Co-translational Interaction Research Articles

Multimodal cotranslational interactions direct assembly of the human multi-tRNA synthetase complex

Amino acid ligation to cognate transfer RNAs (tRNAs) is catalyzed by aminoacyl-tRNA synthetases (aaRSs)-essential interpreters of the genetic code during translation. Mammalian cells harbor 20 cytoplasmic aaRSs, out of which 9 (in 8 proteins), with 3 non-aaRS proteins, AIMPs 1 to 3, form the ∼1.25-MDa multi-tRNA synthetase complex (MSC). The function of MSC remains uncertain, as does its mechanism of assembly. Constituents of multiprotein complexes encounter obstacles during assembly, including inappropriate interactions, topological constraints, premature degradation of unassembled subunits, and suboptimal stoichiometry. To facilitate orderly and efficient complex formation, some complexes are assembled cotranslationally by a mechanism in which a fully formed, mature protein binds a nascent partner as it emerges from the translating ribosome. Here, we show out of the 121 possible interaction events between the 11 MSC constituents, 15 are cotranslational. AIMPs are involved in the majority of these cotranslational interactions, suggesting they are not only critical for MSC structure but also for assembly. Unexpectedly, several cotranslational events involve more than the usual dyad of interacting proteins. We show two modes of cotranslational interaction, namely a "multisite" mechanism in which two or more mature proteins bind the same nascent peptide at distinct sites and a second "piggy-back" mechanism in which a mature protein carries a second fully formed protein and binds to a single site on an emerging peptide. Multimodal mechanisms of cotranslational interaction offer a diversity of pathways for ordered, piecewise assembly of small subcomplexes into larger heteromultimeric complexes such as the mammalian MSC.

Cotranslational interaction of human EBP50 and ezrin overcomes masked binding site during complex assembly.

Multiprotein assemblages are the intracellular workhorses of many physiological processes. Assembly of constituents into complexes can be driven by stochastic, domain-dependent, posttranslational events in which mature, folded proteins specifically interact. However, inaccessibility of interacting surfaces in mature proteins (e.g., due to "buried" domains) can obstruct complex formation. Mechanisms by which multiprotein complex constituents overcome topological impediments remain enigmatic. For example, the heterodimeric complex formed by EBP50 and ezrin must address this issue as the EBP50-interacting domain in ezrin is obstructed by a self-interaction that occupies the EBP50 binding site. Here, we show that the EBP50-ezrin complex is formed by a cotranslational mechanism in which the C terminus of mature, fully formed EBP50 binds the emerging, ribosome-bound N-terminal FERM domain of ezrin during EZR mRNA translation. Consistent with this observation, a C-terminal EBP50 peptide mimetic reduces the cotranslational interaction and abrogates EBP50-ezrin complex formation. Phosphorylation of EBP50 at Ser339 and Ser340 abrogates the cotranslational interaction and inhibits complex formation. In summary, we show that the function of eukaryotic mRNA translation extends beyond "simple" generation of a linear peptide chain that folds into a tertiary structure, potentially for subsequent complex assembly; importantly, translation can facilitate interactions with sterically inaccessible domains to form functional multiprotein complexes.

Homeostatic control of cardiac excitability by an RNA-based mechanism

Coordinated activity of ion channels in the human heart is essential for normal cardiac function and preventing life-threatening arrhythmias. We previously reported a cotranslational interaction between KCNH2 and SCN5A transcripts respectively encoding the human ERG (hERG) potassium channel and Nav1.5 sodium channel. In addition to their association during biogenesis, these transcripts and the corresponding membrane currents are also subject to a curious co-knockdown effect elicited by selective RNAi of either transcript.

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling.

