Mitochondrial AAA‐ATPase Msp1 detects mislocalized tail‐anchored proteins through a dual‐recognition mechanism

Abstract

The conserved AAA‐ATPase Msp1 is embedded in the outer mitochondrial membrane and removes mislocalized tail‐anchored (TA) proteins upon dysfunction of the guided entry of tail‐anchored (GET) pathway. It remains unclear how Msp1 recognizes its substrates. Here, we extensively characterize Msp1 and its substrates, including the mitochondrially targeted Pex15Δ30, and full‐length Pex15, which mislocalizes to mitochondria upon dysfunction of Pex19 but not the GET pathway. Moreover, we identify two new substrates, Frt1 and Ysy6. Our results suggest that mislocalized TA proteins expose hydrophobic surfaces in the cytoplasm and are recognized by Msp1 through conserved hydrophobic residues. Introducing a hydrophobic patch into mitochondrial TA proteins transforms them into Msp1 substrates. In addition, Pex15Δ30 and Frt1 contain basic inter‐membrane space (IMS) residues critical for their mitochondrial mistargeting. Remarkably, Msp1 recognizes this feature through the acidic D12 residue in its IMS domain. This dual‐recognition mechanism involving interactions at the cytoplasmic and IMS domains of Msp1 and substrates greatly facilitates substrate recognition and is required by Msp1 to safeguard mitochondrial functions.

Cooperation of mitochondrial and ER factors in quality control of tail-anchored proteins

“…This raises the question of how Msp1 recognizes its substrates in the OMM membrane. Recent analysis suggested the involvement of a juxtamembrane cytoplasmic hydrophobic patch in Pex15 is important for Msp1-mediated extraction (Li et al, 2019). However, Fmp32 and Gem1 do not show this feature (Figure 6—figure supplement 1A).…”

“…In vitro studies suggest that Msp1 is sufficient to extract TA proteins from proteoliposomes (Wohlever et al, 2017). Although these reconstitution experiments showed that the Msp1 N-terminal TM domain is dispensable for the dislocation reaction, recently Li et al suggested that a conserved negatively charged aspartate residue in the N-terminal portion of Msp1 facing the inter membrane space (IMS) is important for efficient recognition of Msp1 clients in vivo (Li et al, 2019). Moreover, a hydrophobic surface of the AAA-ATPase domain has been implicated in recognition of Msp1 clients (Li et al, 2019).…”

“…Although these reconstitution experiments showed that the Msp1 N-terminal TM domain is dispensable for the dislocation reaction, recently Li et al suggested that a conserved negatively charged aspartate residue in the N-terminal portion of Msp1 facing the inter membrane space (IMS) is important for efficient recognition of Msp1 clients in vivo (Li et al, 2019). Moreover, a hydrophobic surface of the AAA-ATPase domain has been implicated in recognition of Msp1 clients (Li et al, 2019). However, no clear sequence consensus between all known Msp1 clients exists (Chen et al, 2014; Li et al, 2019; Okreglak and Walter, 2014) and the mechanism how Msp1 distinguishes between mislocalized and properly located TA proteins is ill defined.…”

“…Moreover, a hydrophobic surface of the AAA-ATPase domain has been implicated in recognition of Msp1 clients (Li et al, 2019). However, no clear sequence consensus between all known Msp1 clients exists (Chen et al, 2014; Li et al, 2019; Okreglak and Walter, 2014) and the mechanism how Msp1 distinguishes between mislocalized and properly located TA proteins is ill defined. This, taken together with the finding that Cis1 can recruit Msp1 to TOM for degradation of stalled import intermediates (Weidberg and Amon, 2018), indicates that multiple routes can target proteins for Msp1-mediated dislocation.…”

Protein quality control in the endoplasmic reticulum

“…On the other hand, when ER membrane proteins are mislocalised, either to the mitochondria ( Figure 2 ) or the peroxisome, they are extracted and targeted for degradation by the hexametric ATPase Msp1 [ 15 , 16 ]. Recognition of such mislocalised substrates probably involves assessment of oligomeric state [ 17 , 18 ] and detection of characteristic hydrophobic and charged motifs adjacent to the transmembrane domain [ 19 ]. Substrates mistargeted to mitochondria are extracted by Msp1 near ER contact sites before ubiquitination and degradation at the ER membrane in a process involving the traditional ERAD components Doa10 and Cdc48 [ 17 , 20 , 21 ].…”