Hexameric ATPase Research Articles

A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion

The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.

Molecular Basis for ATP-Hydrolysis-Driven DNA Translocation by the CMG Helicase of the Eukaryotic Replisome

SummaryIn the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding.

Motor torque measurement of Halobacterium salinarum archaellar suggests a general model for ATP-driven rotary motors

It is unknown how the archaellum—the rotary propeller used by Archaea for motility—works. To further understand the molecular mechanism by which the hexameric ATPase motor protein FlaI drives rotation of the membrane-embedded archaellar motor, we determined motor torque by imposition of various loads on Halobacterium salinarum archaella. Markers of different sizes were attached to single archaella, and their trajectories were quantified using three-dimensional tracking and high-speed recording. We show that rotation slows as the viscous drag of markers increases, but torque remains constant at 160 pN·nm independent of rotation speed. Notably, the estimated work done in a single rotation is twice the expected energy that would come from hydrolysis of six ATP molecules in the hexamer, indicating that more ATP molecules are required for one rotation of archaellum. To reconcile the apparent contradiction, we suggest a new and general model for the mechanism of ATP-driven rotary motors.

Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cag\u03b1 from the Helicobacter pylori type IV secretion system

Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here present the identification and characterization of inhibitors of Cagα, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein suggesting that they bind Cagα. Several molecules affect binding of ADP and four of them inhibit the ATPase activity. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the active site. Cross-linking suggests that the active molecules change protein conformation and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cagα in vivo. Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins.

Hsp104 and Potentiated Variants Can Operate as Distinct Nonprocessive Translocases

Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel. Here, we apply transient state, single turnover kinetics to investigate the ATP-dependent translocation of soluble polypeptides by Hsp104 and Hsp104A503S, a potentiated variant developed to resolve misfolded conformers implicated in neurodegenerative disease. We establish that Hsp104 and Hsp104A503S can operate as nonprocessive translocases for soluble substrates, indicating a “partial threading” model of translocation. Remarkably, Hsp104A503S exhibits altered coupling of ATP binding to translocation and decelerated dissociation from polypeptide substrate compared to Hsp104. This altered coupling and prolonged substrate interaction likely increases entropic pulling forces, thereby enabling more effective aggregate dissolution by Hsp104A503S.

A Ubiquitin‐dependent Switch during Assembly of the Proteasomal ATPases Mediated by Not4 Ubiquitin Ligase

In the proteasome holoenzyme, the hexameric ATPases (Rpt1‐Rpt6) enable degradation of ubiquitinated proteins by unfolding and translocating them into the proteolytic core particle. During early‐stage proteasome assembly, individual Rpt proteins assemble into the hexameric ‘Rpt ring’ through binding to their cognate chaperones: Nas2, Hsm3, Nas6 and Rpn14. Here, we show that Rpt ring assembly employs a specific ubiquitination‐mediated control. An E3 ligase, Not4, selectively ubiquitinates Rpt5 during Rpt ring assembly. To access Rpt5, Not4 competes with Nas2 until the penultimate step, and then with Hsm3 at the final step of Rpt ring completion. Using the known Rpt‐chaperone co‐crystal structures, we show that Not4‐mediated ubiquitination sites in Rpt5 are obstructed by Nas2 and Hsm3. Thus, Not4 can distinguish a Rpt ring that matures without these chaperones, based on its accessibility to Rpt5. Rpt5 ubiquitination does not destabilize the ring, but hinders incorporation of incoming subunits—Rpn1 ubiquitin receptor and Ubp6 deubiquitinase, thereby blocking progression of proteasome assembly and ubiquitin regeneration from proteasome substrates. Our findings reveal an assembly checkpoint where Not4 monitors chaperone actions during hexameric ATPase ring assembly, thereby ensuring the accuracy of proteasome holoenzyme maturation.Support or Funding InformationBoettcher Webb‐Waring Biomedical Research Award and 1R01GM127688‐01A1 from NIH (S.P.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Oligomeric State of the Plasma Membrane H⁺-ATPase from Kluyveromyces lactis.

The plasma membrane H+-ATPase was purified from the yeast K. lactis. The oligomeric state of the H+-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H+-ATPase hexamer in both MASs as the sole/main oligomeric state—in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H+-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H+-ATPase. It is concluded that the quaternary structure of the H+-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.

The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides.

