Chp1 is a dedicated chaperone at the ribosome that safeguards eEF1A biogenesis.

Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we discover a dedicated ribosome-associated chaperone, Chp1, that rewires the cotranslational folding machinery to assist in the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). Our results indicate that during eEF1A synthesis, Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production in the absence of Chp1 triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult-to-fold protein that necessitates a biogenesis pathway starting with dedicated folding factor Chp1 at the ribosome to protect the eukaryotic cell from proteostasis collapse.

Selective ribosome profiling as a tool to study interactions of translating ribosomes in mammalian cells.

The processing, membrane targeting and folding of newly synthesized polypeptides is closely linked to their synthesis at the ribosome. A network of enzymes, chaperones and targeting factors engages ribosome-nascent chain complexes (RNCs) to support these maturation processes. Exploring the modes of action of this machinery is critical for our understanding of functional protein biogenesis. Selective ribosome profiling (SeRP) is a powerful method for interrogating co-translational interactions of maturation factors with RNCs. It provides proteome-wide information on the factor's nascent chain interactome, the timing of factor binding and release during the progress of translation of individual nascent chain species, and the mechanisms and features controlling factor engagement. SeRP is based on the combination of two ribosome profiling (RP) experiments performed on the same cell population. In one experiment the ribosome-protected mRNA footprints of all translating ribosomes of the cell are sequenced (total translatome), while the other experiment detects only the ribosome footprints of the subpopulation of ribosomes engaged by the factor of interest (selected translatome). The codon-specific ratio of ribosome footprint densities from selected over total translatome reports on the factor enrichment at specific nascent chains. In this chapter, we provide a detailed SeRP protocol for mammalian cells. The protocol includes instructions on cell growth and cell harvest, stabilization of factor-RNC interactions, nuclease digest and purification of (factor-engaged) monosomes, as well as preparation of cDNA libraries from ribosome footprint fragments and deep sequencing data analysis. Purification protocols of factor-engaged monosomes and experimental results are exemplified for the human ribosomal tunnel exit-binding factor Ebp1 and chaperone Hsp90, but the protocols are readily adaptable to other co-translationally acting mammalian factors.

Selective ribosome profiling reveals a role for SecB in the co-translational inner membrane protein biogenesis.

The chaperone SecB has been implicated in de novo protein folding and translocation across the membrane, but it remains unclear which nascent polypeptides SecB binds, when during translation SecB acts, how SecB function is coordinated with other chaperones and targeting factors, and how polypeptide engagement contributes to protein biogenesis. Using selective ribosome profiling, we show that SecB binds many nascent cytoplasmic and translocated proteins generally late during translation and controlled by the chaperone trigger factor. Revealing an uncharted role in co-translational translocation, inner membrane proteins (IMPs) are the most prominent nascent SecB interactors. Unlike other substrates, IMPs are bound early during translation, following the membrane targeting by the signal recognition particle. SecB remains bound until translation is terminated, and contributes to membrane insertion. Our study establishes a role of SecB in the co-translational maturation of proteins from all cellular compartments and functionally implicates cytosolic chaperones in membrane protein biogenesis.

SecA2 Associates with Translating Ribosomes and Contributes to the Secretion of Potent IFN-ß Inducing RNAs.

Protein secretion plays a central role in modulating interactions of the human pathogen Listeria monocytogenes with its environment. Recently, secretion of RNA has emerged as an important strategy used by the pathogen to manipulate the host cell response to its advantage. In general, the Sec-dependent translocation pathway is a major route for protein secretion in L. monocytogenes, but mechanistic insights into the secretion of RNA by these pathways are lacking. Apart from the classical SecA1 secretion pathway, L. monocytogenes also encodes for a SecA paralogue (SecA2) which targets the export of a specific subset of proteins, some of which are involved in virulence. Here, we demonstrated that SecA2 co-sediments with translating ribosomes and provided evidence that it associates with a subset of secreted small non-coding RNAs (sRNAs) that induce high levels of IFN-ß response in host cells. We found that enolase, which is translocated by a SecA2-dependent mechanism, binds to several sRNAs, suggesting a pathway by which sRNAs are targeted to the supernatant of L. monocytogenes.

Protein chain collapse modulation and folding stimulation by GroEL-ES.

The collapse of polypeptides is thought important to protein folding, aggregation, intrinsic disorder, and phase separation. However, whether polypeptide collapse is modulated in cells to control protein states is unclear. Here, using integrated protein manipulation and imaging, we show that the chaperonin GroEL-ES can accelerate the folding of proteins by strengthening their collapse. GroEL induces contractile forces in substrate chains, which draws them into the cavity and triggers a general compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL and is aided by GroES binding to the cavity rim and by the amphiphilic C-terminal tails at the cavity bottom. Collapse modulation is distinct from other proposed GroEL-ES folding acceleration mechanisms, including steric confinement and misfold unfolding. Given the prevalence of collapse throughout the proteome, we conjecture that collapse modulation is more generally relevant within the protein quality control machinery.

