Ataxin-3 interactions with rad23 and valosin-containing protein and its associations with ubiquitin chains and the proteasome are consistent with a role in ubiquitin-mediated proteolysis

Machado-Joseph disease is caused by an expansion of a trinucleotide CAG repeat in the gene encoding the protein ataxin-3. We investigated if ataxin-3 was a proteasome-associated factor that recognized ubiquitinated substrates based on the rationale that (i) it is present with proteasome subunits and ubiquitin in cellular inclusions, (ii) it interacts with human Rad23, a protein that may translocate proteolytic substrates to the proteasome, and (iii) it shares regions of sequence similarity with the proteasome subunit S5a, which can recognize multiubiquitinated proteins. We report that ataxin-3 interacts with ubiquitinated proteins, can bind the proteasome, and, when the gene harbors an expanded repeat length, can interfere with the degradation of a well-characterized test substrate. Additionally, ataxin-3 associates with the ubiquitin- and proteasome-binding factors Rad23 and valosin-containing protein (VCP/p97), findings that support the hypothesis that ataxin-3 is a proteasome-associated factor that mediates the degradation of ubiquitinated proteins.     

The Proteasome-associated Protein Ecm29 Inhibits Proteasomal ATPase Activity and in Vivo Protein Degradation by the Proteasome

Several proteasome-associated proteins regulate degradation by the 26 S proteasome using the ubiquitin chains that mark most substrates for degradation. The proteasome-associated protein Ecm29, however, has no ubiquitin-binding or modifying activity, and its direct effect on substrate degradation is unclear. Here, we show that Ecm29 acts as a proteasome inhibitor. Besides inhibiting the proteolytic cleavage of peptide substrates in vitro, it inhibits the degradation of ubiquitin-dependent and -independent substrates in vivo. Binding of Ecm29 to the proteasome induces a closed conformation of the substrate entry channel of the core particle. Furthermore, Ecm29 inhibits proteasomal ATPase activity, suggesting that the mechanism of inhibition and gate regulation by Ecm29 is through regulation of the proteasomal ATPases. Consistent with this, we identified through chemical cross-linking that Ecm29 binds to, or in close proximity to, the proteasomal ATPase subunit Rpt5. Additionally, we show that Ecm29 preferentially associates with both mutant and nucleotide depleted proteasomes. We propose that the inhibitory ability of Ecm29 is important for its function as a proteasome quality control factor by ensuring that aberrant proteasomes recognized by Ecm29 are inactive.     

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.     

Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.     

Cadmium pyrithione suppresses tumor growth in vitro and in vivo through inhibition of proteasomal deubiquitinase

The ubiquitin–proteasome system (UPS) is indispensable to the protein quality control in eukaryotic cells. Due to the remarkable clinical success of using proteasome inhibitors for clinical treatment of multiple myeloma, it is anticipated that targeting the UPS upstream of the proteasome step be an effective strategy for cancer therapy. Deubiquitinases (DUB) are proteases that remove ubiquitin from target proteins and therefore regulate multiple cellular processes including some signaling pathways altered in cancer cells. Thus, targeting DUB is a promising strategy for cancer drug discovery. Previously, we have reported that metal complexes, such as copper and gold complexes, can disrupt the UPS via suppressing the activity of 19S proteasome-associated DUBs and/or of the 20S proteasomes, thereby inducing cancer cell death. In this study, we found that cadmium pyrithione (CdPT) treatment led to remarkable accumulation of ubiquitinated proteins in cultured cancer cells and primary leukemia cells. CdPT potently inhibited the activity of proteasomal DUBs (USP14 and UCHL5), but slightly inhibited 20S proteasome activity. The anti-cancer activity of CdPT was associated with triggering apoptosis via caspase activation. Moreover, treatment with CdPT inhibited proteasome function and repressed tumor growth in animal xenograft models. Our results show that cadmium-containing complex CdPT may function as a novel proteasomal DUB inhibitor and suggest appealing prospects for cancer treatment.     

Proteasome plasticity

The 26S proteasome is responsible for regulated proteolysis of most intracellular proteins yet the focus of intense regulatory action itself. Proteasome abundance is responsive to cell needs or stress conditions, and dynamically localized to concentrations of substrates. Proteasomes are continually assembled and disassembled, and their subunits subject to a variety of posttranslational modifications. Furthermore, as robust and multi-tasking as this complex is, it does not function alone. A spattering of closely associating proteins enhances complex stability, fine-tunes activity, assists in substrate-binding, recycling of ubiquitin, and more. HEAT repeat caps activate proteasomes, yet share remarkable features with nuclear importins. Fascinating cross talk even occurs with ribosomes through common maturation factors. The dynamics of proteasome configurations and how they relate to diverse activities is the topic of this review.     