HslVU is an ATP-dependent proteolytic complex present in certain bacteria and in the mitochondrion of some primordial eukaryotes, including deadly parasites such as Leishmania. It is formed by the dodecameric protease HslV and the hexameric ATPase HslU, which binds via the C-terminal end of its subunits to HslV and activates it by a yet unclear allosteric mechanism. We undertook the characterization of HslV from Leishmania major (LmHslV), a trypanosomatid that expresses two isoforms for HslU, LmHslU1 and LmHslU2. Using a novel and sensitive peptide substrate, we found that LmHslV can be activated by peptides derived from the C-termini of both LmHslU1 and LmHslU2. Truncations, Ala- and D-scans of the C-terminal dodecapeptide of LmHslU2 (LmC12-U2) showed that five out of the six C-terminal residues of LmHslU2 are essential for binding to and activating HslV. Peptide cyclisation with a lactam bridge allowed shortening of the peptide without loss of potency. Finally, we found that dodecapeptides derived from HslU of other parasites and bacteria are able to activate LmHslV with similar or even higher efficiency. Importantly, using electron microscopy approaches, we observed that the activation of LmHslV was accompanied by a large conformational remodeling, which represents a yet unidentified layer of control of HslV activation.

Structure and Function of the AAA+ ATPase p97, a Key Player in Protein Homeostasis.

p97 belongs to the functional diverse superfamily of AAA+ (ATPases Associated with diverse cellular Activities) ATPases and is characterized by an N-terminal regulatory domain and two stacked hexameric ATPase domains forming a central protein conducting channel. p97 is highly versatile and has key functions in maintaining protein homeostasis including protein quality control mechanisms like the ubiquitin proteasome system (UPS) and autophagy to disassemble polyubiquitylated proteins from chromatin, membranes, macromolecular protein complexes and aggregates which are either degraded by the proteasome or recycled. p97 can use energy derived from ATP hydrolysis to catalyze substrate unfolding and threading through its central channel. The function of p97 in a large variety of different cellular contexts is reflected by its simultaneous association with different cofactors, which are involved in substrate recognition and processing, thus leading to the formation of transient multi-protein complexes. Dysregulation in protein homeostasis and proteotoxic stress are often involved in the development of cancer and neurological diseases and targeting the UPS including p97 in cancer is a well-established pharmacological strategy. In this chapter we will describe structural and functional aspects of the p97 interactome in regulating diverse cellular processes and will discuss the role of p97 in targeted cancer therapy.

Ubiquitin-dependent switch during assembly of the proteasomal ATPases mediated by Not4 ubiquitin ligase

In the proteasome holoenzyme, the hexameric ATPases (Rpt1-Rpt6) enable degradation of ubiquitinated proteins by unfolding and translocating them into the proteolytic core particle. During early-stage proteasome assembly, individual Rpt proteins assemble into the hexameric "Rpt ring" through binding to their cognate chaperones: Nas2, Hsm3, Nas6, and Rpn14. Here, we show that Rpt ring assembly employs a specific ubiquitination-mediated control. An E3 ligase, Not4, selectively ubiquitinates Rpt5 during Rpt ring assembly. To access Rpt5, Not4 competes with Nas2 until the penultimate step and then with Hsm3 at the final step of Rpt ring completion. Using the known Rpt-chaperone cocrystal structures, we show that Not4-mediated ubiquitination sites in Rpt5 are obstructed by Nas2 and Hsm3. Thus, Not4 can distinguish a Rpt ring that matures without these chaperones, based on its accessibility to Rpt5. Rpt5 ubiquitination does not destabilize the ring but hinders incorporation of incoming subunits-Rpn1 ubiquitin receptor and Ubp6 deubiquitinase-thereby blocking progression of proteasome assembly and ubiquitin regeneration from proteasome substrates. Our findings reveal an assembly checkpoint where Not4 monitors chaperone actions during hexameric ATPase ring assembly, thereby ensuring the accuracy of proteasome holoenzyme maturation.

Molecular dynamics simulations of nucleotide release from the circadian clock protein KaiC reveal atomic-resolution functional insights

The cyanobacterial clock proteins KaiA, KaiB, and KaiC form a powerful system to study the biophysical basis of circadian rhythms, because an in vitro mixture of the three proteins is sufficient to generate a robust ∼24-h rhythm in the phosphorylation of KaiC. The nucleotide-bound states of KaiC critically affect both KaiB binding to the N-terminal domain (CI) and the phosphotransfer reactions that (de)phosphorylate the KaiC C-terminal domain (CII). However, the nucleotide exchange pathways associated with transitions among these states are poorly understood. In this study, we integrate recent advances in molecular dynamics methods to elucidate the structure and energetics of the pathway for Mg·ADP release from the CII domain. We find that nucleotide release is coupled to large-scale conformational changes in the KaiC hexamer. Solvating the nucleotide requires widening the subunit interface leading to the active site, which is linked to extension of the A-loop, a structure implicated in KaiA binding. These results provide a molecular hypothesis for how KaiA acts as a nucleotide exchange factor. In turn, structural parallels between the CI and CII domains suggest a mechanism for allosteric coupling between the domains. We relate our results to structures observed for other hexameric ATPases, which perform diverse functions.

