Folding Of Nascent Polypeptide Chains Research Articles

Molecular chaperones in stroke-induced immunosuppression.

Stroke-induced immunosuppression is a process that leads to peripheral suppression of the immune system after a stroke and belongs to the central nervous system injury-induced immunosuppressive syndrome. Stroke-induced immunosuppression leads to increased susceptibility to post-stroke infections, such as urinary tract infections and stroke-associated pneumonia, worsening prognosis. Molecular chaperones are a large class of proteins that are able to maintain proteostasis by directing the folding of nascent polypeptide chains, refolding misfolded proteins, and targeting misfolded proteins for degradation. Various molecular chaperones have been shown to play roles in stroke-induced immunosuppression by modulating the activity of other molecular chaperones, cochaperones, and their associated pathways. This review summarizes the role of molecular chaperones in stroke-induced immunosuppression and discusses new approaches to restore host immune defense after stroke.

Structure and evolution of the 4-helix bundle domain of Zuotin, a J-domain protein co-chaperone of Hsp70.

The J-domain protein Zuotin is a multi-domain eukaryotic Hsp70 co-chaperone. Though it is primarily ribosome-associated, positioned at the exit of the 60S subunit tunnel where it promotes folding of nascent polypeptide chains, Zuotin also has off-ribosome functions. Domains of Zuotin needed for 60S association and interaction with Hsp70 are conserved in eukaryotes. However, whether the 4-helix bundle (4HB) domain is conserved remains an open question. We undertook evolutionary and structural approaches to clarify this issue. We found that the 4HB segment of human Zuotin also forms a bundle of 4 helices. The positive charge of Helix I, which in Saccharomyces cerevisiae is responsible for interaction with the 40S subunit, is particularly conserved. However, the C-termini of fungal and human 4HBs are not similar. In fungi the C-terminal segment forms a plug that folds back into the bundle; in S. cerevisiae it plays an important role in bundle stability and, off the ribosome, in transcriptional activation. In human, C-terminal helix IV of the 4HB is extended, protruding from the bundle. This extension serves as a linker to the regulatory SANT domains, which are present in animals, plants and protists, but not fungi. Further analysis of Zuotin sequences revealed that the plug likely arose as a result of genomic rearrangement upon SANT domain loss early in the fungal lineage. In the lineage leading to S. cerevisiae, the 4HB was subjected to positive selection with the plug becoming increasingly hydrophobic. Eventually, these hydrophobic plug residues were coopted for a novel regulatory function—activation of a recently emerged transcription factor, Pdr1. Our data suggests that Zuotin evolved off-ribosome functions twice—once involving SANT domains, then later in fungi, after SANT domain loss, by coopting the hydrophobic plug. Zuotin serves as an example of complex intertwining of molecular chaperone function and cell regulation.

Implications of HSP 90 Q488H Polymorphism in Long QT Syndrome—A South Indian Study

Long QT syndrome (LQTS) is a rare primary cardiac electrophysiological disorder with characteristic symptoms of syncope, tachyarrhythmia and torsades-de-pointes. It is the outcome of mutations in genes encoding ion channels that function as voltage regulators. Heat shock protein (HSP) 90 is a molecular chaperone that plays vital roles in a variety of cellular processes, one of which is folding of nascent polypeptide chains into their native forms. The main objective of this study was to screen for HSP90 Q488H (C > G) polymorphism in patients with LQTS and identify its risk towards LQTS manifestation.

Role of the osmolyte taurine on the folding of a model protein, hen egg white lysozyme, under a crowding condition.

Taurine is one of the osmolytes that maintain the structure of proteins in cells exposed to denaturing environmental stressors. Recently, cryoelectron tomographic analysis of eukaryotic cells has revealed that their cytoplasms are crowded with proteins. Such crowding conditions would be expected to hinder the efficient folding of nascent polypeptide chains. Therefore, we examined the role of taurine on the folding of denatured and reduced lysozyme, as a model protein, under a crowding condition. The results confirmed that taurine had a better effect on protein folding than did β-alanine, which has a similar chemical structure, when the protein to be folded was present at submillimolar concentration. NMR analyses further revealed that under the crowding condition, taurine had more interactions than did β-alanine with the lysozyme molecule in both the folded and denatured states. We concluded that taurine improves the folding of the reduced lysozyme at submillimolar concentration to allow it to interact more favorably with the lysozyme molecule. Thus, the role of taurine, as an osmolyte in vivo, may be to assist in the efficient folding of proteins.