Mechanisms of Cotranslational Protein Maturation in Bacteria.

Growing cells invest a significant part of their biosynthetic capacity into the production of proteins. To become functional, newly-synthesized proteins must be N-terminally processed, folded and often translocated to other cellular compartments. A general strategy is to integrate these protein maturation processes with translation, by cotranslationally engaging processing enzymes, chaperones and targeting factors with the nascent polypeptide. Precise coordination of all factors involved is critical for the efficiency and accuracy of protein synthesis and cellular homeostasis. This review provides an overview of the current knowledge on cotranslational protein maturation, with a focus on the production of cytosolic proteins in bacteria. We describe the role of the ribosome and the chaperone network in protein folding and how the dynamic interplay of all cotranslationally acting factors guides the sequence of cotranslational events. Finally, we discuss recent data demonstrating the coupling of protein synthesis with the assembly of protein complexes and end with a brief discussion of outstanding questions and emerging concepts in the field of cotranslational protein maturation.

Protein Synthesis in the Developing Neocortex at Near-Atomic Resolution Reveals Ebp1-Mediated Neuronal Proteostasis at the 60S Tunnel Exit.

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

DENR promotes translation reinitiation via ribosome recycling to drive expression of oncogenes including ATF4.

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.

GPD1 Specifically Marks Dormant Glioma Stem Cells with a Distinct Metabolic Profile.

Brain tumor stem cells (BTSCs) are a chemoresistant population that can drive tumor growth and relapse, but the lack of BTSC-specific markers prevents selective targeting that spares resident stem cells. Through a ribosome-profiling analysis of mouse neural stem cells (NSCs) and BTSCs, we find glycerol-3-phosphate dehydrogenase 1 (GPD1) expression specifically in BTSCs and not in NSCs. GPD1 expression is present in the dormant BTSC population, which is enriched at tumor borders and drives tumor relapse after chemotherapy. GPD1 inhibition prolongs survival in mouse models of glioblastoma in part through altering cellular metabolism and protein translation, compromising BTSC maintenance. Metabolomic and lipidomic analyses confirm that GPD1+ BTSCs have a profile distinct from that of NSCs, which is dependent on GPD1 expression. Similar GPD1 expression patterns and prognostic associations are observed in human gliomas. This study provides an attractive therapeutic target for treating brain tumors and new insights into mechanisms regulating BTSC dormancy.

The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity.

In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.

Mechanisms of Cotranslational Maturation of Newly Synthesized Proteins.

The timely production of functional proteins is of critical importance for the biological activity of cells. To reach the functional state, newly synthesized polypeptides have to become enzymatically processed, folded, and assembled into oligomeric complexes and, for noncytosolic proteins, translocated across membranes. Key activities of these processes occur cotranslationally, assisted by a network of machineries that transiently engage nascent polypeptides at distinct phases of translation. The sequence of events is tuned by intrinsic features of the nascent polypeptides and timely association of factors with the translating ribosome. Considering the dynamics of translation, the heterogeneity of cellular proteins, and the diversity of interaction partners, it is a major cellular achievement that these processes are temporally and spatially so precisely coordinated, minimizing the generation of damaged proteins. This review summarizes the current progress we have made toward a comprehensive understanding of the cotranslational interactions of nascent chains, which pave the way to their functional state.

Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling.

The folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, as non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by using molecular chaperones to safeguard folding cotranslationally2. However, although most of the cellular proteome forms oligomeric assemblies3, little is known about the final step of folding: the assembly of polypeptides into complexes. In prokaryotes, a proof-of-concept study showed that the assembly of heterodimeric luciferase is an organized cotranslational process that is facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences-such as the rarity of polycistronic mRNAs and different chaperone constellations-raise the question of whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of the assembly of protein complexes in eukaryotes using ribosome profiling. We determined the in vivo interactions of the nascent subunits from twelve hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find nine complexes assemble cotranslationally; the three complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5-7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit, thereby counteracting its propensity for aggregation. The onset of cotranslational subunit association coincides directly with the full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains and then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for the assembly of hetero-oligomers in yeast and indicates that translation, folding and the assembly of protein complexes are integrated processes in eukaryotes.

Protein Folding Mediated by Trigger Factor and Hsp70: New Insights from Single-Molecule Approaches.

Chaperones assist in protein folding, but what this common phrase means in concrete terms has remained surprisingly poorly understood. We can readily measure chaperone binding to unfolded proteins, but how they bind and affect proteins along folding trajectories has remained obscure. Here we review recent efforts by our labs and others that are beginning to pry into this issue, with a focus on the chaperones trigger factor and Hsp70. Single-molecule methods are central, as they allow the stepwise process of folding to be followed directly. First results have already revealed contrasts with long-standing paradigms: rather than acting only "early" by stabilizing unfolded chain segments, these chaperones can bind and stabilize partially folded structures as they grow to their native state. The findings suggest a fundamental redefinition of the protein folding problem and a more extensive functional repertoire of chaperones than previously assumed.