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis. Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

Analysis of the co-translational assembly of the fungal fatty acid synthase (FAS)

The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex. Upon barrel-formation, two multidomain subunits, each more than 200 kDa large, intertwine to form a heterododecameric complex that buries 170,000 Å2 of protein surface. In spite of the rich knowledge about yeast FAS in structure and function, its assembly remained elusive until recently, when co-translational interaction of the β-subunit with the nascent α-subunit was found to initiate assembly. Here, we characterize the co-translational assembly of yeast FAS at a molecular level. We show that the co-translationally formed interface is sensitive to subtle perturbations, so that the exchange of two amino acids located in the emerging interface can prevent assembly. On the other hand, assembly can also be initiated via the co-translational interaction of the subunits at other sites, which implies that this process is not strictly site or sequence specific. We further highlight additional steps in the biogenesis of yeast FAS, as the formation of a dimeric subunit that orchestrates complex formation and acts as platform for post-translational phosphopantetheinylation. The presented data supports the understanding of the recently discovered prevalence of eukaryotic complexes for co-translational assembly, and is valuable for further harnessing FAS in the biotechnological production of aliphatic compounds.

Co-translational assembly of mammalian nuclear multisubunit complexes

Cells dedicate significant energy to build proteins often organized in multiprotein assemblies with tightly regulated stoichiometries. As genes encoding subunits assembling in a multisubunit complex are dispersed in the genome of eukaryotes, it is unclear how these protein complexes assemble. Here, we show that mammalian nuclear transcription complexes (TFIID, TREX-2 and SAGA) composed of a large number of subunits, but lacking precise architectural details are built co-translationally. We demonstrate that dimerization domains and their positions in the interacting subunits determine the co-translational assembly pathway (simultaneous or sequential). The lack of co-translational interaction can lead to degradation of the partner protein. Thus, protein synthesis and complex assembly are linked in building mammalian multisubunit complexes, suggesting that co-translational assembly is a general principle in mammalian cells to avoid non-specific interactions and protein aggregation. These findings will also advance structural biology by defining endogenous co-translational building blocks in the architecture of multisubunit complexes.

Pathogenic Signal Sequence Mutations in Progranulin Disrupt SRP Interactions Required for mRNA Stability

SUMMARYCells have evolved quality control pathways to prevent the accumulation of improperly localized proteins, which are often toxic. One of these pathways, regulation of aberrant protein production (RAPP), recognizes aberrant secretory proteins during translation and degrades the associated mRNA. Here, we demonstrate endogenous RAPP substrates. Haploinsufficiency of the secretory protein progranulin (GRN) is associated with the neurodegenerative disease frontotemporal lobar degeneration (FTLD). Our results show FTLD-associated GRN mutations W7R and A9D disrupt co-translational interaction with a targeting factor, signal recognition particle (SRP). This triggers RAPP and initiates specific mRNA degradation. Conversely, wild-type GRN and the naturally occurring polymorphism V5L GRN are efficiently expressed and secreted. Thus, RAPP plays a role in the molecular pathology of A9D GRN and W7R GRN.

Co-translational control of protein complex formation: a fundamental pathway of cellular organization?

Analyses of proteomes from a large number of organisms throughout the domains of life highlight the key role played by multiprotein complexes for the implementation of cellular function. While the occurrence of multiprotein assemblies is ubiquitous, the understanding of pathways that dictate the formation of quaternary structure remains enigmatic. Interestingly, there are now well-established examples of protein complexes that are assembled co-translationally in both prokaryotes and eukaryotes, and indications are that the phenomenon is widespread in cells. Here, we review complex assembly with an emphasis on co-translational pathways, which involve interactions of nascent chains with other nascent or mature partner proteins, respectively. In prokaryotes, such interactions are promoted by the polycistronic arrangement of mRNA and the associated co-translation of functionally related cell constituents in order to enhance otherwise diffusion-dependent processes. Beyond merely stochastic events, however, co-translational complex formation may be sensitive to subunit availability and allow for overall regulation of the assembly process. We speculate how co-translational pathways may constitute integral components of quality control systems to ensure the correct and complete formation of hundreds of heterogeneous assemblies in a single cell. Coupling of folding of intrinsically disordered domains with co-translational interaction of binding partners may furthermore enhance the efficiency and fidelity with which correct conformation is attained. Co-translational complex formation may constitute a fundamental pathway of cellular organization, with profound importance for health and disease.