Mycobacterial chaperonins: the tail wags the dog

Molecular chaperones are defined as proteins that assist the noncovalent assembly of other protein-containing structures in vivo, but which are not components of these structures when they are carrying out their normal biological functions. There are numerous families of protein that fit this definition of molecular chaperones, the most ubiquitous of which are the chaperonins and the Hsp70 families, both of which are required for the correct folding of nascent polypeptide chains and thus essential genes for cell viability. The groE genes of Escherichia coli were the first chaperonin genes to be discovered, within an operon comprising two genes, groEL and groES, that function together in the correct folding of nascent polypeptide chains. The identification of multiple groEL genes in mycobacteria, only one of which is operon-encoded with a groES gene, has led to debate about the functions of their encoded proteins, especially as the essential copies are surprisingly often not the operon-encoded genes. Comparisons of these protein sequences reveals a consistent functional homology and identifies an actinomycete-specific chaperonin family, which may chaperone the folding of enzymes involved in mycolic acid synthesis and thus provide a unique target for the development of a new class of broad-spectrum antimycobacterial drugs.

Viewing folding of nascent polypeptide chains from ribosomes

Regulation of gene expression occurs at multiple stages and ultimately determines the protein levels in cells. The last decade has witnessed impressive progress in the development of approaches to measuring changes in cellular transcription and protein complement. However, techniques monitoring protein synthesis have lagged far behind. Current proteomic studies gauge the steady state levels of intracellular protein and rely on mass spectrometry, which has a low dynamic range and rarely samples more than 10% of the proteome [1]. RNA-seq is a sensitive approach for assessing transcriptional changes but the transcriptome correlates poorly with changes in protein levels [2,3]. Recent data suggest that the regulation of gene expression within cells occurs predominantly at the level of translation and that protein abundance correlates closely with translation rates [3]. Therefore, monitoring protein synthesis is crucial to understanding cellular homeostasis. Additionally, regulation of protein synthesis at the level of translation permits the cell to respond swiftly to adverse conditions [4]. Therefore, translational control is an integral part of cellular stress response. An intricate process associated with protein synthesis is folding. Proteins must achieve proper tertiary structure to acquire their designated properties and functions. Our current understanding of protein folding is predominantly based on in vitro refolding of denatured full-length proteins [5]. However, under native intracellular conditions, protein folding could occur concurrently with the synthesis of primary polypeptide chains on the ribosomes [6,7]. Cotranslational folding of partially synthesized nascent chains differs from refolding of full length polypeptides owing to the vectorial nature and the relatively slow elongation speed of the translation process. However, it is challenging to capture cotranslational folding events of nascent chains attached to the translating ribosome. Several approaches have been developed to dissect this process but most of them rely on in vitro systems with limited resolution [7,8]. There is thus an urgent need to establish new approaches to monitor cotranslational folding processes in vivo with high resolution. One solution is to look at the folding status of nascent polypeptide chains from the perspective of translating ribosomes.

Analysis of Two Bradyrhizobium japonicum Strains with Different Symbiotic Matching for Nodulation by Primary Proteomic

The symbiotic matching for nodulation of Bradyrhizobium japonicum strains is a synergy of multi-proteins and plays a key role in symbiotic nitrogen fixation in nature. Studies on mechanism of symbiotic matching are significant in both theory and practice. In this paper, B. japonicum USDA110-A with high symbiotic matching with high-oil content soybean cultivar Suinong 20 and B. japonicum 2178 with low symbiotic matching were selected for proteomic to reveal mechanism of different symbiotic nodulation. The results showed that the amount and categories of proteins identified in this test were different when the two strains were treated by symbiotic nodulation. There were 10 up-regulated proteins and 5 down-regulated proteins with significant difference for B. japonicum USDA110-A. Proteins associated with nodulation and metabolism of energy and material, which were propitious to symbiotic nodulation, were all up-regulated, such as PHDPS synthase, metal-dependent phosphohydrolase, glycosyl transferase family. In contrast, only 5 up-regulated and 7 down-regulated differential proteins were detected in B. japonicum 2178. Molecular chaperones and defensive proteins, which influence the folding of nascent polypeptide chains and the active of azotase were down-regulated. To a certain extent, the different responses of B. japonicum to daidzein were one of the most important reasons that cause varieties in symbiotic matching ability.