A Conserved Unfoldase Activity for the p97 AAA-ATPase in Proteasomal Degradation

The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97^Ufd1/Npl4 complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.     

Identification and proteomic analysis of distinct UBE3A/E6AP protein complexes

The E6AP ubiquitin ligase catalyzes the high-risk human papillomaviruses' E6-mediated ubiquitylation of p53, contributing to the neoplastic progression of cells infected by these viruses. Defects in the activity and the dosage of E6AP are linked to Angelman syndrome and to autism spectrum disorders, respectively, highlighting the need for precise control of the enzyme. With the exception of HERC2, which modulates the ubiquitin ligase activity of E6AP, little is known about the regulation or function of E6AP normally. Using a proteomic approach, we have identified and validated several new E6AP-interacting proteins, including HIF1AN, NEURL4, and mitogen-activated protein kinase 6 (MAPK6). E6AP exists as part of several different protein complexes, including the proteasome and an independent high-molecular-weight complex containing HERC2, NEURL4, and MAPK6. In examining the functional consequence of its interaction with the proteasome, we found that UBE3C (another proteasome-associated ubiquitin ligase), but not E6AP, contributes to proteasomal processivity in mammalian cells. We also found that E6 associates with the HERC2-containing high-molecular-weight complex through its binding to E6AP. These proteomic studies reveal a level of complexity for E6AP that has not been previously appreciated and identify a number of new cellular proteins through which E6AP may be regulated or functioning.     

In vivo function of the proteasome in the ubiquitin pathway.

A major eukaryotic proteolytic system is known to require the covalent attachment of ubiquitin to substrates prior to their degradation, yet the proteinase involved remains poorly defined. The proteasome, a large conserved multi-subunit protein complex of the cytosol and the nucleus, has been implicated in a variety of cellular functions. It is shown here that a yeast mutant with a defective proteasome fails to degrade proteins which are subject to ubiquitin-dependent proteolysis in wild-type cells. Thus, the proteasome is part of the ubiquitin system and mediates the degradation of ubiquitin-protein conjugates in vivo.     