N-Glycan-dependent and -independent Quality Control of Human δ Opioid Receptor N-terminal Variants

Quality control (QC) in the endoplasmic reticulum (ER) scrutinizes newly synthesized proteins and directs them either to ER export or ER-associated degradation (ERAD). Here, we demonstrate that the human δ-opioid receptor (hδOR) is subjected to ERQC in both N-glycan-dependent and -independent manners. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated F27C variants in metabolic pulse-chase assays coupled with flow cytometry and cell surface biotinylation. Both QC mechanisms distinguished the minute one-amino acid difference between the variants, targeting a large fraction of hδOR-Cys(27) to ERAD. However, the N-glycan-independent QC was unable to compensate the N-glycan-dependent pathway, and some incompletely folded non-N-glycosylated hδOR-Cys(27) reached the cell surface in conformation incompatible with ligand binding. The turnover of receptors associating with the molecular chaperone calnexin (CNX) was significantly slower for the hδOR-Cys(27), pointing to an important role of CNX in the hδOR N-glycan-dependent QC. This was further supported by the fact that inhibiting the co-translational interaction of hδOR-Cys(27) precursors with CNX led to their ERAD. Opioid receptor pharmacological chaperones released the CNX-bound receptors to ER export and, furthermore, were able to rescue the Cys(27) variant from polyubiquitination and retrotranslocation to the cytosol whether carrying N-glycans or not. Taken together, the hδOR appears to rely primarily on the CNX-mediated N-glycan-dependent QC that has the capacity to assist in folding, whereas the N-glycan-independent mechanism constitutes an alternative, although less accurate, system for directing misfolded/incompletely folded receptors to ERAD, possibly in altered cellular conditions.

Cotranslational protein-RNA associations predict protein-protein interactions.

BackgroundMost cellular proteins function as part of stable protein complexes. We recently showed that around 38% of proteins associate with mRNAs that encode interacting proteins, reflecting the cotranslational formation of the complex between the bait protein and the nascent peptides encoded by the interacting mRNAs. Here we hypothesise that these cotranslational protein-mRNA associations can be used to predict protein-protein interactions.ResultsWe found that the fission yeast Exo2 protein, which encodes an exonuclease of the XRN1 family, coimmunoprecipitates with the eti1 mRNA, which codes for a protein of unknown function and uninformative sequence. Based on this protein-mRNA association, we predicted that the Exo2 and Eti1 protein are part of the same complex, and confirmed this hypothesis by coimmunoprecipitation and colocalization of the proteins. Similarly, we show that the cotranslational interaction between the Sty1 MAP kinase and the cip2 mRNA, which encodes an RNA-binding protein, predicts a complex between Sty1 and Cip2.ConclusionsOur results demonstrate that cotranslational protein-mRNA associations can be used to identify new components of protein complexes.

Abstract 437: Optimizing a Prokaryotic Cell Free Expression System for the Generation of synthetic ApoB-Containing Lipoprotein Particles

High serum levels of low density lipoproteins (LDL) is associated with increased risk of atherosclerosis. Apolipoprotein B (ApoB) is required for the assembly and secretion of chylomicrons and very low density lipoproteins (VLDL), the precursor of low density lipoproteins (LDL). Despite its clinical significance, the mechanism of the assembly of these ApoB containing lipoproteins is poorly understood. The assembly process is an interplay of several key components including but not limited to nascent ApoB, lipids, ER resident chaperones and importantly, microsomal triglyceride transfer protein (MTP). In the current study, we are trying to understand several unanswered questions in the mechanism of the lipoprotein assembly. We have used a novel prokaryotic cell-free expression system and lipids mimicking the ER membrane to produce particles that represent the early dense initiation particles formed in the ER. After optimizing several different conditions, we were able to make “synthetic” lipoproteins by cotranslational expression of constructs from the first 22% of ApoB tagged with a 6-histidine tag at the C-terminus (ApoB 22-His) with small unilamellar phosphatidylcholine (PC) vesicles and phosphatidylcholine:triolein (PC:TO) emulsions. After cotranslational interaction with lipids, these constructs migrate to a lower density in potassium bromide (KBr) density gradient centrifugation. Here we report a new ApoB 22 construct with a FLAG tag at the N-terminus in addition to the C-terminal His tag. The construct makes significant amount of soluble protein that is soluble in the cell free reaction. The two N- and C-terminal tags allow us to purify full length construct from any truncation products. In addition, the dual-tag approach will allow us to purify the synthetic lipoproteins directly from the cell free system, and thereby avoid the requirement for KBr density gradient centrifugation. This new strategy will provide far more efficient generation and purification of synthetic ApoB containing lipoprotein particles.