Atomic force microscopy captures folded ribosome bound nascent chains

Direct visualization of co-translational folding of nascent polypeptide chains is challenging. Here we present, for the first time, AFM images of large protein constructs based on the membrane binding domain of ankyrin-R, complexed with the ribosome. The characteristic "horse-shoe" shape of ankyrin-R emerging from the ribosome was captured.

EGD1 (β-NAC) mRNA is localized in a novel cytoplasmic structure in Saccharomyces cerevisiae

RNA localization is a common mechanism for recruiting proteins to specific regions of a cell, which causes cell polarization and sometimes asymmetric division. We found that EGD1 mRNA accumulates dose-dependently as a cytoplasmic granule in Saccharomyces cerevisiae. EGD1 encodes a β-subunit of the nascent polypeptide-associated complex (NAC). NAC is a heterodimer consisting of α- and β-subunits, associated with ribosomes and thought to be involved in the folding of nascent polypeptide chains. Analysis of deletion constructs showed that the localization of EGD1 mRNA requires both an upstream region and an ORF of EGD1, suggesting that the translation of Egd1p is important for localization. We also showed that Egd1p and P-body components are co-localized with EGD1 mRNA. This granule, named the EGD1 granule, has features similar to cellular inclusions containing aggregated proteins. Disruption of microtubules by treatment with a drug, benomyl, resulted in loss of the EGD1 granule. When the expression level of EGD2 encoding the αNAC increased, the percentage of cells showing the EGD1 granule decreased, suggesting that the granular distribution of EGD1 depends on the quantitative balance between α- and β-subunits of NAC. Taken together, we propose a novel microtubule-dependent mechanism for controlling NAC through RNA localization.

Cotranslational Folding Increases GFP Folding Yield

Protein sequences evolved to fold in cells, including cotranslational folding of nascent polypeptide chains during their synthesis by the ribosome. The vectorial (N- to C-terminal) nature of cotranslational folding constrains the conformations of the nascent polypeptide chain in a manner not experienced by full-length chains diluted out of denaturant. We are still discovering to what extent these constraints affect later, posttranslational folding events. Here we directly address whether conformational constraints imposed by cotranslational folding affect the partitioning between productive folding to the native structure versus aggregation. We isolated polyribosomes from Escherichia coli cells expressing GFP, analyzed the nascent chain length distribution to determine the number of nascent chains that were long enough to fold to the native fluorescent structure, and calculated the folding yield for these nascent chains upon ribosome release versus the folding yield of an equivalent concentration of full-length, chemically denatured GFP polypeptide chains. We find that the yield of native fluorescent GFP is dramatically higher upon ribosome release of nascent chains versus dilution of full-length chains from denaturant. For kinetically trapped native structures such as GFP, folding correctly the first time, immediately after release from the ribosome, can lead to lifelong population of the native structure, as opposed to aggregation.

