Ribosome-based protein folding systems are structurally divergent but functionally universal across biological kingdoms

In bacteria, Trigger factor (TF) is the first chaperone that interacts with nascent polypeptides as soon as they emerge from the exit tunnel of the ribosome. TF binds to the ribosomal protein L23 located next to the tunnel exit of the large subunit, with which it forms a cradle-like space embracing the polypeptide exit region. It cooperates with the DnaK Hsp70 chaperone system to ensure correct folding of a number of newly translated cytosolic proteins in Escherichia coli. Whereas TF is exclusively found in prokaryotes and chloroplasts, Saccharomyces cerevisiae, a eukaryotic microorganism, has a three-member Hsp70-J protein complex, Ssb-Ssz-Zuo, which could act as a ribosome-associated folding facilitator. In the work reported in this volume of Molecular Microbiology, Rauch et al. (2005, Mol Microbiol, doi:10.1111/j.1365-2958.2005.04690.x) examined the functional similarity of the ribosome-associated chaperones in prokaryotes and eukaryotes. In spite of the fact that TF and the Hsp70-based triad are structurally unrelated, TF can bind to the yeast ribosome via Rpl25 (the L23 counterpart) and can substitute for some, but not all, of the functions assigned to Ssb-Ssz-Zuo in yeast. The functional conservation of the ribosome-associated chaperones without structural similarity is remarkable and suggests that during evolution nature has employed a common design but divergent components to facilitate folding of polypeptides as they emerge from the ribosomal exit, a fundamental process required for the efficient expression of genetic information.     

Association of protein biogenesis factors at the yeast ribosomal tunnel exit is affected by the translational status and nascent polypeptide sequence

Ribosome-associated protein biogenesis factors (RPBs) act during a short but critical period of protein biogenesis. The action of RPBs starts as soon as a nascent polypeptide becomes accessible from the outside of the ribosome and ends upon termination of translation. In yeast, RPBs include the chaperones Ssb1/2 and ribosome-associated complex, signal recognition particle, nascent polypeptide-associated complex (NAC), the aminopeptidases Map1 and Map2, and the Nalpha-terminal acetyltransferase NatA. Here, we provide the first comprehensive analysis of RPB binding at the yeast ribosomal tunnel exit as a function of translational status and polypeptide sequence. We measured the ratios of RPBs to ribosomes in yeast cells and determined RPB occupation of translating and non-translating ribosomes. The combined results imply a requirement for dynamic and coordinated interactions at the tunnel exit. Exclusively, NAC was associated with the majority of ribosomes regardless of their translational status. All other RPBs occupied only ribosomal subpopulations, binding with increased apparent affinity to randomly translating ribosomes as compared with non-translating ones. Analysis of RPB interaction with homogenous ribosome populations engaged in the translation of specific nascent polypeptides revealed that the affinities of Ssb1/2, NAC, and, as expected, signal recognition particle, were influenced by the amino acid sequence of the nascent polypeptide. Complementary cross-linking data suggest that not only affinity of RPBs to the ribosome but also positioning can be influenced in a nascent polypeptide-dependent manner.     

Ribosome-associated chaperones as key players in proteostasis

De novo protein folding is delicate and error-prone and requires the guidance of molecular chaperones. Besides cytosolic and organelle-specific chaperones, cells have evolved ribosome-associated chaperones that support early folding events and prevent misfolding and aggregation. This class of chaperones includes the bacterial trigger factor (TF), the archaeal and eukaryotic nascent polypeptide-associated complex (NAC) and specialized eukaryotic heat shock protein (Hsp) 70/40 chaperones. This review focuses on the cellular activities of ribosome-associated chaperones and highlights new findings indicating additional functions beyond de novo folding. These activities include the assembly of oligomeric complexes, such as ribosomes, modulation of translation and targeting of proteins.     

Dynamics of trigger factor interaction with translating ribosomes

In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of TF-ribosome complexes are modulated by nascent chains, whereby their length, sequence, and folding status are influencing parameters. However, the effect of the folding status is modest, indicating that TF can bind small globular domains and accommodate them within its substrate binding cavity. In general, the presence of a nascent chain causes an up to 9-fold increase in the rate of TF association, which provides a kinetic explanation for the observed ability of TF to efficiently compete with other cytosolic chaperones for binding to nascent chains. Furthermore, a subset of longer nascent polypeptides promotes the stabilization of TF-ribosome complexes, which increases the half-life of these complexes from 15 to 50 s. Nascent chains thus regulate their folding environment generated by ribosome-associated chaperones.     