Widespread Cotranslational Formation of Protein Complexes

Most cellular processes are conducted by multi-protein complexes. However, little is known about how these complexes are assembled. In particular, it is not known if they are formed while one or more members of the complexes are being translated (cotranslational assembly). We took a genomic approach to address this question, by systematically identifying mRNAs associated with specific proteins. In a sample of 31 proteins from Schizosaccharomyces pombe that did not contain RNA–binding domains, we found that ∼38% copurify with mRNAs that encode interacting proteins. For example, the cyclin-dependent kinase Cdc2p associates with the rum1 and cdc18 mRNAs, which encode, respectively, an inhibitor of Cdc2p kinase activity and an essential regulator of DNA replication. Both proteins interact with Cdc2p and are key cell cycle regulators. We obtained analogous results with proteins with different structures and cellular functions (kinesins, protein kinases, transcription factors, proteasome components, etc.). We showed that copurification of a bait protein and of specific mRNAs was dependent on the presence of the proteins encoded by the interacting mRNAs and on polysomal integrity. These results indicate that these observed associations reflect the cotranslational interaction between the bait and the nascent proteins encoded by the interacting mRNAs. Therefore, we show that the cotranslational formation of protein–protein interactions is a widespread phenomenon.

Site-Specific Fluorescent Labeling of Nascent Proteins on the Translating Ribosome

As newly synthesized proteins emerge from the ribosome, they interact with a variety of cotranslational cellular machineries that facilitate their proper folding, maturation, and localization. These interactions are essential for proper function of the cell, and the ability to study these events is crucial to understanding cellular protein biogenesis. To this end, we have developed a highly efficient method to generate ribosome-nascent chain complexes (RNCs) site-specifically labeled with a fluorescent dye on the nascent polypeptide. The fluorescent RNC provides real-time, quantitative information on its cotranslational interaction with the signal recognition particle and will be a valuable tool in elucidating the role of the translating ribosome in numerous biochemical pathways.

Cell‐Free Production of Aggregation‐Prone Proteins in Soluble and Active Forms

We have developed an efficient cell-free protein synthesis system for the production of soluble and active eukaryotic proteins that are predominantly produced as inclusion bodies in bacteria. S30 extracts (indicating the supernatant of cell homogenate when centrifuged at 30,000g) for cell-free protein synthesis were prepared from Escherichia coli that was modified to overexpress a set of chaperones (GroEL/ES or DnaK/J-GrpE) and disulfide isomerase (leader sequence-free mature DsbC expressed in the cytoplasm). The solubility and biological activity concentration (biological activity per unit volume of cell-free protein synthesis reaction mixture) of the protein synthesized by the new cell-free protein synthesis system showed a dramatic improvement. Solubility enhancement was most dramatic with the existence of DnaK/J-GrpE. It shows that the co-translational interaction with DnaK/J-GrpE prior to folding trial is important in maintenance of the aggregation-prone protein in a folding-competent soluble state. For maximizing the biological activity concentration of the expressed protein, the additional presence of GroEL/ES and DsbC was required. When human erythropoietin was expressed in the developed cell-free protein synthesis system including endogenously overexpressed chaperones and/or DsbC, the biological activity concentration of erythropoietin was enhanced by 700%. It implies that the post-translational folding and disulfide bond reshuffling as well as co-translational folding are important in acquiring functionally active protein from cell-free expression system. This is the first report of using S30 extracts including endogenously overexpressed chaperones and/or disulfide isomerase for the efficient production of soluble and active proteins in cell-free protein synthesis. This new cell-free protein synthesis system was capable of introducing much larger amounts of chaperones and disulfide isomerase compared to a conventional method that supplements them separately. The developed cell-free protein synthesis system supported efficient expression of the eukaryotic proteins in soluble and active forms without the need of any exogenous addition or coexpression of folding effectors.