Structure of the EMMPRIN N‐terminal domain 1: Dimerization via β‐strand swapping

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as Hab18G, CD147, Basigin, M6, and neurothelin, is a membrane glycoprotein expressed on the surface of various cell types and many cancer cells. EMMPRIN stimulates adjacent fibroblasts and tumor cells to produce matrix metalloproteinases and plays an important role in tumor invasion and metastasis, angiogenesis, spermatogensis and fertilization, cell-cell adhesion and communication, and other biological processes (reviewed in Ref. 1 and references therein). It was demonstrated that the EMMPRIN extracellular domain (ECD), which structurally belongs to the IgG superfamily, can form homo-oligomers in a cis dependent manner and the N-terminal domain 1 (residues 22-101) was necessary and sufficient to mediate this interaction. The crystal structure of the ECD of recombinant human EMMPRIN (Hab18G/CD147) expressed in E. coli was reported at 2.8 {angstrom} resolution (Yu et al. 2008). The construct consists of residues 22-205 of the mature protein and has both an N-terminal IgC2 domain (ND1, residues 22-101) and a C-terminal IgC2 domain (ND2, residues 107-205). The two domains are joined by a five amino acid residue linker that constitutes a flexible hinge between the two domains. The crystal form has four copies of the molecule in the asymmetric unit, each of which hasmore » a different inter-domain angle that varies from 121{sup o} to 144{sup o}. The two domains each have a conserved disulfide bridge and both are comprised of two {beta}-sheets formed by strands EBA and GFCC, and DEBA and AGFCC for ND1 and ND2, respectively. Based on the crystal packing in this structure, the authors proposed that lateral packing between the two IgG domains of EMMPRIN ECD represents a potential mechanism for cell adhesion. Here we report the 2.0-{angstrom} crystal structure of the N-terminal domain of EMMPRIN ECD (ND1) expressed in mammalian cells. The overall structure of the domain is very similar to that in the full length ECD. Quite unexpectedly, ND1 forms a dimer mediated through the exchange of its last {beta}-strand (strand G). {beta}-strand swapping, which is a subset of 3D domain swapping, has been found to mediate cell-cell adhesion by cadherins. 3D domain swapping has been proposed to be a mechanism of protein oligomerization, aggregation, evolution of oligomeric proteins from single domains and amyloidogenesis. In domain swapped proteins, the same structural elements are involved in the final 3D structure, and so there is little overall energetic difference between the monomer and the swapped oligomers. However, there is often a high energy barrier for the conversion as it often goes through an unfolded state. It is also possible that strand-swapping occurs during folding of nascent polypeptide chains. Frequently, the exchange hinges contain proline-rich motifs which are often in high strain conformations. Domain swapping appears to be a strategy to resolve such local structural strain. The exchange hinge of ND1 contains a Pro-Glu-Pro tripeptide motif. Both of the proline residues adopt extended trans conformations, when compared with cis in the full-length ECD structure. Proline cis-trans isomerization may be the driving force for this exchange. Strand-exchanged dimerization may be a mechanism for the oligomerization of EMMPRIN ECD and its cis-dependent homophilic interactions in cell-cell adhesion.« less

The perspectives of studying multi-domain protein folding.

Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In vitro many of these proteins are well characterized by a reversible two-state folding scheme. However, the majority of proteins in the cell belong to the class of larger multi-domain proteins that often unfold irreversibly under in vitro conditions. This makes folding studies difficult or even impossible. In spite of these problems for many multi-domain proteins, folding has been investigated by classical refolding. Co-translational folding of nascent polypeptide chains when synthesized by ribosomes has also been studied. Single molecule techniques represent a promising approach for future studies on the folding of multi-domain proteins, and tremendous advances have been made in these techniques in recent years. In particular, fluorescence-based methods can contribute significantly to an understanding of the fundamental principles of multi-domain protein folding.

Mycobacterial Heat Shock Proteins as Vaccines - A Model of Facilitated Antigen Presentation

Heat shock proteins (hsps) are a highly conserved family of proteins, first recognized by their upregulated expression in response to host exposure to raised temperatures. Further study has revealed that they have numerous functions in the cell, primarily as chaperones mediating both the correct folding of nascent polypeptide chains and the dissolution of aggregated protein complexes. The energy requirement for this chaperone activity is provided by the ATPase activity found in most families of hsps and thus the peptide binding capacity is controlled by ATP hydrolysis. The structural consequence of this is that hsps isolated in situ are found complexed to chaperoned peptides (hspCs). Much previous work has implicated hsps in the immune response to pathogens and recent studies have shown that the interaction of hsps with antigen presenting cells, such as dendritic cells (DCs), mediates the integration of the innate and acquired immune responses. This central role for hspCs in immunity is facilitated by their dual function in both innate immunity, with the induction of cytokines and the maturation of DCs mediated by the hsp component, and acquired immunity, with the trafficking of antigens chaperoned in hspCs for antigen presentation by the mature DCs.

ER-associated protein degradation is a common mechanism underpinning numerous monogenic diseases including Robinow syndrome

