RESUMO
Mammals encode â¼5,000 integral membrane proteins that need to be inserted in a defined topology at the endoplasmic reticulum (ER) membrane by mechanisms that are incompletely understood. Here, we found that efficient biogenesis of ß1-adrenergic receptor (ß1AR) and other G protein-coupled receptors (GPCRs) requires the conserved ER membrane protein complex (EMC). Reconstitution studies of ß1AR biogenesis narrowed the EMC requirement to the co-translational insertion of the first transmembrane domain (TMD). Without EMC, a proportion of TMD1 inserted in an inverted orientation or failed altogether. Purified EMC and SRP receptor were sufficient for correctly oriented TMD1 insertion, while the Sec61 translocon was necessary for insertion of the next TMD. Enforcing TMD1 topology with an N-terminal signal peptide bypassed the EMC requirement for insertion in vitro and restored efficient biogenesis of multiple GPCRs in EMC-knockout cells. Thus, EMC inserts TMDs co-translationally and cooperates with the Sec61 translocon to ensure accurate topogenesis of many membrane proteins.
Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Peptídeos/metabolismo , Canais de Translocação SEC/metabolismo , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Feminino , Humanos , Domínios Proteicos , Transporte Proteico/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Peptídeos/genética , Canais de Translocação SEC/genética , PerusRESUMO
The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic DNA and induces interferon-stimulated genes (ISGs) to activate the innate immune system. Here, we report the unexpected discovery that cGAS also senses dysfunctional protein production. Purified ribosomes interact directly with cGAS and stimulate its DNA-dependent activity in vitro. Disruption of the ribosome-associated protein quality control (RQC) pathway, which detects and resolves ribosome collision during translation, results in cGAS-dependent ISG expression and causes re-localization of cGAS from the nucleus to the cytosol. Indeed, cGAS preferentially binds collided ribosomes in vitro, and orthogonal perturbations that result in elevated levels of collided ribosomes and RQC activation cause sub-cellular re-localization of cGAS and ribosome binding in vivo as well. Thus, translation stress potently increases DNA-dependent cGAS activation. These findings have implications for the inflammatory response to viral infection and tumorigenesis, both of which substantially reprogram cellular protein synthesis.
Assuntos
Núcleo Celular , Nucleotidiltransferases , Biossíntese de Proteínas , Ribossomos , Transdução de Sinais , Estresse Fisiológico , Transporte Ativo do Núcleo Celular , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Translating ribosomes that slow excessively incur collisions with trailing ribosomes. Persistent collisions are detected by ZNF598, a ubiquitin ligase that ubiquitinates sites on the ribosomal 40S subunit to initiate pathways of mRNA and protein quality control. The collided ribosome complex must be disassembled to initiate downstream quality control, but the mechanistic basis of disassembly is unclear. Here, we reconstitute the disassembly of a collided polysome in a mammalian cell-free system. The widely conserved ASC-1 complex (ASCC) containing the ASCC3 helicase disassembles the leading ribosome in an ATP-dependent reaction. Disassembly, but not ribosome association, requires 40S ubiquitination by ZNF598, but not GTP-dependent factors, including the Pelo-Hbs1L ribosome rescue complex. Trailing ribosomes can elongate once the roadblock has been removed and only become targets if they subsequently stall and incur collisions. These findings define the specific role of ASCC during ribosome-associated quality control and identify the molecular target of its activity.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sistema Livre de Células , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Polirribossomos/genética , Polirribossomos/metabolismo , Coelhos , Subunidades Ribossômicas/genética , Subunidades Ribossômicas/metabolismo , Ribossomos/genética , UbiquitinaçãoRESUMO
The billions of proteins inside a eukaryotic cell are organized among dozens of sub-cellular compartments, within which they are further organized into protein complexes. The maintenance of both levels of organization is crucial for normal cellular function. Newly made proteins that fail to be segregated to the correct compartment or assembled into the appropriate complex are defined as orphans. In this review, we discuss the challenges faced by a cell of minimizing orphaned proteins, the quality control systems that recognize orphans, and the consequences of excess orphans for protein homeostasis and disease.
