Sorting out how Msp1 maintains mitochondrial membrane proteostasis

Accepted Manuscript Sorting out how Msp1 maintains mitochondrial membrane proteostasis

Heidi L. Fresenius, Matthew L. Wohlever PII: DOI: Reference:

S1567-7249(19)30076-5 https://doi.org/10.1016/j.mito.2019.07.011 MITOCH 1392

To appear in:

Mitochondrion

Received date: Accepted date:

4 April 2019 31 July 2019

Please cite this article as: H.L. Fresenius and M.L. Wohlever, Sorting out how Msp1 maintains mitochondrial membrane proteostasis, Mitochondrion, https://doi.org/10.1016/ j.mito.2019.07.011

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ACCEPTED MANUSCRIPT

Sorting Out How Msp1 Maintains Mitochondrial Membrane Proteostasis Heidi L. Fresenius, Matthew L. Wohlever*

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Department of Chemistry & Biochemistry, University of Toledo, Toledo, OH 43606, USA

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* Corresponding Author, [email protected]

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Declarations of interest: none

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Abstract

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Robust membrane proteostasis networks are essential for cells to withstand proteotoxic stress arising from environmental insult and intrinsic errors in protein production1,2. Failures in mitochondrial membrane proteostasis are associated with cancer, aging, and a range of cardiovascular and neurodegenerative diseases3-5. As a result, mitochondria possess numerous pathways to maintain proteostasis6-11. Mitochondrial Sorting of Proteins 1 (Msp1) is a membrane anchored AAA ATPase that extracts proteins from the outer mitochondrial membrane (OMM)12,13. In the past few years, several papers have addressed various aspects of Msp1 function. Here, we summarize these recent advances to build a basic model for how Msp1 maintains mitochondrial membrane proteostasis while also highlighting outstanding questions in the field.

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Key Words

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Msp1, tail-anchored, AAA, proteostasis, mitochondria

MEF = Mouse Embryonic Fibroblasts Msp1 = Mitochondrial Sorting of Proteins 1

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OMM = Outer Mitochondrial Membrane TA = tail-anchored TMD = Transmembrane domain

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MM = Mitochondrial Matrix

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AAA = ATPase Associated with cellular Activities ER = Endoplasmic Reticulum GET = Guided Entry of Tail-Anchored IMM = Inner Mitochondrial Membrane IMS = Intermembrane Space

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Abbreviations

TOM = Translocase of Outer Mitochondrial Membrane

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1. Introduction

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Mitochondria serve as key hubs for metabolism, cellular signaling, and apoptotic regulation in eukaryotic cells14. Mitochondria have at least 1000 proteins in yeast or 1500 proteins in humans, over 99% of which are encoded in the nuclear genome and depend on specific targeting signals to direct them from the site of synthesis in the cytosol to the appropriate subcompartment15-17. As a double membrane organelle, there are four distinct targeting destinations for mitochondrial proteins: the outer mitochondrial membrane (OMM), the inner mitochondrial membrane (IMM), the intermembrane space (IMS), and the mitochondrial matrix (MM). Proper mitochondrial function depends on the ability of each subcompartment to maintain homeostasis of the proteome, a process called proteostasis. Failures in mitochondrial proteostasis are associated with a number of diseases, including cancer, diabetes, cardiovascular, and neurodegenerative diseases 18-21. Proteostasis depends on a dynamic balance of protein synthesis, trafficking to the correct subcompartment, and removal of unnecessary or damaged proteins. Membrane proteins present unique challenges to the proteostatic network as they must be targeted to the correct membrane and overcome substantial thermodynamic barriers to enter and exit the lipid bilayer, all while avoiding the formation of potentially toxic aggregates in the cytosol22. Consequently, specialized machinery has evolved to shield the hydrophobic TMD from the aqueous cytosol, target the nascent protein to the correct membrane, and facilitate membrane insertion17,23-31. These targeting pathways are imperfect, often resulting in proteins being sent to the wrong membrane or failing to properly fold in target membrane. For example, studies of the prion protein PrP in transgenic mice suggest a 3%-5% failure rate for membrane protein targeting32. Thus, cells require dedicated machinery to remove mislocalized or damaged membrane proteins from the lipid bilayer.