Correct folding of nascent polypeptide chains within the ER is critical for function, assembly into multi-subunit complexes and trafficking through the exocytic pathway for secretory and cell surface proteins. This process is rather inefficient, and a substantial proportion of nascent polypeptides is rejected by an ER quality control system and targeted for degradation. In some cases, only a minor fraction of nascent chains is correctly folded, and the smallest alteration to polypeptide primary structure (i.e. point mutation) can result in the complete loss of function with inherent pathological consequences; cystic fibrosis and emphysema result from such mutations. We have taken a bioinformatic approach to parse a large database of known disease susceptibility genes for candidates whose disease-associated alleles are likely prone to misfolding in the ER. Surprisingly, we find that proteins with ER-targeting signals are over represented in this database when compared with all predicted proteins in the human genome (45 versus 30%). We selected a subgroup of proteins that were positive for both an ER-targeting signal and a membrane-anchoring domain and thereby identified several ER-associated degradation diseases candidates. To determine whether our analysis had identified new ER-degradation substrates, we established that ER retention is indeed the mechanism underlying Robinow syndrome (RRS), one of the identified candidates. Specifically, mutant alleles of ROR2 that are associated with RRS are retained within the ER, whereas wild-type and non-pathogenic alleles are exported to the plasma membrane. These data both uncover a major pathogenic factor for RRS and indicate that misfolding of secretory proteins is likely to significantly contribute to human disease and morbidity.

Heat-shock proteins as regulators of apoptosis.

Heat-shock proteins are produced in response to different types of stress conditions making cells resistant to stress-induced cell damage. Under normal conditions, heat-shock proteins play numerous roles in cell function, including modulating protein activity by changing protein conformation, promoting multiprotein complex assembly/disassembly, regulating protein degradation within the proteasome pathway, facilitating protein translocation across organellar membranes, and ensuring proper folding of nascent polypeptide chains during protein translation. When cells are stressed, a common response is to undergo cell death by one of two pathways, either 'necrosis' or 'apoptosis'. Recently, both routes to cell death have been revealed to share similar mechanisms, with heat-shock proteins and their cofactors responsible for inhibiting both apoptotic and necrotic pathways. We therefore briefly summarize recent reports showing molecular evidence of cell death regulation by heat-shock proteins and their cochaperones.

Granulocyte-macrophage colony-stimulating factor gene-transduced tumor cells combined with tumor-derived gp96 inhibit tumor growth in mice.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-based cancer cell vaccines have been shown to be potent inducers of antitumor immunity in several murine models, but the antitumor effects on established tumors have been minimal. Conversely, the major role of the heat shock protein gp96, localized in the endoplasmic reticulum (ER), is to act as a molecular chaperone to assist the folding of nascent polypeptide chains in the ER. gp96 derived from tumor cells elicits specific protective immunity against parental tumors, presumably through the transport of tumor-specific peptides to antigen-presenting cells and the maturation of dendritic cells (DCs). However, the therapeutic effects of tumor-derived gp96 on established tumors have not been promising. The present study analyzes the therapeutic effects of GM-CSF gene-transduced Lewis lung cancer (LLC/GM) cells combined with LLC-derived gp96 on established wild-type LLC tumors in immunocompetent C57BL/6 mice. Therapy with either irradiated LLC/GM cells or LLC-derived gp96 barely affected established LLC tumor growth. The antitumor effect was significantly enhanced when 1 microg of LLC-derived gp96 was administered together with 1 x 10(6) irradiated LLC/GM cells (p < 0.05). The antitumor effects of irradiated LLC/GM cells and LLC-derived gp96 required mainly CD8(+) T cells. Spleen cells obtained from mice vaccinated with irradiated LLC/GM cells and LLC-derived gp96 showed specific CD8 cytotoxic activities against LLC cells (specific lysis rate of approximately 28%). This antibody response was not associated with a synergic effect of the combination therapy. Moreover, draining lymph nodes from mice immunized with irradiated LLC/GM cells and LLC-derived gp96 contained more migrating mature CD11c(+) cells (higher levels of CD86 and major histocompatibility complex [MHC] class II molecules) compared with those from any other immunization protocols. These results suggest that the combination of irradiated LLC/GM cells and tumor-derived gp96 has potential as a new immunogene therapeutic strategy against lung cancer.