Characterization of a yeast mitochondrial ribosomal protein structurally related to the mammalian 68-kDa high affinity laminin receptor.

We have cloned the nuclear gene MRP4 coding for a mitochondrial ribosomal protein of the yeast, Saccharomyces cerevisiae. The gene was isolated by complementation of a respiratory-deficient mutant with a pleiotropic defect in mitochondrial gene expression. The nucleotide sequence of MRP4 revealed that it has sequence similarity with Escherichia coli ribosomal protein S2 and related proteins of chloroplast ribosomes from different plants. Further characterization of the MRP4 protein revealed that it is a component of the 37 S subunit of mitochondrial ribosomes. Moreover, the phenotype of cells carrying a disrupted copy of MRP4 is consistent with the MRP4 protein being an essential component of the mitochondrial protein synthetic machinery. Finally, we note that the MRP4 protein and other members of the S2 family of ribosomal proteins have regions of sequence similarity with the mammalian 68-kDa high affinity laminin receptor.     

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro-synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.     

Ribosome-tethered molecular chaperones: the first line of defense against protein misfolding?

Folding of many cellular proteins is facilitated by molecular chaperones. Analysis of both prokaryotic and lower eukaryotic model systems has revealed the presence of ribosome-associated molecular chaperones, thought to be the first line of defense against protein aggregation as translating polypeptides emerge from the ribosome. However, structurally unrelated chaperones have evolved to carry out these functions in different microbes. In the yeast Saccharomyces cerevisiae, an unusual complex of Hsp70 and J-type chaperones associates with ribosome-bound nascent chains, whereas in Escherichia coli the ribosome-associated peptidyl-prolyl-cis-trans isomerase, trigger factor, plays a predominant role.     

Localization of the trigger factor binding site on the ribosomal 50S subunit

In Escherichia coli, protein folding is undertaken by three distinct sets of chaperones, the DnaK-DnaJ and GroEL-GroES systems and the trigger factor (TF). TF has been proposed to be the first chaperone to interact with the nascent polypeptide chain as it emerges from the tunnel of the 70S ribosome and thus probably plays an important role in co-translational protein folding. We have made complexes with deuterated ribosomes (50S subunits and 70S ribosomes) and protated TF and determined the TF binding site on the respective complexes using the neutron scattering technique of spin-contrast variation. Our data suggest that the TF binds in the form of a homodimer. On both the 50S subunit and the 70S ribosome, the TF position is in proximity to the tunnel exit site, near ribosomal proteins L23 and L29, located on the back of the 50S subunit. The positions deviate from one another, such that the position on the 70S ribosome is located slightly further from the tunnel than that determined for the 50S subunit alone. Nevertheless, from both determined positions interaction between TF and a short nascent chain of 57 amino acid residues would be plausible, compatible with a role for TF participation in co-translational protein folding.     

Trigger factor retards protein export in Escherichia coli

Trigger factor (TF) is a ribosome-associated protein that interacts with a wide variety of nascent polypeptides in Escherichia coli. Previous studies have indicated that TF cooperates with DnaK to facilitate protein folding, but the basis of this cooperation is unclear. In this study we monitored protein export in E. coli that lack or overproduce TF to obtain further insights into its function. Whereas inactivation of genes encoding most molecular chaperones (including dnaK) impairs protein export, inactivation of the TF gene accelerated protein export and suppressed the need for targeting factors to maintain the translocation competence of presecretory proteins. Furthermore, overproduction of TF (but not DnaK) markedly retarded protein export. Manipulation of TF levels produced similar effects on the export of a cytosolic enzyme fused to a signal peptide. The data strongly suggest that TF has a unique ability to sequester nascent polypeptides for a relatively prolonged period. Based on our results, we propose that TF and DnaK promote protein folding by distinct (but complementary) mechanisms.     

