The polySUMOylation axis promotes nucleolar release of Tof2 for mitotic exit.

In budding yeast, the nucleolus serves as the site to sequester Cdc14, a phosphatase essential for mitotic exit. Nucleolar proteins Tof2, Net1, and Fob1 are required for this sequestration. Although it is known that these nucleolar proteins are SUMOylated, how SUMOylation regulates their activity remains unknown. Here, we show that Tof2 exhibits cell-cycle-regulated nucleolar delocalization and turnover. Depletion of the nuclear small ubiquitin-like modifier (SUMO) protease Ulp2 not only causes Tof2 polySUMOylation, nucleolar delocalization, and degradation but also leads to Cdc14 nucleolar release and activation. This outcome depends on polySUMOylation and the activity of downstream enzymes, including SUMO-targeted ubiquitin ligase and Cdc48/p97 segregase. We further developed a system to tether SUMO machinery to Tof2 and generated a SUMO-deficient tof2 mutant, and the results indicate that Tof2 polySUMOylation is necessary and sufficient for its nucleolar delocalization and degradation. Together, our work reveals a polySUMO-dependent mechanism that delocalizes Tof2 from the nucleolus to facilitate mitotic exit.

The mechanosensitive gene arrestin domain containing 2 regulates myotube diameter with direct implications for disuse atrophy with aging.

Arrestin domain containing 2 and 3 (Arrdc2/3) are genes whose mRNA contents are decreased in young skeletal muscle following mechanical overload. Arrdc3 is linked to the regulation of signaling pathways in nonmuscle cells that could influence skeletal muscle size. Despite a similar amino acid sequence, Arrdc2 function remains undefined. The purpose of this study was to further explore the relationship of Arrdc2/Arrdc3 expression with changes in mechanical load in young and aged muscle and define the effect of Arrdc2/3 expression on C2C12 myotube diameter. In young and aged mice, mechanical load was decreased using hindlimb suspension whereas mechanical load was increased by reloading previously unloaded muscle or inducing high-force contractions. Arrdc2 and Arrdc3 mRNAs were overexpressed in C2C12 myotubes using adenoviruses. Myotube diameter was determined 48-h posttransfection, and RNA sequencing was performed on those samples. Arrdc2 and Arrdc3 mRNA content was higher in the unloaded muscle within 1 day of disuse and remained higher up through 10 days. The induction of Arrdc2 mRNA was more pronounced in aged muscle than young muscle in response to unloading. Reloading previously unloaded muscle of young and aged mice restored Arrdc2 and Arrdc3 levels to ambulatory levels. Increasing mechanical load beyond normal ambulatory levels lowered Arrdc2 mRNA, but not Arrdc3 mRNA, in young and aged muscle. Arrdc2 overexpression only was sufficient to lower myotube diameter in C2C12 cells in part by altering the transcriptome favoring muscle atrophy. These data are consistent with Arrdc2 contributing to disuse atrophy, particularly in aged muscle.NEW & NOTEWORTHY We establish Arrdc2 as a novel mechanosensitive gene highly induced in response to mechanical unloading, particularly in aged muscle. Arrdc2 induction in C2C12 myotubes is sufficient to produce thinner myotubes and a transcriptional landscape consistent with muscle atrophy and disuse.

An unstructured proteasome inhibitor comes into focus.

The inhibitory mechanism of an intrinsically disordered proteasome inhibitor identified over 30 years ago has finally been revealed by cryo-electron microscopy by Hsu et al. in a recent report in the Journal of Biological Chemistry. The structure, coupled with biochemical and cell-based experiments, resolves lingering questions about how the inhibitor achieves multisite inhibition of proteasomal protease activity, while raising several exciting new questions on the nature of proteasome subpopulations in the process.

Wiggle and Shake: Managing and Exploiting Conformational Dynamics during Proteasome Biogenesis.

The 26S proteasome is the largest and most complicated protease known, and changes to proteasome assembly or function contribute to numerous human diseases. Assembly of the 26S proteasome from its ~66 individual polypeptide subunits is a highly orchestrated process requiring the concerted actions of both intrinsic elements of proteasome subunits, as well as assistance by extrinsic, dedicated proteasome assembly chaperones. With the advent of near-atomic resolution cryo-electron microscopy, it has become evident that the proteasome is a highly dynamic machine, undergoing numerous conformational changes in response to ligand binding and during the proteolytic cycle. In contrast, an appreciation of the role of conformational dynamics during the biogenesis of the proteasome has only recently begun to emerge. Herein, we review our current knowledge of proteasome assembly, with a particular focus on how conformational dynamics guide particular proteasome biogenesis events. Furthermore, we highlight key emerging questions in this rapidly expanding area.

