The ESCRT protein CHMP2B and the RNA-binding protein TDP-43 are both associated with ALS and FTD. The pathogenicity of CHMP2B has mainly been considered a consequence of autophagy–endolysosomal dysfunction, whereas protein inclusions containing phosphorylated TDP-43 are a pathological hallmark of ALS and FTD. Intriguingly, TDP-43 pathology has not been associated with the FTD-causing CHMP2B^Intron5 mutation. In this study, we identify CHMP2B as a modifier of TDP-43–mediated neurodegeneration in a Drosophila screen. Down-regulation of CHMP2B reduces TDP-43 phosphorylation and toxicity in flies and mammalian cells. Surprisingly, although CHMP2B^Intron5 causes dramatic autophagy dysfunction, disturbance of autophagy does not alter TDP-43 phosphorylation levels. Instead, we find that inhibition of CK1, but not TTBK1/2 (all of which are kinases phosphorylating TDP-43), abolishes the modifying effect of CHMP2B on TDP-43 phosphorylation. Finally, we uncover that CHMP2B modulates CK1 protein levels by negatively regulating ubiquitination and the proteasome-mediated turnover of CK1. Together, our findings propose an autophagy-independent role and mechanism of CHMP2B in regulating CK1 abundance and TDP-43 phosphorylation.

The guided entry of tail-anchored protein (GET) pathway, in which the Get3 ATPase delivers an essential class of tail-anchored membrane proteins (TAs) to the Get1/2 receptor at the endoplasmic reticulum, provides a conserved mechanism for TA biogenesis in eukaryotic cells. The membrane-associated events of this pathway remain poorly understood. Here we show that complex assembly between the cytosolic domains (CDs) of Get1 and Get2 strongly enhances the affinity of the individual subunits for Get3•TA, thus enabling efficient capture of the targeting complex. In addition to the known role of Get1CD in remodeling Get3 conformation, two molecular recognition features (MoRFs) in Get2CD induce Get3 opening, and both subunits are required for optimal TA release from Get3. Mutation of the MoRFs attenuates TA insertion into the ER in vivo. Our results demonstrate extensive cooperation between the Get1/2 receptor subunits in the capture and remodeling of the targeting complex, and emphasize the role of MoRFs in receptor function during membrane protein biogenesis.

Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

The in vivo physiological function of liquid–liquid phase separation (LLPS) that governs non–membrane-bound structures remains elusive. Among LLPS-prone proteins, TAR DNA-binding protein of 43 kD (TDP-43) is under intense investigation because of its close association with neurological disorders. Here, we generated mice expressing endogenous LLPS-deficient murine TDP-43. LLPS-deficient TDP-43 mice demonstrate impaired neuronal function and behavioral abnormalities specifically related to brain function. Brain neurons of these mice, however, did not show TDP-43 proteinopathy or neurodegeneration. Instead, the global rate of protein synthesis was found to be greatly enhanced by TDP-43 LLPS loss. Mechanistically, TDP-43 LLPS ablation increased its association with PABPC4, RPS6, RPL7, and other translational factors. The physical interactions between TDP-43 and translational factors relies on a motif, the deletion of which abolished the impact of LLPS-deficient TDP-43 on translation. Our findings show a specific physiological role for TDP-43 LLPS in the regulation of brain function and uncover an intriguing novel molecular mechanism of translational control by LLPS.

Lysosomes (vacuoles in yeast) are master regulators of metabolism and protein turnover, but how they degrade their own resident proteins is unclear. Recently, multiple models have been proposed explaining yeast vacuole protein sorting, but the role of the ESCRT pathway was unclear. In this JCB issue, work from Yang et al. ( https://doi.org/10.1083/jcb.202012104 ) highlights how the ESCRT pathway localizes to the vacuole surface to execute protein sorting of its resident proteins.

Tail-anchored (TA) proteins fulfill diverse cellular functions within different organellar membranes. Their characteristic C-terminal transmembrane segment renders TA proteins inherently prone to aggregation and necessitates their posttranslational targeting. The guided entry of TA proteins (GET in yeast)/transmembrane recognition complex (TRC in humans) pathway represents a major route for TA proteins to the endoplasmic reticulum (ER). Here, we review important new insights into the capture of nascent TA proteins at the ribosome by the GET pathway pretargeting complex and the mechanism of their delivery into the ER membrane by the GET receptor insertase. Interestingly, several alternative routes by which TA proteins can be targeted to the ER have emerged, raising intriguing questions about how selectivity is achieved during TA protein capture. Furthermore, mistargeting of TA proteins is a fundamental cellular problem, and we discuss the recently discovered quality control machineries in the ER and outer mitochondrial membrane for displacing mislocalized TA proteins.

