Abstract
The mitochondrial AAA protein ATAD1 (in humans; Msp1 in yeast) removes mislocalized membrane proteins, as well as stuck import substrates from the mitochondrial outer membrane, facilitating their re-insertion into their cognate organelles and maintaining mitochondria’s protein import capacity. In doing so, it helps to maintain proteostasis in mitochondria. How ATAD1 tackles the energetic challenge to extract hydrophobic membrane proteins from the lipid bilayer and what structural features adapt ATAD1 for its particular function has remained a mystery. Previously, we determined the structure of Msp1 in complex with a peptide substrate (Wang et al., 2020). The structure showed that Msp1’s mechanism follows the general principle established for AAA proteins while adopting several structural features that specialize it for its function. We used the yeast growth assay to test the function of key amino acids within these structural elements. In this work, we determined the cryo-EM structures of the human ATAD1 in complex with a peptide substrate at near atomic resolution. The structures show that phylogenetically conserved structural elements adapt ATAD1 for its function while generally adopting a conserved mechanism shared by many AAA proteins. ATAD1’s distinguishing features include an abundance of aromatic amino acids that line the central pore proposed to give ATAD1 a firm grip on its substrate and a short α-helix at the C-terminus that strongly facilitates ATAD1’s oligomerization. We also developed a microscopy-based assay reporting on protein mislocalization, with which we directly assessed ATAD1’s activity in live cells.
Competing Interest Statement
The authors have declared no competing interest.
