Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. [Display omitted] •Msp1 is necessary and sufficient to dislocate TA proteins from membranes•The AAA domain of Msp1 forms ATP-dependent hexamers in solution•Conserved residues in the central pore of Msp1 are essential for TA protein extraction•Heterologous TMD sequences can functionally substitute for the Msp1 TMD The AAA ATPase Msp1 plays a central role in clearing mislocalized tail-anchored proteins from the outer mitochondrial membrane. Wohlever et al. use a purified, reconstituted system and structural analysis to show that Msp1 is necessary and sufficient for the ATP-dependent extraction of fully integrated TA proteins from the bilayer.