Long-lived proteins (LLPs) have recently emerged as vital components of intracellular structures whose function is coupled to long-term stability. Mitochondria are multifaceted organelles, and their function hinges on efficient proteome renewal and replacement. Here, using metabolic stable isotope labeling of mice combined with mass spectrometry (MS)–based proteomic analysis, we demonstrate remarkable longevity for a subset of the mitochondrial proteome. We discovered that mitochondrial LLPs (mt-LLPs) can persist for months in tissues harboring long-lived cells, such as brain and heart. Our analysis revealed enrichment of mt-LLPs within the inner mitochondrial membrane, specifically in the cristae subcompartment, and demonstrates that the mitochondrial proteome is not turned over in bulk. Pioneering cross-linking experiments revealed that mt-LLPs are spatially restricted and copreserved within protein OXPHOS complexes, with limited subunit exchange throughout their lifetimes. This study provides an explanation for the exceptional mitochondrial protein lifetimes and supports the concept that LLPs provide key structural stability to multiple large and dynamic intracellular structures.

Epithelial cells lining mucosal surfaces distinctively express the inflammatory bowel disease risk gene INAVA. We previously found that INAVA has dual and competing functions: one at lateral membranes where it affects mucosal barrier function and the other in the cytosol where INAVA enhances IL-1β signal transduction and protein ubiquitination and forms puncta. We now find that IL-1β–induced INAVA puncta are biomolecular condensates that rapidly assemble and physiologically resolve. The condensates contain ubiquitin and the E3 ligase βTrCP2, and their formation correlates with amplified ubiquitination, suggesting function in regulation of cellular proteostasis. Accordingly, a small-molecule screen identified ROS inducers, proteasome inhibitors, and inhibitors of the protein folding chaperone HSP90 as potent agonists for INAVA condensate formation. Notably, inhibitors of the p38α and mTOR pathways enhanced resolution of the condensates, and inhibitors of the Rho–ROCK pathway induced INAVA’s competing function by recruiting INAVA to newly assembled intercellular junctions in cells where none existed before.

The ER is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine cross-linking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.

Degradation by macroautophagy can be highly selective, but given the promiscuity of cargo receptors, questions remain surrounding how this selectivity is achieved. In this issue, Nthiga et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202006128 ) show how the adaptor Calcoco1 distinguishes cargo by how it binds.

In mammals, tail-anchored (TA) proteins that are posttranslationally captured by the chaperone SGTA are triaged by the BAG6 complex into one of two fates: handoff to an ER targeting factor for membrane insertion or polyubiquitination for destruction by the proteasome. In this issue, Culver and Mariappan (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202004086 ) show that a fraction of newly synthesized TA proteins is polyubiquitinated in HEK293 cells independently of the BAG6 complex yet evades proteasomal degradation by undergoing deubiquitination en route to becoming stably inserted into the ER membrane.

Numerous proteins that have hydrophobic transmembrane domains (TMDs) traverse the cytosol and posttranslationally insert into cellular membranes. It is unclear how these hydrophobic membrane proteins evade recognition by the cytosolic protein quality control (PQC), which typically recognizes exposed hydrophobicity in misfolded proteins and marks them for proteasomal degradation by adding ubiquitin chains. Here, we find that tail-anchored (TA) proteins, a vital class of membrane proteins, are recognized by cytosolic PQC and are ubiquitinated as soon as they are synthesized in cells. Surprisingly, the ubiquitinated TA proteins are not routed for proteasomal degradation but instead are handed over to the targeting factor, TRC40, and delivered to the ER for insertion. The ER-associated deubiquitinases, USP20 and USP33, remove ubiquitin chains from TA proteins after their insertion into the ER. Thus, our data suggest that deubiquitinases rescue posttranslationally targeted membrane proteins that are inappropriately ubiquitinated by PQC in the cytosol.

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.

Aging, disease, and environmental stressors are associated with failures in the ubiquitin-proteasome system (UPS), yet a quantitative understanding of how stressors affect the proteome and how the UPS responds is lacking. Here we assessed UPS performance and adaptability in yeast under stressors using quantitative measurements of misfolded substrate stability and stress-dependent UPS regulation by the transcription factor Rpn4. We found that impairing degradation rates (proteolytic stress) and generating misfolded proteins (folding stress) elicited distinct effects on the proteome and on UPS adaptation. Folding stressors stabilized proteins via aggregation rather than overburdening the proteasome, as occurred under proteolytic stress. Still, the UPS productively adapted to both stressors using separate mechanisms: proteolytic stressors caused Rpn4 stabilization while folding stressors increased RPN4 transcription. In some cases, adaptation completely prevented loss of UPS substrate degradation. Our work reveals the distinct effects of proteotoxic stressors and the versatility of cells in adapting the UPS.