Corrigendum

Corrigendum16 December 2002free access Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP4 Katja Schröder Katja Schröder Search for more papers by this author Bruno Martoglio Bruno Martoglio Search for more papers by this author Michael Hofmann Michael Hofmann Search for more papers by this author Christina Hölscher Christina Hölscher Search for more papers by this author Enno Hartmann Enno Hartmann Search for more papers by this author Siegfried Prehn Siegfried Prehn Search for more papers by this author Tom A. Rapoport Tom A. Rapoport Search for more papers by this author Bernhard Dobberstein Bernhard Dobberstein Search for more papers by this author Katja Schröder Katja Schröder Search for more papers by this author Bruno Martoglio Bruno Martoglio Search for more papers by this author Michael Hofmann Michael Hofmann Search for more papers by this author Christina Hölscher Christina Hölscher Search for more papers by this author Enno Hartmann Enno Hartmann Search for more papers by this author Siegfried Prehn Siegfried Prehn Search for more papers by this author Tom A. Rapoport Tom A. Rapoport Search for more papers by this author Bernhard Dobberstein Bernhard Dobberstein Search for more papers by this author Author Information Katja Schröder, Bruno Martoglio, Michael Hofmann, Christina Hölscher, Enno Hartmann, Siegfried Prehn, Tom A. Rapoport and Bernhard Dobberstein The EMBO Journal (2002)21:6954-6954https://doi.org/10.1093/emboj/cdf693 This article corrects the following: Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP401 September 1999 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The EMBO Journal, 18, 4804–4815, 1999 In this paper we had identified a region in nascent invariant chain (Ii) that could be cross-linked to RAMP4 during translocation across the endoplasmic reticulum membrane. Mutants in Ii that prevented cross-linking to RAMP4 showed reduced glycosylation. Based on these observations, we had proposed that the RAMP4 interaction with Ii affects its N-glycosylation. Recently we have discovered that the reduced level of N-glycosylation of the Ii mutants is due to an enzymatic activity occurring during the native immunoprecipitation. When cells expressing the mutant invariant chains are lysed in the presence of N-ethylmaleimide (NEM), or denatured prior to immunoprecipitation, no reduction in N-glycosylation is observed in the mutants relative to the wild-type invariant chain. This NEM-sensitive activity most likely results from the cytoplasmic N-glycanase (Suzuki et al., 1995). The differential sensitivity of the wild-type Ii protein and the mutants may be due to a conformational change leading to increased accessibility of Ii mutants to the glycanase (Ii mutants affecting the RAMP4 interaction are in a region of Ii implicated in trimerization). In conclusion, our data do not support a role for RAMP4 in mediating the co-translational N-glycosylation of Ii. Rather, mutants affecting the RAMP4 interaction must be interpreted as affecting Ii conformation or assembly. Thus, the functional significance of the persistent co-translational interaction between the invariant chain and RAMP4 remains to be elucidated. Jeannie Barrett, Klaus Meese and Bernhard Dobberstein References Suzuki T, Kitajima K, Inoue Y and Inoue S (1995) Carbohydrate-binding property of peptide: N-glycanase from mouse fibroblast L-929 cells as evaluated by inhibition and binding experiments using various oligosaccharides. J Biol Chem, 270, 15181–15186.CrossrefCASPubMedWeb of Science®Google Scholar Next ArticlePrevious Article Read MoreAbout the coverClose modalView large imageVolume 21,Issue 24,December 16, 2002Cover. Stirling University campus is located on the outskirts of Stirling in Scotland and is surrounded by 360 acres of breathtaking scenery. The University was opened in 1967 and has won several awards for architecture. Lindsay Pirrit is a postdoctoral fellow working on cytoadhesion in Plasmodium falciparum at the Pasteur Institute in Paris. Volume 21Issue 2416 December 2002In this issue ReferencesRelatedDetailsLoading ...

Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY.

Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane-targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co-translational targeting and assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co-translational targeting and membrane assembly of NG. The co-translational interaction of FtsY with the membrane was confirmed by in vitro experiments.

Physical Evidence for Cotranslational Regulation of β-Tubulin mRNA Degradation

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.

Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation.

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.