, the protein is also found in mitochondria, although the reason for its mitochondrial localization and the mechanism of its mitochondrial targeting remain 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 demonstrate that the N-terminal 73 residues of ANKZF1 negatively regulate its mitochondrial targeting, and this portion of the protein is sufficient to hinder mitochondrial targeting of Δ73-ANKZF1 upon co-expression. Using a series of truncation mutants of ANKZF1, we further demonstrate that the iMTS-Ls comprised of residues 231-240 of ANKZF1 are essential for its mitochondrial localization. Furthermore, the sequence of this region is well conserved across different organisms, indicating the structural and functional importance and role in maintaining the mitochondrial targeting of the protein. Importantly, residues 231-324 of ANKZF1 form two consecutive predicted iMTS-Ls, which together constitute an independent mitochondrial signal sequence that can target green fluorescent protein (GFP) to the mitochondria when fused to the N terminus. Furthermore, by molecular dynamics simulation, we show that the deletion of the N-terminal 74 amino acids of ANKZF1 leads to a massive structural rearrangement within the protein, causing the opening of its C-terminal part and solvent exposure of the 231-240 residues. We postulate that these structural rearrangements and exposure of the internal MTS in the absence of the N-terminal segment of ANKZF1 lead to its mitochondrial translocation.