ACCEPTED MANUSCRIPT A common theme in membrane proteostasis is the use of AAA (ATPase Associated with cellular Activities) proteins to extract membrane proteins from the lipid bilayer6,7,12,33-38. AAA proteins form hexameric rings and undergo ATP-dependent conformational changes to remodel substrates, often by translocating substrates through a central pore37,39. Msp1 is a membrane anchored AAA protein found in the OMM and peroxisomes that maintains membrane proteostasis by extracting proteins from the lipid bilayer12,13. Over the past few years, a series of papers have illuminated the role of Msp1 in protein quality control, yet many of the key molecular details of Msp1 function remain obscure. Here, we summarize these papers with a focus on the role of Msp1 in mitochondrial membrane proteostasis. In the later part of the article, we present competing models for less well-understood aspects of Msp1 function, including: the molecular basis for substrate selectivity, the structure of the Msp1 hexamer, and the mechanism of substrate extraction.

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2.1 Msp1 removes mislocalized tail-anchored proteins from the OMM

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2. Mps1 in mitochondrial membrane proteostasis

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Msp1 has a single TMD at the N-terminus which anchors it to the OMM or the peroxisome membrane with the AAA domain facing the cytosol. Msp1 was originally identified in a genetic selection that looked for mutants which caused the missorting of an OMM protein to the inter membrane space and hence was named Mitochondrial Sorting of Proteins 1 40. Although it was identified as a membrane anchored ATPase, the function of Msp1 would remain mysterious for another two decades, likely because deletion of Msp1 in S. cerevisiae has a mild effect on yeast physiology.

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In 2014, papers from the Rutter and Walter labs demonstrated that Msp1 removes mislocalized tail-anchored (TA) proteins from the OMM12,13. Deletion of both Msp1 and components of the Guided Entry of Tail-Anchored (GET) pathway in S. cerevisiae led to the accumulation of mislocalized TA proteins in the OMM and severe mitochondrial defects such as: failures in oxidative phosphorylation, altered mitochondrial morphology and loss of mitochondrial DNA. TA proteins contain a single transmembrane domain (TMD) at the very C-terminus of the protein with the N-terminus facing the cytosol. This unique topology necessitates post-translational membrane insertion into the Endoplasmic Reticulum (ER) by the GET pathway or the OMM by a still uncharacterized pathway41-44.

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There are several factors which contribute to the mistargeting of TA proteins. First, an emerging theme in membrane protein targeting is that targeting factors have overlapping specificities. These overlaps provide partial redundancy when the primary targeting pathway is compromised but also result in intrinsic mistargeting, even under normal circumstances26,45,2. Second, the ultimate destination of a TA protein is determined by the biophysical characteristics of the TMD rather than a specific recognition sequence46-48. ER-TA proteins generally contain a more hydrophobic TMD than mitochondrial TA proteins, although the overlap is substantial, which increases the challenges of targeting fidelity22. Third, the fidelity of TA protein targeting is constrained by the dual functionality of the targeting signal. For single pass membrane proteins, the TMD often serves as both a targeting signal and a key motif for protein-protein interactions and may not be optimized for perfect targeting fidelity2. Thus, even under non-stress conditions, TA proteins are often mislocalized to the OMM2. Indeed, deletion of the Msp1 homolog ATAD1 in mouse embryonic fibroblasts results in severe mitochondrial defects independent of a compromised GET pathway12. Thus, the OMM requires a robust quality control system to remove these mislocalized TA proteins and Msp1 appears to be a key component of this quality control pathway.

