Abstract
The AAA protein Msp1 extracts mislocalized tail-anchored membrane proteins and targets them for degradation, thus maintaining proper cell organization. How Msp1 selects its substrates and firmly engages them during the energetically unfavorable extraction process remains a mystery. To address this question, we solved cryo-EM structures of Msp1-substrate complexes at near-atomic resolution. Akin to other AAA proteins, Msp1 forms hexameric spirals that translocate substrates through a central pore. A singular hydrophobic substrate recruitment site is exposed at the spiral's seam, which we propose positions the substrate for entry into the pore. There, a tight web of aromatic amino acids grips the substrate in a sequence-promiscuous, hydrophobic milieu. Elements at the intersubunit interfaces coordinate ATP hydrolysis with the subunits' positions in the spiral. We present a comprehensive model of Msp1's mechanism, which follows general architectural principles established for other AAA proteins yet specializes Msp1 for its unique role in membrane protein extraction.
Highlights
ATPases associated with diverse cellular activities (AAA proteins) utilize the energy of ATP hydrolysis to facilitate numerous functions in the cell, such as degrading proteins (Pickart and Cohen, 2004), dissolving protein aggregates (Sanchez and Lindquist, 1990), or moving proteins across membranes (Ye et al, 2001; Gardner et al, 2018)
Pore loops projecting from the ATPases engage with the polypeptide and with each M6fiM1 conversion cycle translocate it in steps of two amino acids as the spiral crawls along the substrate (Gates et al, 2017; Monroe et al, 2017; Puchades et al, 2017; de la Pena et al, 2018; Dong et al, 2019)
To obtain a homogeneous sample suitable for structural studies, we expressed the cytosolic domain of Msp1/ATAD1 both X1 and X2 are aromatic (Msp1) lacking its 30 amino acid N-terminal membrane anchor (D30-Msp1) from the thermophilic yeast Chaetomium thermophilum (Figure 1—figure supplement 2)
Summary
ATPases associated with diverse cellular activities (AAA proteins) utilize the energy of ATP hydrolysis to facilitate numerous functions in the cell, such as degrading proteins (Pickart and Cohen, 2004), dissolving protein aggregates (Sanchez and Lindquist, 1990), or moving proteins across membranes (Ye et al, 2001; Gardner et al, 2018). Pore-loop three is short and, likewise, does not contact the substrate directly Despite these common architectural features, AAA proteins comprise a diverse superfamily. TA proteins that escape this reaction are mistargeted to the mitochondrial outer membrane (MOM), necessitating their removal This proofreading function is performed by Msp1/ATAD1, which extracts the mistargeted TA proteins from the MOM to facilitate their subsequent proteasomal degradation (Dederer et al, 2019; Matsumoto et al, 2019). If and how Msp1’s pore-loop two contacts the substrate remained unknown Another common feature among the AAAMC proteins lies in sequences, referred to as intersubunit signaling (ISS) motifs (Augustin et al, 2009), ISS motifs transmit information regarding the adenosine nucleotide-bound state between adjacent subunits and synchronize ATP hydrolysis with poreloop movement through allosteric conformational changes. In Msp the most N-terminal region of the N-domain
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