正常ヒト末梢血単核球膜表面のＨＳＣ７０蛋白質について

71kDa heat shock cognate protein (HSC70) is a member of the Hsp70 family and is constitutively expressed in organisms as diverse as slime molds, bacteria, plants and higher eukaryotes. Proteins in the Hsp70 family are known as chaperones that involve quality control of cellular proteins, folding of nascent polypeptide chains and trafficking of proteins via membrane structure. Recently, it was revealed that Hsp/HSC70 proteins were presented on surface of virus- or bacteria-infected cells, immortalized cells and blood cells of autoimmune disease patients. We previously reported that the HTLV-1 receptor consists of HSC70, β-actin and phosphatidylglycerol on the surface of MOLT-4 cells. In this study, we examined the induction of HSC70 protein on the surface of peripheral blood mononuclear cells (PBMCs) from healthy individuals. Little HSC70 protein was observed on the surface of PBMCs before cultivation. However, after 72 hours culture, 41±9.1% of PBMCs presented HSC70 proteins on the cell surface. On the other hand, in culture with phytohemagglutinin (PHA), 70±14% of PBMCs exposed HSC70 protein on the cell surface after 24 hours, and 84±5.0% after 72 hours. PHA stimulated the presentation of HSC70 protein on the cell surface of CD4+ T cells, which are the target cells of HTLV-1 in vivo, as well as that on the surface of PBMCs. Susceptibility to HTLV-1 of target cells was parallel to the expression level of HSC70 molecules on the cell surface. These results demonstrate that HSC70 protein on the surface of PBMCs and CD4+ T cells exposes the domains which are functional for HTLV-1 infection, and that stimulation by cultivation and mitogen enhances the trafficking of HSC70 proteins to the surface of plasma membrane. This is the first finding that HSC70 on the surface of CD4+ T cells functions as a receptor for cell-to-cell transmission of HTLV-1.

The unfolded protein response represses nitrogen-starvation induced developmental differentiation in yeast.

Diploid budding yeast exhibits two developmental programs in response to nitrogen starvation, pseudohyphal growth, and sporulation. Here we show that both programs are repressed by activation of the unfolded protein response (UPR), a stress-signal transduction pathway responsible for induction of endoplasmic reticulum (ER)-resident chaperones when protein folding in the ER is impaired. Pseudohyphal growth was derepressed in ire1Delta/ire1Delta and hac1Delta/hac1Delta strains. Activation of the UPR or overexpression of the transcription factor Hac1(i)p, the product of an unconventional splicing reaction regulated by the UPR, was sufficient for repression of pseudohyphal growth and meiosis. HAC1 splicing occurred in a nitrogen-rich environment but ceased rapidly on nitrogen starvation. Further, addition of ammonium salts to nitrogen-starved cells was sufficient to rapidly reactivate HAC1 splicing. We propose that high translation rates in a nitrogen-rich environment are coupled to limited protein unfolding in the ER, thereby activating the UPR. An activated UPR then represses pseudohyphal growth and meiosis. Nitrogen starvation slows translation rates, allowing for more efficient folding of nascent polypeptide chains, down-regulation of the UPR, and subsequent derepression of pseudohyphal growth and meiosis. These findings significantly broaden the range of physiological functions of the UPR and define a role for the UPR in nitrogen sensing.

Structure and folding of nascent polypeptide chains during protein translocation in the endoplasmic reticulum.

To investigate the role of protein folding and chaperone-nascent chain interactions in translocation across the endoplasmic reticulum membrane, the translocation of wild type and mutant forms of preprolactin were studied in vivo and in vitro. The preprolactin mutant studied contains an 18-amino acid substitution at the amino terminus of the mature protein, eliminating a disulfide-bonded loop domain. In COS-7 cells, mutant prolactin accumulated in the endoplasmic reticulum as stable protein-protein and disulfide-bonded aggregates, whereas wild type prolactin was efficiently secreted. In vitro, wild type and mutant preprolactin translocated with equal efficiency although both translation products were recovered as heterogeneous aggregates. Studies with translocation intermediates indicated that aggregation occurred co-translationally. To evaluate the contribution of lumenal chaperones to translocation and folding, in vitro studies were performed with native and reconstituted, chaperone-deficient membranes. The absence of lumenal chaperones was associated with a decrease in translocation efficiency and pronounced aggregation of the translation products. These studies suggest that chaperone-nascent chain interactions significantly enhance translocation and indicate that in the absence of such interactions, aggregation can serve as the predominant in vitro protein folding end point. The ramifications of these observations on investigations into the mechanism of translocation are discussed.

Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones.

The folding of polypeptides emerging from ribosomes was analysed in a mammalian translation system using firefly luciferase as a model protein. The growing polypeptide interacts with a specific set of molecular chaperones, including Hsp70, the DnaJ homologue Hsp40 and the chaperonin TRiC. The ordered assembly of these components on the nascent chain forms a high molecular mass complex that allows the cotranslational formation of protein domains and the completion of folding once the chain is released from the ribosome.