2.2. Reconstitution of Msp1 activity These groundbreaking studies by the Rutter and Walter labs firmly established a role for Msp1 in mitochondrial proteostasis but left many important questions unanswered. Does Msp1 actually extract mislocalized proteins from the lipid bilayer or simply disperse aggregates on the surface of the membrane. Does Msp1 function autonomously or does it require other factors to remove substrates? How does Msp1 recognize substrates for extraction? To address these questions, we reconstituted Msp1 activity with fully purified components49. The assay consists of co-reconstituting Msp1 and a model TA protein (sumo-Sec22) into liposomes with lipid content that mimics the OMM50-52. SGTA, a chaperone for ER-TA proteins, is added to the reaction to serve as a sink for

ACCEPTED MANUSCRIPT extracted TA proteins53. The assay is initiated by addition of ATP and substrate removal is monitored by immunoprecipitation of SGTA and subsequent western blotting for the TA protein (Figure 1).

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Figure 1

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Figure 1: Reconstitution of Msp1 activity. A) Design of reconstituted assay. Following co-reconstitution, non-integrated TA proteins are removed by SGTA pull down to generate pre-cleared proteoliposomes. TA protein extraction is monitored by SGTA pull down, SDS-PAGE, and western blotting. B) Cartoon of extraction assay.

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Because this assay was trying to distinguish between disaggregase activity and membrane extraction, it is essential that the reconstituted material contain only proteins which are fully-integrated into the lipid bilayer. To remove proteins that are not integrated into the bilayer, we incubated the reconstituted material with SGTA and then did an SGTA immunoprecipitation prior to the extraction assay. This “pre-clearing” step significantly reduced the amount of non-integrated sumo-Sec22 and allowed us to determine that Msp1 indeed extracts substrates from the lipid bilayer49. Parallel work by the Denic lab used protease protection assays on isolated mitochondria to show that the mislocalized TA proteins are fully integrated into the lipid bilayer 54.

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Using this assay, we demonstrated that Msp1 is sufficient to remove TA proteins from a lipid bilayer. No additional proteins are required and the substrate does not need to be modified in any way for extraction to occur. This assay provides a powerful tool for studying Msp1 activity, but care needs to be taken in interpreting the results. Currently, the assay is only semi-quantitative. The concentration of enzyme and substrate on the surface of the proteoliposome are not precisely known due to inefficiencies in reconstitution, but they are likely above physiological concentrations. Thus, although no adaptor proteins are required for substrate extraction, this does not preclude the possibility that adaptor proteins are required for substrate selectivity or regulation of Msp1 activity. Similarly, our results do not preclude the possibility that Msp1 is also acting as a disaggregase. Given that many AAA proteins can moonlight as disaggresases55-57, we consider this highly likely. Further optimization of the reconstituted system will provide a powerful tool for examining the mechanistic details and thermodynamics of Msp1 activity.

2.3. Beyond tail-anchored proteins: a broader role in quality control It is clear that Msp1 removes mislocalized TA proteins from the OMM, but is this the primary function of Msp1 in proteostasis? Attempts to identify other proteins that interact with Msp1 yielded only a few additional TA proteins, but surprisingly identified IMM proteins12 58. Recent work by the Amon lab provides a ready explanation for this unexpected result while simultaneously showing that Msp1 has a broader substrate range than just mislocalized TA proteins8. Overexpression of IMM proteins that rely on a bipartite signal sequence can overwhelm the mitochondrial protein import machinery and lead to stalling of IMM proteins in the translocase of the outer mitochondrial membrane (TOM) complex. Protease protection and carbonate extraction assays confirmed that the substrates are indeed stalled in the TOM complex rather than aggregated on the membrane surface. This import stress induces expression of the protein Cis1 as part of the MitoCPR

ACCEPTED MANUSCRIPT transcriptional response. Cis1 then recruits Msp1 to the TOM complex to clear stalled substrates and restore TOM function.