Profiling Ssb-Nascent Chain Interactions Reveals Principles of Hsp70-Assisted Folding.

The yeast Hsp70 chaperone Ssb interacts with ribosomes and nascent polypeptides to assist protein folding. To reveal its working principle, we determined the nascent chain-binding pattern of Ssb at near-residue resolution by in vivo selective ribosome profiling. Ssb associates broadly with cytosolic, nuclear, and hitherto unknown substrate classes of mitochondrial and endoplasmic reticulum (ER) nascent proteins, supporting its general chaperone function. Ssb engages most substrates by multiple binding-release cycles to a degenerate sequence enriched in positively charged and aromatic amino acids. Timely association with this motif upon emergence at the ribosomal tunnel exit requires ribosome-associated complex (RAC) but not nascent polypeptide-associated complex (NAC). Ribosome footprint densities along orfs reveal faster translation at times of Ssb binding, mainly imposed by biases in mRNA secondary structure, codon usage, and Ssb action. Ssb thus employs substrate-tailored dynamic nascent chain associations to coordinate co-translational protein folding, facilitate accelerated translation, and support membrane targeting of organellar proteins.

SecA Cotranslationally Interacts with Nascent Substrate Proteins In Vivo.

SecA is an essential component of the Sec machinery in bacteria, which is responsible for transporting proteins across the cytoplasmic membrane. Recent work from our laboratory indicates that SecA binds to ribosomes. Here, we used two different approaches to demonstrate that SecA also interacts with nascent polypeptides in vivo and that these polypeptides are Sec substrates. First, we photo-cross-linked SecA to ribosomes in vivo and identified mRNAs that copurify with SecA. Microarray analysis of the copurifying mRNAs indicated a strong enrichment for proteins containing Sec-targeting sequences. Second, we used a 2-dimensional (2-D) gel approach to analyze radioactively labeled nascent polypeptides that copurify with SecA, including maltose binding protein, a well-characterized SecA substrate. The interaction of SecA with nascent chains was not strongly affected in cells lacking SecB or trigger factor, both of which also interact with nascent Sec substrates. Indeed, the ability of SecB to interact with nascent chains was disrupted in strains in which the interaction between SecA and the ribosome was defective. Analysis of the interaction of SecA with purified ribosomes containing arrested nascent chains in vitro indicates that SecA can begin to interact with a variety of nascent chains when they reach a length of ∼110 amino acids, which is considerably shorter than the length required for interaction with SecB. Our results suggest that SecA cotranslationally recognizes nascent Sec substrates and that this recognition could be required for the efficient delivery of these proteins to the membrane-embedded Sec machinery. IMPORTANCE: SecA is an ATPase that provides the energy for the translocation of proteins across the cytoplasmic membrane by the Sec machinery in bacteria. The translocation of most of these proteins is uncoupled from protein synthesis and is frequently described as "posttranslational." Here, we show that SecA interacts with nascent Sec substrates. This interaction is not dependent on SecB or trigger factor, which also interact with nascent Sec substrates. Moreover, the interaction of SecB with nascent polypeptides is dependent on the interaction of SecA with the ribosome, suggesting that interaction of the nascent chain with SecA precedes interaction with SecB. Our results suggest that SecA could recognize substrate proteins cotranslationally in order to efficiently target them for uncoupled protein translocation.

Alternative modes of client binding enable functional plasticity of Hsp70.

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70-substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR.

Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA-adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes. A distinction of the respective pathways depends largely on the hydrophobicity of the recognition sequence. Hydrophobic transmembrane domains stabilize SRP binding, whereas less hydrophobic signal sequences, typical for periplasmic and outer membrane proteins, stimulate SecA binding and disfavor SRP interactions. In this context, the formation of helical structures of signal peptides within the ribosome was considered to be an important factor. We applied dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance to investigate the conformational states of the disulfide oxidoreductase A (DsbA) signal peptide stalled within the exit tunnel of the ribosome. Our results suggest that the nascent chain comprising the DsbA signal sequence adopts an extended structure in the ribosome with only minor populations of helical structure.

Global profiling of SRP interaction with nascent polypeptides.

Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons. The existence of parallel co- and post-translational transport pathways, however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.

Co-translational mechanisms of protein maturation.

Protein biogenesis integrates multiple finely regulated mechanisms, ensuring nascent polypeptide chains are correctly enzymatically processed, targeted to membranes and folded to native structure. Recent studies show that the cellular translation machinery serves as hub that coordinates the maturation events in space and time at various levels. The ribosome itself serves as docking site for a multitude of nascent chain-interacting factors. The movement of ribosomes along open reading frames is non-uniformous and includes pausing sites, which facilitates nascent chain folding and perhaps factor engagement. Here we summarize current knowledge and discuss emerging concepts underlying the critical interplay between translation and protein maturation in E. coli.