Proteasomes: Isolation and Activity Assays.

In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5-MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purification schemes for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae. A gel filtration step can be added to further enhance purity. We also describe assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Purification of active 26S proteasomes Support Protocol 1: Growth of yeast strains and production of yeast cell powder Support Protocol 2: Regeneration of anti-flag M2 affinity gel Basic Protocol 2: Purification of the 19S regulatory particle (RP) Basic Protocol 3: Purification of active 20S CP Basic Protocol 4: In-gel peptidase activity assay for 20S CP and 26S proteasomes Basic Protocol 5: In-solution peptidase activity assay for 20S and 26S proteasomes Basic Protocol 6: Measuring degradation of polyubiquitinated SIC1PY Basic Protocol 7: Gel filtration of purified proteasomes and subcomplexes.

Assembly chaperone Nas6 selectively destabilizes 26S proteasomes with defective regulatory particle-core particle interfaces.

The 26S proteasome is a 66-subunit-chambered protease present in all eukaryotes that maintains organismal health by degrading unneeded or defective proteins. Defects in proteasome function or assembly are known to contribute to the development of various cancers, neurodegeneration, and diabetes. During proteasome biogenesis, a family of evolutionarily conserved chaperones assembles a hexameric ring of AAA+ family ATPase subunits contained within the proteasomal regulatory particle (RP) and guide their docking onto the surface of the proteolytic core particle (CP). This RP-CP interaction couples the substrate capture and unfolding process to proteolysis. We previously reported a mutation in the proteasome that promoted dissociation of the RP and CP by one of these chaperones, Nas6. However, the nature of the signal for Nas6-dependent proteasome disassembly and the generality of this postassembly proteasome quality control function for Nas6 remain unknown. Here, we use structure-guided mutagenesis and in vitro proteasome disassembly assays to demonstrate that Nas6 more broadly destabilizes 26S proteasomes with a defective RP-CP interface. We show that Nas6 can promote dissociation of mature proteasomes into RP and CP in cells harboring defects on either side of the RP-CP interface. This function is unique to Nas6 and independent from other known RP assembly chaperones. Further biochemical experiments suggest that Nas6 may exploit a weakened RP-CP interface to dissociate the RP from the CP. We propose that this postassembly role of Nas6 may fulfill a quality control function in cells by promoting the recycling of functional subcomplexes contained within defective proteasomes.

Proteasome Inhibition Is an Effective Treatment Strategy for Microsporidia Infection in Honey Bees.

The microsporidia Nosema ceranae is an obligate intracellular parasite that causes honey bee mortality and contributes to colony collapse. Fumagillin is presently the only pharmacological control for N. ceranae infections in honey bees. Resistance is already emerging, and alternative controls are critically needed. Nosema spp. exhibit increased sensitivity to heat shock, a common proteotoxic stress. Thus, we hypothesized that targeting the Nosema proteasome, the major protease removing misfolded proteins, might be effective against N. ceranae infections in honey bees. Nosema genome analysis and molecular modeling revealed an unexpectedly compact proteasome apparently lacking multiple canonical subunits, but with highly conserved proteolytic active sites expected to be receptive to FDA-approved proteasome inhibitors. Indeed, N. ceranae were strikingly sensitive to pharmacological disruption of proteasome function at doses that were well tolerated by honey bees. Thus, proteasome inhibition is a novel candidate treatment strategy for microsporidia infection in honey bees.

Androgen depletion alters the diurnal patterns to signals that regulate autophagy in the limb skeletal muscle.

Hypogonadism contributes to limb skeletal muscle atrophy by increasing rates of muscle protein breakdown. Androgen depletion increases markers of the autophagy protein breakdown pathway in the limb muscle that persist throughout the diurnal cycle. However, the regulatory signals underpinning the increase in autophagy markers remain ill-defined. The purpose of this study was to characterize changes to autophagy regulatory signals in the limb skeletal muscle following androgen depletion. Male mice were subjected to a castration surgery or a sham surgery as a control. Seven weeks post-surgery, a subset of mice from each group was sacrificed every 4 hr over a 24 hr period. Protein and mRNA from the Tibialis Anterior (TA) were subjected to Western blot and RT-PCR. Consistent with an overall increase in autophagy, the phosphorylation pattern of Uncoordinated Like Kinase 1 (ULK1) (Ser555) was elevated throughout the diurnal cycle in the TA of castrated mice. Factors that induce the progression of autophagy were also increased in the TA following androgen depletion including an increase in the phosphorylation of c-Jun N-terminal Kinase (JNK) (Thr183/Tyr185) and an increase in the ratio of BCL-2 Associated X (BAX) to B-cell lymphoma 2 (BCL-2). Moreover, we observed an increase in the protein expression pattern of p53 and the mRNA of the p53 target genes Cyclin-Dependent Kinase Inhibitor 1A (p21) and Growth Arrest and DNA Damage Alpha (Gadd45a), which are known to increase autophagy and induce muscle atrophy. These data characterize novel changes to autophagy regulatory signals in the limb skeletal muscle following androgen deprivation.