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. How its signaling activity is fine-tuned in response to fluctuated Hh gradient is less known. Here, we identify protein phosphatase V (PpV), the catalytic subunit of protein phosphatase 6, as a homeostatic regulator of Hh signaling. PpV is genetically upstream of widerborst (wdb), which encodes a regulatory subunit of PP2A that modulates high-level Hh signaling. We show that PpV negatively regulates Wdb stability independent of phosphatase activity of PpV, by competing with the catalytic subunit of PP2A for Wdb association, leading to Wdb ubiquitination and subsequent proteasomal degradation. Thus, regulated Wdb stability, maintained through competition between two closely related phosphatases, ensures graded Hh signaling. Interestingly, PpV expression is regulated by Hh signaling. Therefore, PpV functions as a Hh activity sensor that regulates Wdb-mediated PP2A activity through feedback mechanisms to maintain Hh signaling homeostasis.

While it is well-known that E3 ubiquitin ligases can selectively ubiquitinate membrane proteins in response to specific environmental cues, the underlying mechanisms for the selectivity are poorly understood. In particular, the role of transmembrane regions, if any, in target recognition remains an open question. Here, we describe how Ssh4, a yeast E3 ligase adaptor, recognizes the PQ-loop lysine transporter Ypq1 only after lysine starvation. We show evidence of an interaction between two transmembrane helices of Ypq1 (TM5 and TM7) and the single transmembrane helix of Ssh4. This interaction is regulated by the conserved PQ motif. Strikingly, recent structural studies of the PQ-loop family have suggested that TM5 and TM7 undergo major conformational changes during substrate transport, implying that transport-associated conformational changes may determine the selectivity. These findings thus provide critical information concerning the regulatory mechanism through which transmembrane domains can be specifically recognized in response to changing environmental conditions.

The heat shock response (HSR) is a gene expression program that protects cells from heat and proteotoxic stressors. In this issue, Feder et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.202005165 ) show that subcellular relocalization of the cochaperone Sis1 drives the HSR by de-suppressing the transcription factor Hsf1.

Cells exposed to heat shock induce a conserved gene expression program, the heat shock response (HSR), encoding protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1, a cofactor for the chaperone Hsp70, controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm, where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the endoplasmic reticulum. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.

Dendrite pathology is frequently observed in various neurodegenerative diseases (NDs). Although previous studies identified several pathogenic mediators of dendrite defects that act through loss of function in NDs, the underlying pathogenic mechanisms remain largely unexplored. Here, our search for additional pathogenic contributors to dendrite defects in NDs identifies Relish/NF-κB as a novel gain-of-toxicity–based mediator of dendrite defects in animal models for polyglutamine (polyQ) diseases and amyotrophic lateral sclerosis (ALS). In a Drosophila model for polyQ diseases, polyQ-induced dendrite defects require Dredd/Caspase-8–mediated endoproteolytic cleavage of Relish to generate the N-terminal fragment, Rel68, and subsequent Charon-mediated nuclear localization of Rel68. Rel68 alone induced neuronal toxicity causing dendrite and behavioral defects, and we identify two novel transcriptional targets, Tup and Pros, that mediate Rel68-induced neuronal toxicity. Finally, we show that Rel68-induced toxicity also contributes to dendrite and behavioral defects in a Drosophila model for ALS. Collectively, our data propose disinhibition of latent toxicity of Relish/NF-κB as a novel pathogenic mechanism underlying dendrite pathology in NDs.

Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, where individual peptides, recognized by a consensus motif, are translocated directly across the lysosomal membrane. CMA regulates the abundance of many disease-related proteins, with causative roles in neoplasia, neurodegeneration, hepatosteatosis, and other pathologies relevant to human health and aging. At the lysosomal membrane, CMA is inhibited by Akt-dependent phosphorylation of the CMA regulator GFAP. The INS-PI3K-PDPK1 pathway regulates Akt, but its role in CMA is unclear. Here, we report that inhibition of class I PI3K or PDPK1 activates CMA. In contrast, selective inhibition of class III PI3Ks does not activate CMA. Isolated liver lysosomes from mice treated with either of two orally bioavailable class I PI3K inhibitors, pictilisib or buparlisib, display elevated CMA activity, and decreased phosphorylation of lysosomal GFAP, with no change in macroautophagy. The findings of this study represent an important first step in repurposing class I PI3K inhibitors to modulate CMA in vivo.