Versatility of Trigger Factor Interactions with Ribosome-Nascent Chain Complexes

Trigger factor (TF) is the first molecular chaperone that interacts with nascent chains emerging from bacterial ribosomes. TF is a modular protein, consisting of an N-terminal ribosome binding domain, a PPIase domain, and a C-terminal domain, all of which participate in polypeptide binding. To directly monitor the interactions of TF with nascent polypeptide chains, TF variants were site-specifically labeled with an environmentally sensitive NBD fluorophore. We found a marked increase in TF-NBD fluorescence during translation of firefly luciferase (Luc) chains, which expose substantial regions of hydrophobicity, but not with nascent chains lacking extensive hydrophobic segments. TF remained associated with Luc nascent chains for 111 ± 7 s, much longer than it remained bound to the ribosomes (t_½ ∼ 10–14 s). Thus, multiple TF molecules can bind per nascent chain during translation. The Escherichia coli cytosolic proteome was classified into predicted weak and strong interactors for TF, based on the occurrence of continuous hydrophobic segments in the primary sequence. The residence time of TF on the nascent chain generally correlated with the presence of hydrophobic regions and the capacity of nascent chains to bury hydrophobicity. Interestingly, TF bound the signal sequence of a secretory protein, pOmpA, but not the hydrophobic signal anchor sequence of the inner membrane protein FtsQ. On the other hand, proteins lacking linear hydrophobic segments also recruited TF, suggesting that TF can recognize hydrophobic surface features discontinuous in sequence. Moreover, TF retained significant affinity for the folded domain of the positively charged, ribosomal protein S7, indicative of an alternative mode of TF action. Thus, unlike other chaperones, TF appears to employ multiple mechanisms to interact with a wide range of substrate proteins.     

Three-state equilibrium of Escherichia coli trigger factor

Trigger Factor (TF) is the first chaperone that interacts with nascent chains of cytosolic proteins in Escherichia coli. Although its chaperone activity requires association with ribosomes, TF is present in vivo in a 2-3 fold molar excess over ribosomes and a fraction of it is not ribosome-associated after cell lysis. Here we show that TF follows a three-state equilibrium. Size exclusion chromatography, crosslinking and analytical ultracentrifugation revealed that uncomplexed TF dimerizes with an apparent Kd of 18 microM. Dimerization is mediated by the N-terminal ribosome binding domain and the C-terminal domain of TF, whereas the central peptidyl prolyl isomerase (PPlase) and substrate binding domain does not contribute to dimerization. Crosslinking experiments showed that TF is monomeric in its ribosome-associated state. Quantitative analysis of TF binding to ribosomes revealed a dissociation constant for the TF-ribosome complex of approximately 1.2 microM. From these data we estimate that in vivo most of the ribosomes are in complex with monomeric TF. Uncomplexed TF, however, is in a monomer-dimer equilibrium with approximately two thirds of TF existing in a dimeric state.     

Molecular mechanism and structure of Trigger Factor bound to the translating ribosome

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome-nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors.     

A conserved motif is prerequisite for the interaction of NAC with ribosomal protein L23 and nascent chains

In eukaryotes, newly synthesized proteins interact co-translationally with a multitude of different ribosome-bound factors and chaperones including the conserved heterodimeric nascent polypeptide-associated complex (NAC) and a Hsp40/70-based chaperone system. These factors are thought to play an important role in protein folding and targeting, yet their specific ribosomal localizations, which are prerequisite for their functions, remain elusive. This study describes the ribosomal localization of NAC and the molecular details by which NAC is able to contact the ribosome and gain access to nascent polypeptides. We identified a conserved RRK(X)nKK ribosome binding motif within the beta-subunit of NAC that is essential for the entire NAC complex to attach to ribosomes and allow for its interaction with nascent polypeptide chains. The motif localizes within a potential loop region between two predicted alpha-helices in the N terminus of betaNAC. This N-terminal betaNAC ribosome-binding domain was completely portable and sufficient to target an otherwise cytosolic protein to the ribosome. NAC modified with a UV-activatable cross-linker within its ribosome binding motif specifically cross-linked to L23 ribosomal protein family members at the exit site of the ribosome, providing the first evidence of NAC-L23 interaction in the context of the ribosome. Mutations of L23 reduced NAC ribosome binding in vivo and in vitro, whereas other eukaryotic ribosome-associated factors such as the Hsp70/40 chaperones Ssb or Zuotin were unaffected. We conclude that NAC employs a conserved ribosome binding domain to position itself on the L23 ribosomal protein adjacent to the nascent polypeptide exit site.     