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Msp1 is well conserved in eukaryotes with the human homolog ATAD1/Thorase sharing 45% sequence identity with S. cerevisiae Msp1. Unlike yeast where deletion of Msp1 is generally well tolerated, loss of ATAD1 is highly detrimental as mice with compromised ATAD1 function exhibit impaired fear conditioning, seizures, and increased injury following stroke59-62. Patients who have homozygous ATAD1 mutations have severe, lethal encephalopathy associated with stiffness and arthrogryposis63. Despite its clear importance, the primary function of ATAD1 is unclear. Similar to Msp1, ATAD1 localizes predominantly to mitochondria and peroxisomes and has been shown to remove mislocalized TA proteins12. Indeed, human ATAD1 can functionally substitute for Msp1 in S. cerevisiae12. However, ATAD1 has also been implicated in recycling of the neuroreceptor AMPAR from the postsynaptic membrane61. Consistent with this, ATAD1-/- mice have excess AMPAR-mediated excitatory signaling and respond well to the AMPAR inhibitor perampanel60.

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Determining if the primary function of ATAD1 is in neuroreceptor recycling or proteostasis will require further investigation into a few divergences between the neurobiology and proteostasis studies. One of the biggest questions is how a mitochondrial/peroxisomal protein mediates recycling of a neuroreceptor on the postsynaptic membrane. Fluorescent microscopy studies suggest that a small amount of ATAD1 is diffuse throughout the cytosol, so perhaps this is sufficient to mediate neuroreceptor recycling12. Biochemical studies of ATAD1 in neuroreceptor recycling do not characterize ATAD1 as a membrane protein and no detergent is used in the purification of full-length ATAD1. This is inconsistent with biochemical studies on Msp1 and ATAD149,61,63. Disease associated mutants of ATAD1 which impair AMPAR recycling have no significant effect on mitochondrial function in mouse embryonic fibroblasts (MEFs) whereas deletion of ATAD1 in MEFs results in mitochondrial damage12,63. While it is now clear that Msp1/ATAD1 has a broader substrate range than just mislocalized TA proteins, the full role of Msp1/ATAD1 in proteostasis remains undefined. We consider it likely that Msp1/ATAD1 is playing additional, undiscovered roles in OMM proteostasis. Fully defining the substrate repertoire and role of Msp1/ATAD1 in OMM proteostasis are high priority goals for the field.

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3. Substrate selectivity

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Figure 2

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Msp1 activity alters the mitochondrial proteome. As OMM proteins include the BCL2 family of apoptotic regulators46,64 the selection of substrates for extraction must be tightly regulated to avoid potentially catastrophic consequences for the cell. The challenges of achieving specificity are magnified considering that mislocalized or damaged proteins are likely present in the membrane at much lower concentrations than endogenous mitochondrial proteins. Current evidence suggests that Msp1 employs three different strategies for substrate recognition: 1) direct recognition of a motif within the substrate; 2) regulation by substrate complex formation; and 3) regulation by adaptor proteins (Figure 2). Although we present these as three distinct models, they are not mutually exclusive and there is experimental evidence supporting each model.

Figure 2: Models for substrate selectivity. A) Msp1 directly recognizes a motif within the ER-TA protein and/or a hydrophobic mismatch between the mislocalized protein and the lipid membrane. Recent results

ACCEPTED MANUSCRIPT suggest an exposed hydrophobic patch on the cytosolic side of the membrane and positively charged residues in the IMS are key motifs for recognition of substrates by Msp1. B) Substrate selectivity is regulated by complex formation. Newly synthesized or mislocalized proteins (Pex15) fail to find a binding partner (Pex3) and are preferentially extracted by Msp1. Complex formation may prevent extraction by increasing the thermodynamic stability of the substrate or masking a motif recognized by Msp1. C) Adaptor proteins, such as Cis1, modulate the substrate preference of Msp1. Cis1 recruits Msp1 to the TOM complex to clear proteins stalled during translocation. This reduces the amount of Msp1 available to remove mislocalized TA proteins.

3.1 Direct recognition of a motif

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Our previous work showed that Msp1 can extract TA proteins in vitro without accessory proteins or substrate modification49. The simplest explanation for these results is that Msp1 is directly recognizing a motif within mislocalized TA proteins. Recent work from the Jiang lab using in vivo photo-cross-linking demonstrates that Msp1 directly recognizes two motifs within the mislocalized TA protein Pex15: 1) an exposed hydrophobic patch on the cytosolic side of the membrane; and 2) positively charged residues in the IMS58. The hydrophobic patch is recognized by the Msp1 N-domain whereas the positively charged residues in the IMS are recognized by the conserved residue D12 in Msp1. The TMD of Msp1 cross-links with Pex15 but does not appear to be essential for substrate recognition, consistent with previous studies49,54,58.