Assuntos
Transporte Proteico/fisiologia , Proteínas/metabolismo , Proteostase/fisiologia , Biossíntese de Proteínas/fisiologia , ProteóliseRESUMO
Aberrantly slow translation elicits quality control pathways initiated by the ubiquitin ligase ZNF598. How ZNF598 discriminates physiologic from pathologic translation complexes and ubiquitinates stalled ribosomes selectively is unclear. Here, we find that the minimal unit engaged by ZNF598 is the collided di-ribosome, a molecular species that arises when a trailing ribosome encounters a slower leading ribosome. The collided di-ribosome structure reveals an extensive 40S-40S interface in which the ubiquitination targets of ZNF598 reside. The paucity of 60S interactions allows for different ribosome rotation states, explaining why ZNF598 recognition is indifferent to how the leading ribosome has stalled. The use of ribosome collisions as a proxy for stalling allows the degree of tolerable slowdown to be tuned by the initiation rate on that mRNA; hence, the threshold for triggering quality control is substrate specific. These findings illustrate how higher-order ribosome architecture can be exploited by cellular factors to monitor translation status.
Assuntos
Proteínas de Transporte/fisiologia , Biossíntese de Proteínas/fisiologia , Ribossomos/fisiologia , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , RNA Mensageiro , Ubiquitina , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
Diverse cellular stressors have been observed to trigger site-specific ubiquitination on several ribosomal proteins. However, the ubiquitin ligases, biochemical consequences, and physiologic pathways linked to these modifications are not known. Here, we show in mammalian cells that the ubiquitin ligase ZNF598 is required for ribosomes to terminally stall during translation of poly(A) sequences. ZNF598-mediated stalling initiated the ribosome-associated quality control (RQC) pathway for degradation of nascent truncated proteins. Biochemical ubiquitination reactions identified two sites of mono-ubiquitination on the 40S protein eS10 as the primary ribosomal target of ZNF598. Cells lacking ZNF598 activity or containing ubiquitination-resistant eS10 ribosomes failed to stall efficiently on poly(A) sequences. In the absence of stalling, read-through of poly(A) produces a poly-lysine tag, which might alter the localization and solubility of the associated protein. Thus, ribosome ubiquitination can modulate translation elongation and impacts co-translational quality control to minimize production of aberrant proteins.
Assuntos
Proteínas de Transporte/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteínas de Transporte/genética , Células HEK293 , Humanos , Mutação , Proteólise , Interferência de RNA , RNA Mensageiro/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , TransfecçãoRESUMO
The eukaryotic endoplasmic reticulum (ER) membrane contains essential complexes that oversee protein biogenesis and lipid metabolism, impacting nearly all aspects of cell physiology. The ER membrane protein complex (EMC) is a newly described transmembrane domain (TMD) insertase linked with various phenotypes, but whose clients and cellular responsibilities remain incompletely understood. We report that EMC deficiency limits the cellular boundaries defining cholesterol tolerance, reflected by diminished viability with limiting or excessive extracellular cholesterol. Lipidomic and proteomic analyses revealed defective biogenesis and concomitant loss of the TMD-containing ER-resident enzymes sterol-O-acyltransferase 1 (SOAT1) and squalene synthase (SQS, also known as FDFT1), which serve strategic roles in the adaptation of cells to changes in cholesterol availability. Insertion of the weakly hydrophobic tail-anchor (TA) of SQS into the ER membrane by the EMC ensures sufficient flux through the sterol biosynthetic pathway while biogenesis of polytopic SOAT1 promoted by the EMC provides cells with the ability to store free cholesterol as inert cholesteryl esters. By facilitating insertion of TMDs that permit essential mammalian sterol-regulating enzymes to mature accurately, the EMC is an important biogenic determinant of cellular robustness to fluctuations in cholesterol availability.This article has an associated First Person interview with the first author of the paper.