The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics.

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.

Interaction between the AAA ATPase p97/VCP and a concealed UBX domain in the copper transporter ATP7A is associated with motor neuron degeneration

The copper-transporting ATPase ATP7A contains eight transmembrane domains and is required for normal human copper homeostasis. Mutations in the ATP7A gene may lead to infantile-onset cerebral degeneration (Menkes disease); occipital horn syndrome (OHS), a related but much milder illness; or an adult-onset isolated distal motor neuropathy. The ATP7A missense mutation T994I is located in the sixth transmembrane domain of ATP7A, represents one of the variants associated with the latter phenotype, and is associated with an abnormal interaction with p97/valosin-containing protein (VCP), a hexameric AAA ATPase (ATPase associated with diverse cellular activities) with multiple biological functions. In this study, we further characterized this interaction and discovered a concealed UBX domain in the third lumenal loop of ATP7A, between its fifth and sixth transmembrane domains. We show that the T994I substitution results in conformational exposure of the UBX domain, which then binds the N-terminal domain of p97/VCP. We also show that this abnormal interaction occurs at or near the cell plasma membrane. The UBX domain has a conserved hydrophobic FP (Phe-Pro) motif, and substitution with di-alanine abrogated the interaction and restored the proper intracellular localization of ATP7A in the trans-Golgi network. Using protein MS, we identified potential coordinating components of the ATP7AT994I-p97 complex, including NSFL1 cofactor (NSF1C or p47) that may be relevant to the pathophysiology and clinical effects associated with ATP7AT994I Our study represents the first report of p97/VCP binding to a UBX domain that is not normally exposed, resulting in an aberrant protein-protein interaction leading to motor neuron degeneration.

Mechanochemical coupling and bi-phasic force-velocity dependence in the ultra-fast ring ATPase SpoIIIE.

Multi-subunit ring-shaped ATPases are molecular motors that harness chemical free energy to perform vital mechanical tasks such as polypeptide translocation, DNA unwinding, and chromosome segregation. Previously we reported the intersubunit coordination and stepping behavior of the hexameric ring-shaped ATPase SpoIIIE (Liu et al., 2015). Here we use optical tweezers to characterize the motor's mechanochemistry. Analysis of the motor response to external force at various nucleotide concentrations identifies phosphate release as the likely force-generating step. Analysis of SpoIIIE pausing indicates that pauses are off-pathway events. Characterization of SpoIIIE slipping behavior reveals that individual motor subunits engage DNA upon ATP binding. Furthermore, we find that SpoIIIE's velocity exhibits an intriguing bi-phasic dependence on force. We hypothesize that this behavior is an adaptation of ultra-fast motors tasked with translocating DNA from which they must also remove DNA-bound protein roadblocks. Based on these results, we formulate a comprehensive mechanochemical model for SpoIIIE.

VPS4 is a dynamic component of the centrosome that regulates centrosome localization of \u03b3-tubulin, centriolar satellite stability and ciliogenesis

The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4EQ, which can’t hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4EQ mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4EQ mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer’s vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.

Structural characterization of the bacterial proteasome homolog BPH reveals a tetradecameric double-ring complex with unique inner cavity properties

Eukaryotic and archaeal proteasomes are paradigms for self-compartmentalizing proteases. To a large extent, their function requires interplay with hexameric ATPases associated with diverse cellular activities (AAA+) that act as substrate unfoldases. Bacteria have various types of self-compartmentalizing proteases; in addition to the proteasome itself, these include the proteasome homolog HslV, which functions together with the AAA+ HslU; the ClpP protease with its partner AAA+ ClpX; and Anbu, a recently characterized ancestral proteasome variant. Previous bioinformatic analysis has revealed a novel bacterial member of the proteasome family Betaproteobacteria proteasome homolog (BPH). Using cluster analysis, we here affirmed that BPH evolutionarily descends from HslV. Crystal structures of the Thiobacillus denitrificans and Cupriavidus metallidurans BPHs disclosed a homo-oligomeric double-ring architecture in which the active sites face the interior of the cylinder. Using small-angle X-ray scattering (SAXS) and electron microscopy averaging, we found that BPH forms tetradecamers in solution, unlike the dodecamers seen in HslV. Although the highly acidic inner surface of BPH was in striking contrast to the cavity characteristics of the proteasome and HslV, a classical proteasomal reaction mechanism could be inferred from the covalent binding of the proteasome-specific inhibitor epoxomicin to BPH. A ligand-bound structure implied that the elongated BPH inner pore loop may be involved in substrate recognition. The apparent lack of a partner unfoldase and other unique features, such as Ser replacing Thr as the catalytic residue in certain BPH subfamilies, suggest a proteolytic function for BPH distinct from those of known bacterial self-compartmentalizing proteases.