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Li et. al. propose that the hydrophobic patch of Pex15 becomes solvent exposed upon mislocalization to the OMM due to inefficient protein folding. Removal of the hydrophobic patch decreased Pex15 binding to Msp1. Attaching a hydrophobic patch of Pex15 to the protein Fis1, which is an OMM-TA protein and thus not extracted by Msp1, is sufficient to make the Fis1 chimera a substrate for Msp1. However, the Fis1 chimera has a half-life of ~4 h whereas mislocalized Pex15 has a half-life of <15 minutes58. Furthermore, newly identified Msp1 substrates Frt1 and Ysy6 lack hydrophobic patches, which suggests there may be additional motifs recognized by Msp158. Given the evidence that Msp1 recognizes hydrophobic residues, we propose that Msp1 may also recognize solvent exposed hydrophobic residues in the TMD caused by a hydrophobic mismatch between the mislocalized substrate and the OMM. ER-TA proteins generally have more hydrophobic TMDs 65 66 than OMM-TA proteins46-48. Furthermore, the lipid composition of the ER and OMM differ , so there may be a hydrophobic mismatch between ER-TA proteins and the OMM. We anticipate that our in vitro extraction assay will provide a powerful tool for studying how the biophysical properties of the substrate TMD and the lipid composition of the membrane affect Msp1 activity and substrate recognition.

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The overall significance of the ionic interaction in the IMS is unclear. Mutation of D12 in Msp1 reduces binding to Pex15 and modestly impairs mitochondrial function when combined with a compromised GET pathway. However, this effect is mitigated by increased expression of Msp1 or mislocalized TA proteins58. Furthermore, this interaction is unlikely to distinguish between OMM-TA and ER-TA proteins as positively charged residues in the IMS are a hallmark of OMM-TA protein46-48, which are not considered substrates for Msp1. The mechanism of binding between the Msp1 N-domain and the hydrophobic patch is also unclear. The side chains of the key residues in the Msp1 N-domain that recognize the hydrophobic patch are not solvent exposed in the crystal structure49,58, so substrate recognition requires a conformational change. Structural data showing a direct interaction between Msp1 and substrate would provide important insights into the mechanism of substrate recognition.

3.2 Regulation by substrate complex formation A second model for substrate recognition, originally proposed by the Denic lab, is that complex formation prevents extraction54. This is an attractive model because endogenous OMM proteins will readily assemble into physiological complexes whereas a protein mislocalized to the OMM is unlikely to encounter binding partners required for complex formation, thereby leading to preferential extraction of the mislocalized protein. This was elegantly demonstrated for the TA protein Pex15, which is significantly more resistant to Msp1 mediated extraction in the peroxisome than the OMM. Using quantitative live cell imaging, Weir et. al. showed that Pex15 forms a complex with Pex3 that renders it resistant to Msp1-mediated extraction54. Consistent with this model, degradation of Pex3 decreases Pex15 stability whereas overexpression of Pex3 further increases the stability of Pex15.

ACCEPTED MANUSCRIPT While this paper convincingly shows that substrate complex formation regulates substrate selectivity, there are two competing explanations for how complex formation regulates extraction. Complex formation could sterically occlude a recognition motif (similar to model 1). Alternatively, Msp1 may use thermodynamic stability as the main criteria for substrate selectivity. Complex formation would make a substrate more thermodynamically stable and thus resistant to extraction. We anticipate that careful substrate design and thermodynamic measurements can help distinguish between these two models.

3.3 Regulation by adaptor proteins

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A third model is that Msp1 substrate selectivity is regulated by adaptor proteins. One example is Cis1, which recruits Msp1 to the TOM complex to remove proteins stalled in translocation across the OMM8. Several lines of evidence suggest that Cis1 changes Msp1 substrate preference from mislocalized TA proteins to IMM proteins stalled in the TOM complex. First, in vitro49 and in vivo58 studies show that Cis1 is not required for extraction of ER-TA proteins. Second, Mito-CPR, and hence Cis1 expression, is not induced by the accumulation of mislocalized ER-TA proteins on the OMM. Third, overexpression of Cis1 actually enhances the toxicity of ER-TA protein mislocalization to the OMM8. This suggests that Cis1 reduces the pool of Msp1 available to extract mislocalized TA proteins. Given that the full substrate repertoire of Msp1 is incompletely defined, it is likely that that there are additional adaptor proteins that regulate Msp1 activity.

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4. Mechanism of substrate extraction 4.1 Structure and oligomeric state

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Msp1 is a member of the meiotic clade of AAA ATPase, which include Spastin, Vps4, and Fidgetin67. Similar to other meiotic clade members, soluble (TMD) Msp1 is not a constitutive hexamer, but instead transitions between a hexamer in the presence of ATP and a mixture of monomers and dimers in the absence of nucleotide49. Blue-native page gels of digitonin solubilized Msp1 demonstrates that the full-length construct also undergoes nucleotide dependent oligomerization58. Currently, there is only one crystal structure of Msp1, which shows a soluble (TMD) construct as a nucleotide free monomer49. The crystal structure has a malformed ATP binding site and is in a conformation that is incompatible with hexamer formation. Although we cannot exclude the possibility that the unique conformation of Msp1 observed in the crystal structure is an artifact of the crystallization conditions, it is worth noting that the conformation is consistent with the biochemical observations on nucleotide dependent oligomerization.

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Thus far, there is no structural data on the Msp1 hexamer. Initial attempts to use Cryo-EM to study the soluble Msp1 hexamer resulted in preferential orientation of the molecules in the vitreous ice49. The resulting class averages clearly showed a hexameric ring with a diameter of ~13 nm, but a 3D reconstruction was not possible due to the lack of side views. To gain insights into the Msp1 hexamer, we generated a model by aligning the large and small domains of the Msp1 crystal structure to the D2 ring of P9768 (PDB 5C18), which results in a 6fold symmetric ring (Figure 3A). With the explosion of cryo-EM data, it is now readily apparent that many AAA proteins form lock washer hexamers with subunits in a spiral arrangement rather than a symmetric hexamer. To generate a model of the Msp1 hexamer as a lock washer, we aligned the large and small domains of Msp1 onto the cryo-EM structure of Vps469 (PDB 6AP1), a member of the meiotic clade of AAA proteins (Figure 3B). Both models have a diameter of ~13 nm and predict that L122 and L123 are at the subunit interface (Figure 3C & D). Consistent with these models, the L122D, L123D double mutant results in hexamer disruption and a loss of activity49. Given the available data, it appears that both the planar and lock-washer models must be seriously considered. Structural data of the Msp1 hexamer will be essential for distinguishing between these two models.

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4.2. Accessing the axial pore

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Figure 3: Models of Msp1 hexamer. Model of a symmetric, planar Msp1 hexamer (A) or lock washer Msp1 hexamer (B) viewed from the top (cytosol) and side, colored by subunit. Planar hexamer and lock washer models were generated by individually aligning the large and small domains of Msp1 onto the corresponding subdomains of the D2 ring of P97 (PDB 5C18) or Vps4 (PDB 6AP1) respectively. Note that the transition subunit in the lock washer model is not fully engaged with neighboring subunits. L122 and L123 lie at the interface between subunits in both the planar hexamer model (C) and the lock washer model (D).

Figure 4

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Recent work has shown that similar to other AAA proteins, Msp1 translocates substrates through the axial pore58. This raises the question of how a membrane anchored substrate gains access to the axial pore of a membrane anchored ring? We can envision two basic models for how substrates access the axial pore of Msp1, which we term the processive and non-processive models.