Wake-sleep cycles are severely disrupted by diseases affecting cytoplasmic homeostasis.

The circadian clock is based on a transcriptional feedback loop with an essential time delay before feedback inhibition. Previous work has shown that PERIOD (PER) proteins generate circadian time cues through rhythmic nuclear accumulation of the inhibitor complex and subsequent interaction with the activator complex in the feedback loop. Although this temporal manifestation of the feedback inhibition is the direct consequence of PER's cytoplasmic trafficking before nuclear entry, how this spatial regulation of the pacemaker affects circadian timing has been largely unexplored. Here we show that circadian rhythms, including wake-sleep cycles, are lengthened and severely unstable if the cytoplasmic trafficking of PER is disrupted by any disease condition that leads to increased congestion in the cytoplasm. Furthermore, we found that the time delay and robustness in the circadian clock are seamlessly generated by delayed and collective phosphorylation of PER molecules, followed by synchronous nuclear entry. These results provide clear mechanistic insight into why circadian and sleep disorders arise in such clinical conditions as metabolic and neurodegenerative diseases and aging, in which the cytoplasm is congested.

The Cdc48 Complex Alleviates the Cytotoxicity of Misfolded Proteins by Regulating Ubiquitin Homeostasis.

The accumulation of misfolded proteins is associated with multiple neurodegenerative disorders, but it remains poorly defined how this accumulation causes cytotoxicity. Here, we demonstrate that the Cdc48/p97 segregase machinery drives the clearance of ubiquitinated model misfolded protein Huntingtin (Htt103QP) and limits its aggregation. Nuclear ubiquitin ligase San1 acts upstream of Cdc48 to ubiquitinate Htt103QP. Unexpectedly, deletion of SAN1 and/or its cytosolic counterpart UBR1 rescues the toxicity associated with Cdc48 deficiency, suggesting that ubiquitin depletion, rather than compromised proteolysis of misfolded proteins, causes the growth defect in cells with Cdc48 deficiency. Indeed, Cdc48 deficiency leads to elevated protein ubiquitination levels and decreased free ubiquitin, which depends on San1/Ubr1. Furthermore, enhancing free ubiquitin levels rescues the toxicity in various Cdc48 pathway mutants and restores normal turnover of a known Cdc48-independent substrate. Our work highlights a previously unappreciated function for Cdc48 in ensuring the regeneration of monoubiquitin that is critical for normal cellular function.

A suite of polymerase chain reaction-based peptide tagging plasmids for epitope-targeted enzymatic functionalization of yeast proteins.

The budding yeast and model eukaryote Saccharomyces cerevisiae has been invaluable for purification and analysis of numerous evolutionarily conserved proteins and multisubunit complexes that cannot be readily reconstituted in Escherichia coli. For many studies, it is desirable to functionalize a particular protein or subunit of a complex with a ligand, fluorophore or other small molecule. Enzyme-catalysed site-specific modification of proteins bearing short peptide tags is a powerful strategy to overcome the limitations associated with traditional nonselective labelling chemistries. Towards this end, we developed a suite of template plasmids for C-terminal tagging with short peptide sequences that can be site-specifically functionalized with high efficiency and selectivity. We have also combined these sequences with the FLAG tag as a handle for purification or immunological detection of the modified protein. We demonstrate the utility of these plasmids by site-specifically labelling the 28-subunit core particle subcomplex of the 26S proteasome with the small molecule fluorophore Cy5. The full set of plasmids has been deposited in the non-profit plasmid repository Addgene (http://www.addgene.org).

Proteasome subunit α1 overexpression preferentially drives canonical proteasome biogenesis and enhances stress tolerance in yeast.