The AAA ATPase Vps4 binds ESCRT-III substrates through a repeating array of dipeptide-binding pockets.

The hexameric AAA ATPase Vps4 drives membrane fission by remodeling and disassembling ESCRT-III filaments. Building upon our earlier 4.3 Å resolution cryo-EM structure (Monroe et al., 2017), we now report a 3.2 Å structure of Vps4 bound to an ESCRT-III peptide substrate. The new structure reveals that the peptide approximates a β-strand conformation whose helical symmetry matches that of the five Vps4 subunits it contacts directly. Adjacent Vps4 subunits make equivalent interactions with successive substrate dipeptides through two distinct classes of side chain binding pockets formed primarily by Vps4 pore loop 1. These pockets accommodate a wide range of residues, while main chain hydrogen bonds may help dictate substrate-binding orientation. The structure supports a 'conveyor belt' model of translocation in which ATP binding allows a Vps4 subunit to join the growing end of the helix and engage the substrate, while hydrolysis and release promotes helix disassembly and substrate release at the lagging end.

Architecture of the type IV coupling protein complex of Legionella pneumophila.

Many bacteria, including Legionella pneumophila, rely on the type IV secretion system to translocate a repertoire of effector proteins into the hosts for their survival and growth. Type IV coupling protein (T4CP) is a hexameric ATPase that links translocating substrates to the transenvelope secretion conduit. Yet, how a large number of effector proteins are selectively recruited and processed by T4CPs remains enigmatic. DotL, the T4CP of L. pneumophila, contains an ATPase domain and a C-terminal extension whose function is unknown. Unlike T4CPs involved in plasmid DNA translocation, DotL appeared to function by forming a multiprotein complex with four other proteins. Here, we show that the C-terminal extension of DotL interacts with DotN, IcmS, IcmW and an additionally identified subunit LvgA, and that this pentameric assembly binds Legionella effector proteins. We determined the crystal structure of this assembly and built an architecture of the T4CP holocomplex by combining a homology model of the ATPase domain of DotL. The holocomplex is a hexamer of a bipartite structure composed of a membrane-proximal ATPase domain and a membrane-distal substrate-recognition assembly. The presented information demonstrates the architecture and functional dissection of the multiprotein T4CP complexes and provides important insights into their substrate recruitment and processing.

Mycobacterium tuberculosis has a proteasome system including a hexameric ATPase Mpa that directly recognizes and unfolds pupylated substrates for delivery into the 20S proteosome core particle (CP) for degradation. This cut through diagram illustrates wit

Molecular MicrobiologyVolume 105, Issue 2 p. i-i Front CoverFree Access Mycobacterium tuberculosis has a proteasome system including a hexameric ATPase Mpa that directly recognizes and unfolds pupylated substrates for delivery into the 20S proteosome core particle (CP) for degradation. This cut through diagram illustrates with red lines the path available for a protein substrate going through the Mpa axial channel to reach the proteolytic sites inside the beta rings of the CP. For details, see the article by Wu et al. on pp. 227–241 of this issue. First published: 04 July 2017 https://doi.org/10.1111/mmi.13513AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume105, Issue2July 2017Pages i-i RelatedInformation

Molecular Mechanism of Substrate Processing by the Cdc48 ATPase Complex

The Cdc48 ATPase and its cofactors Ufd1/Npl4 (UN) extract polyubiquitinated proteins from membranes or macromolecular complexes, but how they perform these functions is unclear. Cdc48 consists of an N-terminal domain that binds UN and two stacked hexameric ATPase rings (D1 and D2) surrounding a central pore. Here, we use purified components to elucidate how the Cdc48 complex processes substrates. After interaction of the polyubiquitin chain with UN, ATP hydrolysis by the D2 ring moves the polypeptide completely through the double ring, generating a pulling force on the substrate and causing its unfolding. ATP hydrolysis by the D1 ring is important for subsequent substrate release from the Cdc48 complex. This release requires cooperation of Cdc48 with a deubiquitinase, which trims polyubiquitin to an oligoubiquitin chain that is then also translocated through the pore. Together, these results lead to a new paradigm for the function of Cdc48 and its mammalian ortholog p97/VCP.