Assuntos
Colesterol/biossíntese , Retículo Endoplasmático/enzimologia , Farnesil-Difosfato Farnesiltransferase/metabolismo , Membranas Intracelulares/enzimologia , Complexos Multienzimáticos/metabolismo , Esterol O-Aciltransferase/metabolismo , Linhagem Celular Tumoral , Colesterol/genética , Retículo Endoplasmático/genética , Farnesil-Difosfato Farnesiltransferase/genética , Humanos , Complexos Multienzimáticos/genética , Esterol O-Aciltransferase/genéticaRESUMO
In eukaryotic cells, half of all proteins function as subunits within multiprotein complexes. Imbalanced synthesis of subunits leads to unassembled intermediates that must be degraded to minimize cellular toxicity. Here, we found that excess PSMC5, a subunit of the proteasome base, was targeted for degradation by the HERC1 ubiquitin ligase in mammalian cells. HERC1 identified unassembled PSMC5 by its cognate assembly chaperone PAAF1. Because PAAF1 only dissociates after assembly, HERC1 could also engage later assembly intermediates such as the PSMC4-PSMC5-PAAF1 complex. A missense mutant of HERC1 that causes neurodegeneration in mice was impaired in the recognition and ubiquitination of the PSMC5-PAAF1 complex. Thus, proteasome assembly factors can serve as adaptors for ubiquitin ligases to facilitate elimination of unassembled intermediates and maintain protein homeostasis.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Calmodulina/metabolismo , Humanos , Células MCF-7 , Camundongos , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Translation of aberrant mRNAs can cause ribosomes to stall, leading to collisions with trailing ribosomes. Collided ribosomes are specifically recognised by ZNF598 to initiate protein and mRNA quality control pathways. Here we found using quantitative proteomics of collided ribosomes that EDF1 is a ZNF598-independent sensor of ribosome collisions. EDF1 stabilises GIGYF2 at collisions to inhibit translation initiation in cis via 4EHP. The GIGYF2 axis acts independently of the ZNF598 axis, but each pathway's output is more pronounced without the other. We propose that the widely conserved and highly abundant EDF1 monitors the transcriptome for excessive ribosome density, then triggers a GIGYF2-mediated response to locally and temporarily reduce ribosome loading. Only when collisions persist is translation abandoned to initiate ZNF598-dependent quality control. This tiered response to ribosome collisions would allow cells to dynamically tune translation rates while ensuring fidelity of the resulting protein products.
Assuntos
Proteínas de Transporte/metabolismo , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo , Retroalimentação Fisiológica , Células HEK293 , Humanos , Proteômica , RNA Mensageiro/metabolismoRESUMO
Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC's cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate's transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC's proposed chaperone function.
Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Citosol/química , Citosol/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Domínios Proteicos , Canais de Translocação SEC/química , Canais de Translocação SEC/metabolismoRESUMO
Faulty or damaged messenger RNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein degradation and ribosome recycling. The most common mRNA defect in eukaryotes is probably inappropriate polyadenylation at near-cognate sites within the coding region. How ribosomes stall selectively when they encounter poly(A) is unclear. Here, we use biochemical and structural approaches in mammalian systems to show that poly-lysine, encoded by poly(A), favors a peptidyl-transfer RNA conformation suboptimal for peptide bond formation. This conformation partially slows elongation, permitting poly(A) mRNA in the ribosome's decoding center to adopt a ribosomal RNA-stabilized single-stranded helix. The reconfigured decoding center clashes with incoming aminoacyl-tRNA, thereby precluding elongation. Thus, coincidence detection of poly-lysine in the exit tunnel and poly(A) in the decoding center allows ribosomes to detect aberrant mRNAs selectively, stall elongation and trigger downstream quality control pathways essential for cellular homeostasis.
Assuntos
Peptídeos/metabolismo , Poli A/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Peptídeos/química , Poli A/química , Poliadenilação , Polilisina/química , Polilisina/metabolismo , Estabilidade de RNA , RNA Mensageiro/química , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/químicaRESUMO
Many nascent proteins are assembled into multiprotein complexes of defined stoichiometry. Imbalances in the synthesis of individual subunits result in orphans. How orphans are selectively eliminated to maintain protein homeostasis is poorly understood. Here, we found that the conserved ubiquitin-conjugating enzyme UBE2O directly recognized juxtaposed basic and hydrophobic patches on unassembled proteins to mediate ubiquitination without a separate ubiquitin ligase. In reticulocytes, where UBE2O is highly up-regulated, unassembled α-globin molecules that failed to assemble with ß-globin were selectively ubiquitinated by UBE2O. In nonreticulocytes, ribosomal proteins that did not engage nuclear import factors were targets for UBE2O. Thus, UBE2O is a self-contained quality control factor that comprises substrate recognition and ubiquitin transfer activities within a single protein to efficiently target orphans of multiprotein complexes for degradation.