The 26S proteasome conducts the majority of regulated protein catabolism in eukaryotes. At the heart of the proteasome is the barrel-shaped 20S core particle (CP), which contains two ß-rings sandwiched between two α-rings. Whereas canonical CPs contain α-rings with seven subunits arranged α1-α7, a non-canonical CP in which a second copy of the α4 subunit replaces the α3 subunit occurs in both yeast and humans. The mechanisms that control canonical versus non-canonical CP biogenesis remain poorly understood. Here, we have repurposed a split-protein reporter to identify genes that can enhance canonical proteasome assembly in mutant yeast producing non-canonical α4-α4 CPs. We identified the proteasome subunit α1 as an enhancer of α3 incorporation, and find that elevating α1 protein levels preferentially drives canonical CP assembly under conditions that normally favor α4-α4 CP formation. Further, we demonstrate that α1 is stoichiometrically limiting for α-ring assembly, and that enhancing α1 levels is sufficient to increase proteasome abundance and enhance stress tolerance in yeast. Together, our data indicate that the abundance of α1 exerts multiple impacts on proteasome assembly and composition, and we propose that the limited α1 levels observed in yeast may prime cells for alternative proteasome assembly following environmental stimuli.

Disruptions to the limb muscle core molecular clock coincide with changes in mitochondrial quality control following androgen depletion.

Androgen depletion in humans leads to significant atrophy of the limb muscles. However, the pathways by which androgens regulate limb muscle mass are unclear. Our laboratory previously showed that mitochondrial degradation was related to the induction of autophagy and the degree of muscle atrophy following androgen depletion, implying that decreased mitochondrial quality contributes to muscle atrophy. To increase our understanding of androgen-sensitive pathways regulating decreased mitochondrial quality, total RNA from the tibialis anterior of sham and castrated mice was subjected to microarray analysis. Using this unbiased approach, we identified significant changes in the expression of genes that compose the core molecular clock. To assess the extent to which androgen depletion altered the limb muscle clock, the tibialis anterior muscles from sham and castrated mice were harvested every 4 h throughout a diurnal cycle. The circadian expression patterns of various core clock genes and known clock-controlled genes were disrupted by castration, with most genes exhibiting an overall reduction in phase amplitude. Given that the core clock regulates mitochondrial quality, disruption of the clock coincided with changes in the expression of genes involved with mitochondrial quality control, suggesting a novel mechanism by which androgens may regulate mitochondrial quality. These events coincided with an overall increase in mitochondrial degradation in the muscle of castrated mice and an increase in markers of global autophagy-mediated protein breakdown. In all, these data are consistent with a novel conceptual model linking androgen depletion-induced limb muscle atrophy to reduced mitochondrial quality control via disruption of the molecular clock.

Engineered disulfide crosslinking to measure conformational changes in the 26S proteasome.

The 26S proteasome is a multisubunit ATP-dependent peptidase complex mediating most regulated protein degradation in eukaryotes. The proteasome undergoes several coordinated conformational changes during catalysis that activate it for substrate processing and functionally couple distinct enzymatic activities during substrate degradation. Understanding the impact of substrate interactions and individual ATP binding events on these conformational changes is currently a major bottleneck in the study of proteasome function. Here, we describe a simple biochemical reporter based on engineered disulfide crosslinking for measuring the conformational distribution of the Saccharomyces cerevisiae 26S proteasome. We demonstrate its use to investigate the impact of ATP analogs and proteasome inhibitors on proteasome conformational equilibria. This reporter allows simultaneous and rapid comparison of multiple treatments or conditions on the steady-state conformational distribution of the proteasome and can be readily extended to the study of other multisubunit complexes for which multiple conformational states are known at near-atomic resolution.

The anaphase-promoting complex regulates the degradation of the inner nuclear membrane protein Mps3.

The nucleus is enclosed by the inner nuclear membrane (INM) and the outer nuclear membrane (ONM). While the ONM is continuous with the endoplasmic reticulum (ER), the INM is independent and separates the nucleoplasm from the ER lumen. Turnover of ER proteins has been well characterized by the ER-associated protein degradation (ERAD) pathway, but very little is known about turnover of resident INM proteins. Here we show that the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase, regulates the degradation of Mps3, a conserved integral protein of the INM. Turnover of Mps3 requires the ubiquitin-conjugating enzyme Ubc7, but was independent of the known ERAD ubiquitin ligases Doa10 and Hrd1 as well as the recently discovered Asi1-Asi3 complex. Using a genetic approach, we have found that Cdh1, a coactivator of APC/C, modulates Mps3 stability. APC/C controls Mps3 degradation through Mps3's N terminus, which resides in the nucleoplasm and possesses two putative APC/C-dependent destruction motifs. Accumulation of Mps3 at the INM impairs nuclear morphological changes and cell division. Our findings therefore reveal an unexpected mechanism of APC/C-mediated protein degradation at the INM that coordinates nuclear morphogenesis and cell cycle progression.