The ribosome-bound chaperones RAC and Ssb1/2p are required for accurate translation in Saccharomyces cerevisiae

The chaperone homologs RAC (ribosome-associated complex) and Ssb1/2p are anchored to ribosomes; Ssb1/2p directly interacts with nascent polypeptides. The absence of RAC or Ssb1/2p results in a similar set of phenotypes, including hypersensitivity against the aminoglycoside paromomycin, which binds to the small ribosomal subunit and compromises the fidelity of translation. In order to understand this phenomenon we measured the frequency of translation termination and misincorporation in vivo and in vitro with a novel reporter system. Translational fidelity was impaired in the absence of functional RAC or Ssb1/2p, and the effect was further enhanced by paromomycin. The mutant strains suffered primarily from a defect in translation termination, while misincorporation was compromised to a lesser extent. Consistently, a low level of soluble translation termination factor Sup35p enhanced growth defects in the mutant strains. Based on the combined data we conclude that RAC and Ssb1/2p are crucial in maintaining translational fidelity beyond their postulated role as chaperones for nascent polypeptides.     

Chaperone-assisted folding of newly synthesized proteins in the cytosol

The way in which a newly synthesized polypeptide chain folds into its unique three-dimensional structure remains one of the fundamental questions in molecular biology. Protein folding in the cell is a problematic process and, in many cases, requires the assistance of a network of molecular chaperones to support productive protein foldingin vivo. During protein biosynthesis, ribosome-associated chaperones guide the folding of the nascent polypeptide emerging from the ribosomal tunnel. In this review we summarize the basic principles of the protein-folding process and the involved chaperones, and focus on the role of ribosome-associated chaperones. Our discussion emphasizes the bacterial Trigger Factor, which is the best studied chaperone of this type. Recent advances have determined the atomic structure of the Trigger Factor, providing new, exciting insights into the role of ribosome-associated chaperones in co-translational protein folding.     

Zuotin, a ribosome-associated DnaJ molecular chaperone.

Correct folding of newly synthesized polypeptides is thought to be facilitated by Hsp70 molecular chaperones in conjunction with DnaJ cohort proteins. In Saccharomyces cerevisiae, SSB proteins are ribosome-associated Hsp70s which interact with the newly synthesized nascent polypeptide chain. Here we report that the phenotypes of an S.cerevisiae strain lacking the DnaJ-related protein Zuotin (Zuo1) are very similar to those of a strain lacking Ssb, including sensitivities to low temperatures, certain protein synthesis inhibitors and high osmolarity. Zuo1, which has been shown previously to be a nucleic acid-binding protein, is also a ribosome-associated protein localized predominantly in the cytosol. Analysis of zuo1 deletion and truncation mutants revealed a positive correlation between the ribosome association of Zuo1 and its ability to bind RNA. We propose that Zuo1 binds to ribosomes, in part, by interaction with ribosomal RNA and that Zuo1 functions with Ssb as a chaperone on the ribosome.     

Chaperone-Assisted Folding of Newly Synthesized Proteins in the Cytosol

The way in which a newly synthesized polypeptide chain folds into its unique three-dimensional structure remains one of the fundamental questions in molecular biology. Protein folding in the cell is a problematic process and, in many cases, requires the assistance of a network of molecular chaperones to support productive protein folding in vivo. During protein biosynthesis, ribosome-associated chaperones guide the folding of the nascent polypeptide emerging from the ribosomal tunnel. In this review we summarize the basic principles of the protein-folding process and the involved chaperones, and focus on the role of ribosome-associated chaperones. Our discussion emphasizes the bacterial Trigger Factor, which is the best studied chaperone of this type. Recent advances have determined the atomic structure of the Trigger Factor, providing new, exciting insights into the role of ribosome-associated chaperones in co-translational protein folding.     

Nascent polypeptide-associated complex stimulates protein import into yeast mitochondria

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide-associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.