The AAA+ protein Msp1 selects substrates by recognizing a hydrophobic mismatch between the substrate transmembrane domain and the lipid bilayer.

An essential aspect of protein quality control is enzymatic removal of membrane proteins from the lipid bilayer. Failures in this essential cellular process are associated with neurodegenerative diseases and cancer. Msp1 is a AAA+ ( A TPases A ssociated with diverse cellular A ctivities) protein that removes mistargeted proteins from the outer mitochondrial membrane (OMM). How Msp1 selectively recognizes and extracts substrates within the complex OMM ecosystem, and the role of the lipid bilayer on these processes is unknown. Here, we describe the development of fully defined, rapid, and quantitative extraction assay that retains physiological substrate selectivity. Using this new assay, we systematically modified both substrates and the lipid environment to demonstrate that Msp1 recognizes substrates by a hydrophobic mismatch between the substrate TMD and the lipid bilayer. We further demonstrate that the rate limiting step in Msp1 activity is extraction of the TMD from the lipid bilayer. Together, these results provide foundational insights into how the lipid bilayer influences AAA+ mediated membrane protein extraction.

Collateral deletion of the mitochondrial AAA+ ATPase ATAD1 sensitizes cancer cells to proteasome dysfunction.

The tumor suppressor gene PTEN is the second most commonly deleted gene in cancer. Such deletions often include portions of the chromosome 10q23 locus beyond the bounds of PTEN itself, which frequently disrupts adjacent genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here, we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis triggered by proteasome dysfunction and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic protein BIM from mitochondria to inactivate it. Cultured cells and mouse xenografts lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which activate BIM and trigger apoptosis. This work furthers our understanding of mitochondrial protein homeostasis and could lead to new therapeutic options for the hundreds of thousands of cancer patients who have tumors with chromosome 10q23 deletion.

Cancer cells have often lost genetic sequences that control when and how cell division takes place. Deleting these genes, however, is not an exact art, and neighboring sequences regularly get removed in the process. For example, the loss of the tumor suppressor gene PTEN, the second most deleted gene in cancer, frequently involves the removal of the nearby ATAD1 gene. While hundreds of thousands of human tumors completely lack ATAD1, individuals born without a functional version of this gene do not survive past early childhood. How can tumor cells cope without ATAD1 ­ and could these coping strategies become the target for new therapies? Winter et al. aimed to answer these questions by examining a variety of cancer cells lacking ATAD1 in the laboratory. Under normal circumstances, the enzyme that this gene codes for sits at the surface of mitochondria, the cellular compartments essential for energy production. There, it extracts any faulty, defective proteins that may otherwise cause havoc and endanger mitochondrial health. Experiments revealed that without ATAD1, cancer cells started to rely more heavily on an alternative mechanism to remove harmful proteins: the process centers on MARCH5, an enzyme which tags molecules that require removal so the cell can recycle them. Drugs that block the pathway involving MARCH5 already exist, but they have so far been employed to treat other types of tumors. Winter et al. showed that using these compounds led to the death of cancerous ATAD1-deficient cells, including in human tumors grown in mice. Overall, this work demonstrates that cancer cells which have lost ATAD1 become more vulnerable to disruptions in the protein removal pathway mediated by MARCH5, including via already existing drugs. If confirmed by further translational work, these findings could have important clinical impact given how frequently PTEN and ATAD1 are lost together in cancer.

Reconstitution of Msp1 Extraction Activity with Fully Purified Components.

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Sorting out how Msp1 maintains mitochondrial membrane proteostasis.

Robust membrane proteostasis networks are essential for cells to withstand proteotoxic stress arising from environmental insult and intrinsic errors in protein production (Labbadia and Morimoto, 2015; Hegde and Zavodszky, 2019). Failures in mitochondrial membrane proteostasis are associated with cancer, aging, and a range of cardiovascular and neurodegenerative diseases (Wallace et al., 2010; Martin, 2012; Gustafsson and Gottlieb, 2007). As a result, mitochondria possess numerous pathways to maintain proteostasis (Avci and Lemberg, 2015; Shi et al., 2016; Weidberg and Amon, 2018; Shpilka and Haynes, 2018; Quirós et al., 2016; Sorrentino et al., 2017). Mitochondrial Sorting of Proteins 1 (Msp1) is a membrane anchored AAA ATPase that extracts proteins from the outer mitochondrial membrane (OMM) (Chen et al., 2014; Okreglak and Walter, 2014). 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.

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.

Ubiquilins Chaperone and Triage Mitochondrial Membrane Proteins for Degradation.