Trimming of Ubiquitin Chains by Proteasome-associated Deubiquitinating Enzymes

The proteasome generally recognizes substrate via its multiubiquitin chain followed by ATP-dependent unfolding and translocation of the substrate from the regulatory particle into the proteolytic core particle to be degraded. Substrate-bound ubiquitin groups are for the most part not delivered to the core particle and broken down together with substrate but instead recovered as intact free ubiquitin and ubiquitin chains. Substrate deubiquitination on the proteasome is mediated by three distinct deubiquitinating enzymes associated with the regulatory particle: RPN11, UCH37, and USP14. RPN11 cleaves at the base of the ubiquitin chain where it is linked to the substrate, whereas UCH37 and apparently USP14 mediate a stepwise removal of ubiquitin from the substrate by disassembling the chain from its distal tip. In contrast to UCH37 and USP14, RPN11 shows degradation-coupled activity; RPN11-mediated deubiquitination is apparently delayed until the proteasome is committed to degrade the substrate. Accordingly, RPN11-mediated deubiquitination promotes substrate degradation. In contrast, removal of ubiquitin prior to commitment could antagonize substrate degradation by promoting substrate dissociation from the proteasome. Emerging evidence suggests that USP14 and UCH37 can both suppress substrate degradation in this way. One line of study has shown that small molecule USP14 inhibitors can enhance proteasome function in cells, which is consistent with this model. Enhancing protein degradation could potentially have therapeutic applications for diseases involving toxic proteins that are proteasome substrates. However, the responsiveness of substrates to inhibition of proteasomal deubiquitinating enzymes may vary substantially. This substrate specificity and its mechanistic basis should be addressed in future studies.The eukaryotic proteasome is dedicated primarily to the degradation of proteins tagged by ubiquitin (1). Proteasomes strongly prefer multiubiquitinated protein substrates. The successive addition of ubiquitin groups to the substrate by ubiquitin ligases is usually accomplished through the formation of ubiquitin chains. The proteasome has much in common with the simple ATP-dependent proteases of prokaryotes and mitochondria (2, 3), although only the proteasome recognizes the ubiquitin modification. In all cases, the ATPases form a hexameric ring complex. These rings are homomeric in the case of the prokaryotic and mitochondrial proteases, whereas in eukaryotic proteasomes, the ATPase ring is heteromeric. Proteasomes and the simple ATP-dependent proteases are fundamentally similar in that they all have an ATPase ring (found within the regulatory particle [RP]1 in proteasomes, also known as the 19S particle and PA700) abutting a proteolytic complex (the core particle [CP] in proteasomes, also known as the 20S particle), although in some cases, the ATPase and protease domains are present on the same polypeptide chain (Fig. 1). Furthermore, this ancient organization of ATP-dependent proteases involves stacked ring complexes. Substrates are translocated from one ring to the next via the central pore within each ring. For most substrates, movement from ring to ring is driven by ATP hydrolysis. Thus, the substrate is captured by the ATPase ring of the RP and then translocated into the central cavity of the CP where it is hydrolyzed.Open in a separate windowFig. 1.Deubiquitinating enzymes of proteasome. In metazoans, three DUBs associate with the proteasome as shown. Each is associated with the 19-subunit RP. The detailed positioning of these enzymes on the RP is not known and is represented here schematically. RPN11 cuts at the base of the chain to release the chain en bloc. As shown, this is coupled (by an unknown mechanism) to translocation of the substrate from the RP to the CP to be degraded. In contrast, the action of USP14 and UCH37 is thought to promote substrate release from the proteasome rather than degradation. However, it should be noted that the attack of these enzymes on a substrate does not guarantee release, especially as their action on the chain is gradual, proceeding stepwise over time from the distal tip of the ubiquitin chain. Some substrates may carry more than one ubiquitin chain and thus be processed in a more complex manner. Moreover, more than one DUB might act on a given chain. The proteasome icon, adapted from Ref. 30 with permission, is based on cryo-EM imaging.The pathway of translocation contains a series of narrow constrictions through which folded proteins cannot pass. The inability of a typical folded protein to pass through these “filters” defines in part the selectivity of such proteases. However, the ATPases can exert a pulling force on the substrate that is strong enough to unfold the protein, which allows for passage through the series of constrictions. This force is exerted within the central channel of the ATPase complex. Thus, translocation and unfolding of the substrate are generally coupled events (1–3).Although not departing from this paradigm, the eukaryotic proteasome interacts with substrate in a more complex manner as a result of interactions involving the ubiquitin tag. Thus, many of the 13 subunits that were added to the evolutionarily ancient ATPase complex to form the RP in the eukaryotic lineage participate in recognition and processing of the ubiquitin tag (1). For example, the yeast proteasome has five and probably more distinct ubiquitin receptors, two that are integral subunits and three that are reversibly proteasome-associated (4). In addition, proteasomes of mammals have three distinct deubiquitinating enzymes (DUBs). The multiplicity of DUBs points to a surprisingly complex role of deubiquitination in proteasome function.     

The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.     

Rad23 promotes the targeting of proteolytic substrates to the proteasome

Rad23 contains a ubiquitin-like domain (UbL(R23)) that interacts with catalytically active proteasomes and two ubiquitin (Ub)-associated (UBA) sequences that bind Ub. The UBA domains can bind Ub in vitro, although the significance of this interaction in vivo is poorly understood. Rad23 can interfere with the assembly of multi-Ub chains in vitro, and high-level expression caused stabilization of proteolytic substrates in vivo. We report here that Rad23 interacts with ubiquitinated cellular proteins through the synergistic action of its UBA domains. Rad23 plays an overlapping role with Rpn10, a proteasome-associated multi-Ub chain binding protein. Mutations in the UBA domains prevent efficient interaction with ubiquitinated proteins and result in poor suppression of the growth and proteolytic defects of a rad23 Delta rpn10 Delta mutant. High-level expression of Rad23 revealed, for the first time, an interaction between ubiquitinated proteins and the proteasome. This increase was not observed in rpn10 Delta mutants, suggesting that Rpn10 participates in the recognition of proteolytic substrates that are delivered by Rad23. Overexpression of UbL(R23) caused stabilization of a model substrate, indicating that an unregulated UbL(R23)-proteasome interaction can interfere with the efficient delivery of proteolytic substrates by Rad23. Because the suppression of a rad23 Delta rpn10 Delta mutant phenotype required both UbL(R23) and UBA domains, our findings support the hypothesis that Rad23 encodes a novel regulatory factor that translocates ubiquitinated substrates to the proteasome.     

hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37

The 26S proteasome catalyzes the degradation of most proteins in mammalian cells. To better define its composition and associated regulatory proteins, we developed affinity methods to rapidly purify 26S proteasomes from mammalian cells. By this approach, we discovered a novel 46-kDa (407 residues) subunit of its 19S regulatory complex (previously termed ADRM1 or GP110). As its N-terminal half can be incorporated into the 26S proteasome and is homologous to Rpn13, a 156-residue subunit of the 19S complex in budding yeast, we renamed it human Rpn13 (hRpn13). The C-terminal half of hRpn13 binds directly to the proteasome-associated deubiquitinating enzyme, UCH37, and enhances its isopeptidase activity. Knockdown of hRpn13 in 293T cells increases the cellular levels of ubiquitin conjugates and decreases the degradation of short-lived proteins. Surprisingly, an overproduction of hRpn13 also reduced their degradation. Furthermore, transfection of the C-terminal half of hRpn13 slows proteolysis and induces cell death, probably by acting as a dominant-negative form. Thus in human 26S proteasomes, hRpn13 appears to be important for the binding of UCH37 to the 19S complex and for efficient proteolysis.     

Analysis of ubiquitination in vivo using a transgenic mouse model

The primary pathway for the proteolytic destruction of cellular proteins is through ubiquitin-mediated targeting to the proteasome. This pathway is pivotal not only in the elimination of damaged or misfolded proteins but also in the temporal, developmental, or signal-mediated destruction of normal cellular substrates. The list of known substrates of the ubiquitin/proteasome pathway is long, but most substrates have been identified in yeast or, more recently, in cultured mammalian cells. It is likely that many mammalian substrates with developmental or disease relevance have yet to be identified because their ubiquitination occurs in tissue or organ systems that cannot be adequately modeled in vitro. We have developed a transgenic mouse model that will allow the isolation and identification of these substrates. The human UbC promoter was used to drive expression of a hexahistidine-tagged version of human ubiquitin in a variety of mouse tissues from early embryonic stages, as assessed by a green fluorescent protein marker. Cleavage of the fusion protein by endogenous enzymes produced epitope-tagged ubiquitin that was detected both in monomeric form and conjugated to cellular proteins. This mouse model should facilitate in the analysis of normal and disease-related ubiquitination events in vivo.     

Illuminating the ubiquitin/proteasome system

The ubiquitin/proteasome system (UPS) is responsible for the regulated processive degradation of proteins residing in the cytosol, nucleus, and endoplasmic reticulum. The two central players are ubiquitin, a small protein that is conjugated to substrates, and the proteasome, a large multi-subunit proteolytic complex that executes degradation of ubiquitylated proteins. Ubiquitylation and proteasomal degradation are highly dynamic processes. During the last decade, many researchers have started taking advantage of fluorescent proteins, which allow studying the dynamic nature of this system in the context of its natural environment: the living cell. In this review, we will summarize studies that have implemented this approach to examine the UPS and discuss novel insights in the dynamic organization of the UPS.     

Substrate selection by the proteasome through initiation regions

Proteins in the cell have to be eliminated once their function is no longer desired or they become damaged. Most regulated protein degradation is achieved by a large enzymatic complex called the proteasome. Many proteasome substrates are targeted for degradation by the covalent attachment of ubiquitin molecules. Ubiquitinated proteins can be bound by the proteasome, but for proteolysis to occur the proteasome needs to find a disordered tail somewhere in the target at which it initiates degradation. The initiation step contributes to the specificity of proteasomal degradation. Here, we review how the proteasome selects initiation sites within its substrates and discuss how the initiation step affects physiological processes.     

Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway

The ubiquitin proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10delta) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimdelta)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10delta cells, whereas only one-fifth as many accumulated in uimdelta cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS.     

Identification of substrates of the Mycobacterium tuberculosis proteasome

The putative proteasome-associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co-factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome-dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice.     

The mycobacterial Mpa–proteasome unfolds and degrades pupylated substrates by engaging Pup's N‐terminus

Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin‐like protein (Pup) that is recognized by the N‐terminal coiled‐coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa–proteasome complex unfolds and degrades Pup‐tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N‐terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant.     

COP9 Signalosome Interacts ATP-dependently with p97/Valosin-containing Protein (VCP) and Controls the Ubiquitination Status of Proteins Bound to p97/VCP

Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/VCP seems to control the ubiquitination status of recruited substrates. The COP9 signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways.