ABSTRACT
Cells respond to environmental cues by remodeling their inventories of multiprotein complexes. Cellular repertoires of SCF (SKP1-CUL1-F box protein) ubiquitin ligase complexes, which mediate much protein degradation, require CAND1 to distribute the limiting CUL1 subunit across the family of â¼70 different F box proteins. Yet, how a single factor coordinately assembles numerous distinct multiprotein complexes remains unknown. We obtained cryo-EM structures of CAND1-bound SCF complexes in multiple states and correlated mutational effects on structures, biochemistry, and cellular assays. The data suggest that CAND1 clasps idling catalytic domains of an inactive SCF, rolls around, and allosterically rocks and destabilizes the SCF. New SCF production proceeds in reverse, through SKP1-F box allosterically destabilizing CAND1. The CAND1-SCF conformational ensemble recycles CUL1 from inactive complexes, fueling mixing and matching of SCF parts for E3 activation in response to substrate availability. Our data reveal biogenesis of a predominant family of E3 ligases, and the molecular basis for systemwide multiprotein complex assembly.
Subject(s)
Cullin Proteins , F-Box Proteins , SKP Cullin F-Box Protein Ligases , Transcription Factors , Humans , Cullin Proteins/chemistry , Cullin Proteins/metabolism , F-Box Proteins/metabolism , Molecular Conformation , SKP Cullin F-Box Protein Ligases/chemistry , SKP Cullin F-Box Protein Ligases/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Cullin-RING ubiquitin ligases (CRLs) are dynamic modular platforms that regulate myriad biological processes through target-specific ubiquitylation. Our knowledge of this system emerged from the F-box hypothesis, posited a quarter century ago: Numerous interchangeable F-box proteins confer specific substrate recognition for a core CUL1-based RING E3 ubiquitin ligase. This paradigm has been expanded through the evolution of a superfamily of analogous modular CRLs, with five major families and over 200 different substrate-binding receptors in humans. Regulation is achieved by numerous factors organized in circuits that dynamically control CRL activation and substrate ubiquitylation. CRLs also serve as a vast landscape for developing small molecules that reshape interactions and promote targeted ubiquitylation-dependent turnover of proteins of interest. Here, we review molecular principles underlying CRL function, the role of allosteric and conformational mechanisms in controlling substrate timing and ubiquitylation, and how the dynamics of substrate receptor interchange drives the turnover of selected target proteins to promote cellular decision-making.
Subject(s)
Cullin Proteins/chemistry , Cullin Proteins/metabolism , F-Box Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , F-Box Proteins/chemistry , Feedback, Physiological , Host-Pathogen Interactions/physiology , Humans , NEDD8 Protein/metabolism , Plant Growth Regulators/metabolism , Protein Domains , Protein Processing, Post-Translational , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/chemistry , UbiquitinationABSTRACT
Our understanding of the ubiquitin code has greatly evolved from conventional E1, E2 and E3 enzymes that modify Lys residues on specific substrates with a single type of ubiquitin chain to more complex processes that regulate and mediate ubiquitylation. In this Review, we discuss recently discovered endogenous mechanisms and unprecedented pathways by which pathogens rewrite the ubiquitin code to promote infection. These processes include unconventional ubiquitin modifications involving ester linkages with proteins, lipids and sugars, or ubiquitylation through a phosphoribosyl bridge involving Arg42 of ubiquitin. We also introduce the enzymatic pathways that write and reverse these modifications, such as the papain-like proteases of severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Furthermore, structural studies have revealed that the ultimate functions of ubiquitin are mediated not simply by straightforward recognition by ubiquitin-binding domains. Instead, elaborate multivalent interactions between ubiquitylated targets or ubiquitin chains and their readers (for example, the proteasome, the MLL1 complex or DOT1L) can elicit conformational changes that regulate protein degradation or transcription. The newly discovered mechanisms provide opportunities for innovative therapeutic interventions for diseases such as cancer and infectious diseases.
Subject(s)
COVID-19 , Ubiquitin , Humans , Ubiquitin/metabolism , SARS-CoV-2 , Ubiquitination , Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Proteasome Endopeptidase Complex/metabolismABSTRACT
For our special issue on stress, we asked scientists about recovering from the stress of the pandemic, including some who shared insights with us in mid-2020. They discuss the importance of teamwork, reassessing priorities, and the added stresses of the cost-of-living crisis, funding cuts, and retaining scientists in academia.
Subject(s)
COVID-19 , Humans , COVID-19/epidemiology , PandemicsABSTRACT
Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.
Subject(s)
Cell Proliferation , Hydroxymethylglutaryl-CoA Synthase , Mechanistic Target of Rapamycin Complex 1 , Proteolysis , Ubiquitin-Protein Ligases , Ubiquitination , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 1/genetics , Humans , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , HEK293 Cells , Hydroxymethylglutaryl-CoA Synthase/metabolism , Hydroxymethylglutaryl-CoA Synthase/genetics , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/genetics , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , Mevalonic Acid/metabolism , Multiprotein Complexes/metabolism , Multiprotein Complexes/genetics , Signal Transduction , Degrons , Adaptor Proteins, Signal TransducingABSTRACT
Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.
Subject(s)
Cullin Proteins , Ubiquitin-Protein Ligases , Humans , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Cullin Proteins/genetics , Cullin Proteins/metabolism , Ubiquitination , Ubiquitin/metabolism , Polyubiquitin/metabolism , Carrier Proteins/metabolismABSTRACT
Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including those for RING-family GID/CTLH E3 ubiquitin ligases and their dedicated E2, Ubc8/UBE2H (yeast/human nomenclature). GID/CTLH-Ubc8/UBE2H-mediated ubiquitylation regulates biological processes ranging from yeast metabolic signaling to human development. Here, cryoelectron microscopy (cryo-EM), biochemistry, and cell biology reveal this exquisitely specific E3-E2 pairing through an unconventional catalytic assembly and auxiliary interactions 70-100 Å away, mediated by E2 multisite phosphorylation. Rather than dynamic polyelectrostatic interactions reported for other ubiquitylation complexes, multiple Ubc8/UBE2H phosphorylation sites within acidic CK2-targeted sequences specifically anchor the E2 C termini to E3 basic patches. Positions of phospho-dependent interactions relative to the catalytic domains correlate across evolution. Overall, our data show that phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, rigidifies the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites.
Subject(s)
Saccharomyces cerevisiae , Ubiquitin-Conjugating Enzymes , Humans , Ubiquitin-Conjugating Enzymes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Phosphorylation , Cryoelectron Microscopy , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.
Subject(s)
Nicotinamide-Nucleotide Adenylyltransferase , Prodrugs , Ubiquitin-Protein Ligases , Ubiquitination , Humans , Cryoelectron Microscopy , HEK293 Cells , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Prodrugs/metabolism , Protein Binding , Substrate Specificity , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolismABSTRACT
Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation.
Subject(s)
Carrier Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Ubiquitination , Ubiquitins/metabolism , Carrier Proteins/genetics , Cullin Proteins/metabolism , Gene Knockdown Techniques , HEK293 Cells , Humans , Mutation , NEDD8 Protein , Polyubiquitin/metabolism , Proteomics , Substrate Specificity , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.
Subject(s)
Anaphase-Promoting Complex-Cyclosome/chemistry , Anaphase-Promoting Complex-Cyclosome/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin/metabolism , Amino Acid Sequence , Biocatalysis , Cryoelectron Microscopy , Humans , Models, Molecular , Saccharomyces cerevisiae Proteins/chemistry , Structure-Activity Relationship , UbiquitinationABSTRACT
Tsai et al.1 in this issue and Mark et al.2 in Cell reveal how the E3 ligase UBR5 mediates broad regulation by selectively targeting agonist-bound nuclear hormone receptors, MYC, and other transcriptional regulators not incorporated into active gene expression complexes.
Subject(s)
Ubiquitin-Protein Ligases , Ubiquitination , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Gene ExpressionABSTRACT
E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.
Subject(s)
Proteins , Ubiquitin-Protein Ligases , Humans , Carrier Proteins , Proteins/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
The TFE3 and MITF master transcription factors maintain metabolic homeostasis by regulating lysosomal, melanocytic, and autophagy genes. Previous studies posited that their cytosolic retention by 14-3-3, mediated by the Rag GTPases-mTORC1, was key for suppressing transcriptional activity in the presence of nutrients. Here, we demonstrate using mammalian cells that regulated protein stability plays a fundamental role in their control. Amino acids promote the recruitment of TFE3 and MITF to the lysosomal surface via the Rag GTPases, activating an evolutionarily conserved phospho-degron and leading to ubiquitination by CUL1ß-TrCP and degradation. Elucidation of the minimal functional degron revealed a conserved alpha-helix required for interaction with RagA, illuminating the molecular basis for a severe neurodevelopmental syndrome caused by missense mutations in TFE3 within the RagA-TFE3 interface. Additionally, the phospho-degron is recurrently lost in TFE3 genomic translocations that cause kidney cancer. Therefore, two divergent pathologies converge on the loss of protein stability regulation by nutrients.
Subject(s)
Amino Acids , Microphthalmia-Associated Transcription Factor , Animals , Mechanistic Target of Rapamycin Complex 1/metabolism , Microphthalmia-Associated Transcription Factor/genetics , Microphthalmia-Associated Transcription Factor/metabolism , Amino Acids/metabolism , Nutrients , Protein Stability , Lysosomes/genetics , Lysosomes/metabolism , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Mammals/metabolismABSTRACT
Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.
Subject(s)
RNA , Ubiquitin-Protein Ligases , Humans , RNA/metabolism , Ubiquitination , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Formaldehyde/toxicity , Aldehydes/toxicity , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Specificity of eukaryotic protein degradation is determined by E3 ubiquitin ligases and their selective binding to protein motifs, termed "degrons," in substrates for ubiquitin-mediated proteolysis. From the discovery of the first substrate degron and the corresponding E3 to a flurry of recent studies enabled by modern systems and structural methods, it is clear that many regulatory pathways depend on E3s recognizing protein termini. Here, we review the structural basis for recognition of protein termini by E3s and how this recognition underlies biological regulation. Diverse E3s evolved to harness a substrate's N and/or C terminus (and often adjacent residues as well) in a sequence-specific manner. Regulation is achieved through selective activation of E3s and also through generation of degrons at ribosomes or by posttranslational means. Collectively, many E3 interactions with protein N and C termini enable intricate control of protein quality and responses to cellular signals.
Subject(s)
Ubiquitin-Protein Ligases , Ubiquitin , Amino Acid Motifs , Proteins/metabolism , Proteolysis , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Neurodevelopmental cognitive disorders provide insights into mechanisms of human brain development. Here, we report an intellectual disability syndrome caused by the loss of APC7, a core component of the E3 ubiquitin ligase anaphase promoting complex (APC). In mechanistic studies, we uncover a critical role for APC7 during the recruitment and ubiquitination of APC substrates. In proteomics analyses of the brain from mice harboring the patient-specific APC7 mutation, we identify the chromatin-associated protein Ki-67 as an APC7-dependent substrate of the APC in neurons. Conditional knockout of the APC coactivator protein Cdh1, but not Cdc20, leads to the accumulation of Ki-67 protein in neurons in vivo, suggesting that APC7 is required for the function of Cdh1-APC in the brain. Deregulated neuronal Ki-67 upon APC7 loss localizes predominantly to constitutive heterochromatin. Our findings define an essential function for APC7 and Cdh1-APC in neuronal heterochromatin regulation, with implications for understanding human brain development and disease.
Subject(s)
Apc7 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Brain/enzymology , Heterochromatin/metabolism , Intellectual Disability/enzymology , Neural Stem Cells/enzymology , Neurogenesis , Adolescent , Animals , Antigens, CD , Apc7 Subunit, Anaphase-Promoting Complex-Cyclosome/genetics , Behavior, Animal , Brain/growth & development , Cadherins/genetics , Cadherins/metabolism , Cell Line , Child , Child, Preschool , Disease Models, Animal , Female , Heterochromatin/genetics , Humans , Infant , Intellectual Disability/pathology , Intellectual Disability/physiopathology , Intellectual Disability/psychology , Intelligence , Ki-67 Antigen/genetics , Ki-67 Antigen/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Mitosis , Mutation , Neural Stem Cells/pathology , Proteolysis , Signal Transduction , Syndrome , Ubiquitination , Young AdultABSTRACT
Autophagy is directed by numerous distinct autophagy-related (Atg) proteins. These transmit starvation-induced signals to lipids and regulatory proteins and assemble a double-membrane autophagosome sequestering bulk cytoplasm and/or selected cargos destined for degradation upon autophagosome fusion with a vacuole or lysosome. This Review discusses the structural mechanisms by which Atg proteins sense membrane curvature, mediate a PI(3)P-signaling cascade, and utilize autophagy-specific ubiquitin-like protein cascades to tether proteins to autophagosomal membranes. Recent elucidation of molecular interactions enabling vesicle nucleation, elongation, and cargo recruitment provides insights into how dynamic protein-protein and protein-membrane interactions may dictate size, shape, and contents of autophagosomes.
Subject(s)
Autophagy , Animals , Cell Membrane/chemistry , Cell Membrane/metabolism , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Phagosomes/metabolismABSTRACT
Most E3 ligases use a RING domain to activate a thioester-linked E2â¼ubiquitin-like protein (UBL) intermediate and promote UBL transfer to a remotely bound target protein. Nonetheless, RING E3 mechanisms matching a specific UBL and acceptor lysine remain elusive, including for RBX1, which mediates NEDD8 ligation to cullins and >10% of all ubiquitination. We report the structure of a trapped RING E3-E2â¼UBL-target intermediate representing RBX1-UBC12â¼NEDD8-CUL1-DCN1, which reveals the mechanism of NEDD8 ligation and how a particular UBL and acceptor lysine are matched by a multifunctional RING E3. Numerous mechanisms specify cullin neddylation while preventing noncognate ubiquitin ligation. Notably, E2-E3-target and RING-E2â¼UBL modules are not optimized to function independently, but instead require integration by the UBL and target for maximal reactivity. The UBL and target regulate the catalytic machinery by positioning the RING-E2â¼UBL catalytic center, licensing the acceptor lysine, and influencing E2 reactivity, thereby driving their specific coupling by a multifunctional RING E3.
Subject(s)
Ubiquitins/chemistry , Ubiquitins/metabolism , Amino Acid Sequence , Carrier Proteins/metabolism , Catalytic Domain , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Crystallography, X-Ray , Cullin Proteins/chemistry , Cullin Proteins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , NEDD8 Protein , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.