We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions.

Roles of the N domain of the AAA+ Lon protease in substrate recognition, allosteric regulation and chaperone activity.

Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation. The sul20 degron from the cell-division inhibitor SulA is shown here to bind to the N domain of Escherichia coli Lon, and the recognition site is identified by cross-linking and scanning for mutations that prevent sul20-peptide binding. These N-domain mutations limit the rates of proteolysis of model sul20-tagged substrates and ATP hydrolysis by an allosteric mechanism. Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber. Lon-mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis. In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP-dependent biological activities do not require translocation.

A mutation in the N domain of Escherichia coli lon stabilizes dodecamers and selectively alters degradation of model substrates.

Escherichia coli Lon, an ATP-dependent AAA(+) protease, recognizes and degrades many different substrates, including the RcsA and SulA regulatory proteins. More than a decade ago, the E240K mutation in the N domain of Lon was shown to prevent degradation of RcsA but not SulA in vivo. Here, we characterize the biochemical properties of the E240K mutant in vitro and present evidence that the effects of this mutation are complex. For example, Lon(E240K) exists almost exclusively as a dodecamer, whereas wild-type Lon equilibrates between hexamers and dodecamers. Moreover, Lon(E240K) displays degradation defects in vitro that do not correlate in any simple fashion with degron identity, substrate stability, or dodecamer formation. The Lon sequence segment near residue 240 is known to undergo nucleotide-dependent conformational changes, and our results suggest that this region may be important for coupling substrate binding with allosteric activation of Lon protease and ATPase activity.

Modular aspects of kinesin force generation machinery.

The motor head of kinesin carries out microtubule binding, ATP hydrolysis, and force generation. Despite a high level of sequence and structural conservation, subtle variations in subdomains of the motor head determine family-specific properties. In particular, both Kinesin-1 (Kin-1) and Kinesin-5 (Kin-5) walk processively to the microtubule plus-end, yet show distinct motility characteristics suitable for their functions. We studied chimeric Kin-1/Kin-5 constructs with a combination of single molecule motility assays and molecular dynamics simulations to demonstrate that Kin-5 possesses a force-generating element similar to Kin-1, i.e., the cover-neck bundle. Furthermore, the Kin-5 neck linker makes additional contacts with the core of the motor head via loop L13, which putatively compensates for the shorter cover-neck bundle of Kin-5. Our results indicate that Kin-1 is mechanically optimized for individual cargo transport, whereas Kin-5 does not necessarily maximize its mechanical performance. Its biochemical rates and enhanced force sensitivity may instead be beneficial for operation in a group of motors. Such variations in subdomains would be a strategy for achieving diversity in motility with the conserved motor head.

Distinct quaternary structures of the AAA+ Lon protease control substrate degradation.

Lon is an ATPase associated with cellular activities (AAA+) protease that controls cell division in response to stress and also degrades misfolded and damaged proteins. Subunits of Lon are known to assemble into ring-shaped homohexamers that enclose an internal degradation chamber. Here, we demonstrate that hexamers of Escherichia coli Lon also interact to form a dodecamer at physiological protein concentrations. Electron microscopy of this dodecamer reveals a prolate structure with the protease chambers at the distal ends and a matrix of N domains forming an equatorial hexamer-hexamer interface, with portals of ∼45 Šproviding access to the enzyme lumen. Compared with hexamers, Lon dodecamers are much less active in degrading large substrates but equally active in degrading small substrates. Our results support a unique gating mechanism that allows the repertoire of Lon substrates to be tuned by its assembly state.

Engineering fluorescent protein substrates for the AAA+ Lon protease.

AAA+ proteases, such as Escherichia coli Lon, recognize protein substrates by binding to specific peptide degrons and then unfold and translocate the protein into an internal degradation chamber for proteolysis. For some AAA+ proteases, attaching specific degrons to the N- or C-terminus of green fluorescent protein (GFP) generates useful substrates, whose unfolding and degradation can be monitored by loss of fluorescence, but Lon fails to degrade appropriately tagged GFP variants at a significant rate. Here, we demonstrate that Lon catalyzes robust unfolding and degradation of circularly permuted variants of GFP with a ß20 degron appended to the N terminus or a sul20 degron appended to the C terminus. Lon degradation of non-permuted GFP-sul20 is very slow, in part because the enzyme cannot efficiently extract the degron-proximal C-terminal ß-strand to initiate denaturation. The circularly permuted GFP substrates described here allow convenient high-throughput assays of the kinetics of Lon degradation in vitro and also permit assays of Lon proteolysis in vivo.