ACCEPTED MANUSCRIPT Figure 4: Models for substrate extraction. A) The processive model predicts that the substrate engages with the closed, Msp1 hexamer. The substrate gains access to the axial pore by going between the AAA core of Msp1 and the lipid bilayer. Membrane extraction requires that the entire substrate is then translocated through the axial pore. B) The non-processive model has Msp1 hexamers assemble around the substrate or allows a substrate to laterally diffuse into the Msp1 pore. Membrane extraction requires that only part of the substrate is translocated through the axial pore.

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In the processive model, Msp1 engages a substrate via the N- or C-terminus and translocates the entire substrate through the axial pore (Figure 4A). This model requires that the substrate travel between the AAA core of Msp1 and the lipid bilayer to engage with the axial pore loops. Consistent with this model, recent work by the Jiang lab has demonstrated that mislocalized TA proteins bind the membrane proximal face of the Ndomain, adjacent to the axial pore loops58. This model also predicts that there should be a flexible linker between the AAA core of Msp1 and the lipid bilayer to allow space for a substrate to pass through. Residues 33-49 of Msp1 are immediately adjacent to the Msp1 TMD and are likely flexible as they are not visible in the crystal structure49. However, deletion of this flexible linker resulted in only a minor defect in degradation when high concentrations of Msp1 and substrate are used49.

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In the non-processive model, Msp1 initially engages the middle of a substrate. The substrate can gain access to the axial pore via lateral diffusion into the ring or de novo ring assembly around a substrate (Figure 4B). There are hints that such models could be correct. First, the crystal structure of soluble Msp1 is in a conformation that is incompatible with hexamer formation49. Second, Msp1 is not a constitutive hexamer but instead assembles upon ATP binding49,58. Third, models of the Msp1 hexamer as a lock washer show that the transition subunit is not fully engaged with neighboring subunits (Figure 3B). This could provide a potential avenue for lateral diffusion of a substrate into axial pore. Distinguishing between these two models will provide important insights into the mechanism of substrate extraction.

5. Conclusions and outlook

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Over the past 5 years a basic framework for the role of Msp1 in OMM quality control has emerged, but many questions remain. Thus far Msp1 has been shown to extract mislocalized tail-anchored proteins and IMM proteins that stall during translocation through the TOM complex. Msp1 also localizes to the peroxisomes where it removes the TA protein Pex15 that is in stoichiometric excess compared to its binding partner Pex3, but otherwise the role of Msp1 in peroxisomal proteostasis is obscure54. The metazoan homolog, ATAD1, has been implicated in neuroreceptor recycling in addition to its roles in proteostasis61. We consider it likely that Msp1 is playing additional roles in proteostasis and fully defining the substrate repertoire of Msp1 is a high priority for the field.

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A full substrate profile for Msp1 will provide a holistic view of how Msp1 integrates with other components of the proteostasis network to promote mitochondrial function and health. In S. cerevisiae, deletion of Msp1 has a mild phenotype under standard growth conditions, suggesting that other components of the quality control network are partially redundant for Msp1 function. Indeed, previous work has shown that Cdc48 can extract some proteins from the OMM34. Little is known about the fate of substrates following extraction from the lipid bilayer. A significant portion are eventually degraded58, but the fate of a substrate between extraction and degradation is unclear. We hypothesize that a chaperone engages substrates after extraction to prevent aggregation, but the identity of any such chaperone and if/how it interacts with Msp1 is unknown. There are several important unanswered questions at the molecular level. One of the most pressing questions is the mechanism of substrate selectivity. There is currently evidence for three different models of substrate recognition (Figure 2). As additional Msp1 substrates are identified, we expect Msp1 to recognize these new substrates by using some variation of these three models. There is currently no structure of the active Msp1 hexamer, although we have built two competing models for the active hexamer based on our crystal structure of the inactive monomer. High resolution structural studies will help distinguish between these models and provide insights into the mechanism of substrate extraction. Lastly, it is unclear how Msp1 overcomes substantial thermodynamic barriers to remove a membrane protein from the lipid bilayer. We believe that the reconstituted system offers a powerful tool to study this important biophysical question

ACCEPTED MANUSCRIPT Acknowledgements This work was supported by startup funds from the University of Toledo.

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