An Allosteric Interaction Network Promotes Conformation State-Dependent Eviction of the Nas6 Assembly Chaperone from Nascent 26S Proteasomes.

The 26S proteasome is the central ATP-dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation-specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP binding by the ATPase subunit Rpt3 promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly-coupled allosteric signals promote chaperone eviction and provides a framework for understanding the eviction of other chaperones from this biomedically important molecular machine.

Expanded Coverage of the 26S Proteasome Conformational Landscape Reveals Mechanisms of Peptidase Gating.

The proteasome is the central protease for intracellular protein breakdown. Coordinated binding and hydrolysis of ATP by the six proteasomal ATPase subunits induces conformational changes that drive the unfolding and translocation of substrates into the proteolytic 20S core particle for degradation. Here, we combine genetic and biochemical approaches with cryo-electron microscopy and integrative modeling to dissect the relationship between individual nucleotide binding events and proteasome conformational dynamics. We demonstrate unique impacts of ATP binding by individual ATPases on the proteasome conformational distribution and report two conformational states of the proteasome suggestive of a rotary ATP hydrolysis mechanism. These structures, coupled with functional analyses, reveal key roles for the ATPases Rpt1 and Rpt6 in gating substrate entry into the core particle. This deepened knowledge of proteasome conformational dynamics reveals key elements of intersubunit communication within the proteasome and clarifies the regulation of substrate entry into the proteolytic chamber.

Autophagic clearance of proteasomes in yeast requires the conserved sorting nexin Snx4.

Turnover of the 26S proteasome by autophagy is an evolutionarily conserved process that governs cellular proteolytic capacity and eliminates inactive particles. In most organisms, proteasomes are located in both the nucleus and cytoplasm. However, the specific autophagy routes for nuclear and cytoplasmic proteasomes are unclear. Here, we investigate the spatial control of autophagic proteasome turnover in budding yeast (Saccharomyces cerevisiae). We found that nitrogen starvation-induced proteasome autophagy is independent of known nucleophagy pathways but is compromised when nuclear protein export is blocked. Furthermore, via pharmacological tethering of proteasomes to chromatin or the plasma membrane, we provide evidence that nuclear proteasomes at least partially disassemble before autophagic turnover, whereas cytoplasmic proteasomes remain largely intact. A targeted screen of autophagy genes identified a requirement for the conserved sorting nexin Snx4 in the autophagic turnover of proteasomes and several other large multisubunit complexes. We demonstrate that Snx4 cooperates with sorting nexins Snx41 and Snx42 to mediate proteasome turnover and is required for the formation of cytoplasmic proteasome puncta that accumulate when autophagosome formation is blocked. Together, our results support distinct mechanistic paths in the turnover of nuclear versus cytoplasmic proteasomes and point to a critical role for Snx4 in cytoplasmic agglomeration of proteasomes en route to autophagic destruction.

Proteasomes, caught in the act.

Although energy-dependent protein destruction by the proteasome has been known for over 30 years, how this intricate molecular machine uses ATP to power protein degradation has remained very poorly understood. In a recently published paper, Ding et al. present a snapshot of the proteasome mid-catalysis, yielding new and unexpected insights into the catalytic mechanism of this ATP-powered multisubunit machine.

A Single α Helix Drives Extensive Remodeling of the Proteasome Lid and Completion of Regulatory Particle Assembly.

Most short-lived eukaryotic proteins are degraded by the proteasome. A proteolytic core particle (CP) capped by regulatory particles (RPs) constitutes the 26S proteasome complex. RP biogenesis culminates with the joining of two large subcomplexes, the lid and base. In yeast and mammals, the lid appears to assemble completely before attaching to the base, but how this hierarchical assembly is enforced has remained unclear. Using biochemical reconstitutions, quantitative cross-linking/mass spectrometry, and electron microscopy, we resolve the mechanistic basis for the linkage between lid biogenesis and lid-base joining. Assimilation of the final lid subunit, Rpn12, triggers a large-scale conformational remodeling of the nascent lid that drives RP assembly, in part by relieving steric clash with the base. Surprisingly, this remodeling is triggered by a single Rpn12 α helix. Such assembly-coupled conformational switching is reminiscent of viral particle maturation and may represent a commonly used mechanism to enforce hierarchical assembly in multisubunit complexes.