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.     

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.     

The interactions of molecular chaperones with client proteins: why are they so weak?

The major classes of molecular chaperones have highly variable sequences, sizes, and shapes, yet they all bind to unfolded proteins, limit their aggregation, and assist in their folding. Despite the central importance of this process to protein homeostasis, it has not been clear exactly how chaperones guide this process or whether the diverse families of chaperones use similar mechanisms. For the first time, recent advances in NMR spectroscopy have enabled detailed studies of how unfolded, “client” proteins interact with both ATP-dependent and ATP-independent classes of chaperones. Here, we review examples from four distinct chaperones, Spy, Trigger Factor, DnaK, and HscA-HscB, highlighting the similarities and differences between their mechanisms. One striking similarity is that the chaperones all bind weakly to their clients, such that the chaperone–client interactions are readily outcompeted by stronger, intra- and intermolecular contacts in the folded state. Thus, the relatively weak affinity of these interactions seems to provide directionality to the folding process. However, there are also key differences, especially in the details of how the chaperones release clients and how ATP cycling impacts that process. For example, Spy releases clients in a largely folded state, while clients seem to be unfolded upon release from Trigger Factor or DnaK. Together, these studies are beginning to uncover the similarities and differences in how chaperones use weak interactions to guide 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.     

The role of ATPase activity in the function of molecular chaperones and some regulatory proteins]

It has been suggested that the ATPase activity of molecular chaperones depends on the structure of the recognizable determinant in the target protein. The role of molecular chaperones in polypeptide chain folding and protein association into oligomeric complexes is discussed. The putative regulatory role of the determinant ATPase activity of molecular chaperones and those of some regulatory proteins are discussed. A hypothesis is proposed that determinant ATPases play a part in the increasing specificity of intermacromolecular interactions.     

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.     

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.     

Trigger factor is involved in GroEL-dependent protein degradation in Escherichia coli and promotes binding of GroEL to unfolded proteins.

In Escherichia coli, the molecular chaperones of hsp60/hsp10 (GroEL/GroES) families are required not only for protein folding but also for the rapid degradation of certain abnormal proteins. The rate-limiting step in the degradation of the fusion protein CRAG by protease ClpP appears to be the formation of a complex with GroEL. We have isolated these complexes and found that each GroEL 14mer contained a short-lived fragment of CRAG plus a 50 kDa polypeptide, which we identified by sequencing and immunological methods as Trigger Factor (TF). Upon ATP addition, GroEL and TF dissociated together from CRAG but remained tightly associated with each other even upon gel filtration. TF was originally proposed to function in protein translocation across membranes but altering cellular content of TF did not affect this process in vivo. By contrast, low levels of TF expression markedly reduced CRAG degradation, while an overproduction of TF greatly stimulated this process. Furthermore, in extracts of cells expressing high levels of TF, the capacity of GroEL to bind to CRAG is greatly increased. Overproduction of TF also stimulated GroEL's ability to bind to other unfolded proteins (fetuin and histone). Thus, TF is a rate-limiting factor for CRAG degradation; it appears to regulate GroEL function and to promote the formation of TF-GroEL-CRAG complexes which are critical for proteolysis.     

Protein folding in vitro and in the cellular environment

The main concepts concerning protein folding have been developed from in vitro refolding studies. They state that the folding of a polypeptide chain is a spontaneous process depending only on the amino-acid sequence in a given environment. It is thermodynamically controlled and driven by the hydrophobic effect. Consequently, it has been accepted that the in vitro refolding process is a valuable model to understand the mechanisms involved during the folding of a nascent polypeptide chain in the cell. Although it does not invalidate the main rules deduced from the in vitro studies, the discovery of molecular chaperones has led to a re-evaluation of this last point. Indeed, in cells molecular chaperones are able to mediate the folding of polypeptide chains and the assembly of subunits in oligomeric proteins. The possible mechanisms by which these folding helpers act are discussed in the light of the data available in the literature. The folding process is assisted in the cell in different ways, preventing premature folding of the polypeptide chain and suppressing the incorrectly folded species and aggregates. Molecular chaperones bind to incompletely folded proteins in a conformation which suggests that the latter are in the "molten globule" state. However, very little is known about the recognition process.     

Molecular chaperones: assisting assembly in addition to folding

The common perception that molecular chaperones are involved primarily with assisting the folding of newly synthesized and stress-denatured polypeptide chains ignores the fact that this term was invented to describe the function of a protein that assists the assembly of folded subunits into oligomeric structures and only later was extended to embrace protein folding. Recent work has clarified the role of nuclear chaperones in the assembly of nucleosomes and has identified a cytosolic chaperone required for mammalian proteasome assembly, suggesting that the formation of other oligomeric complexes might be assisted by chaperones.     

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.     

Molecular chaperones in cellular protein folding

The discovery of “molecular chaperones” has dramatically changed our concept of cellular protein folding. Rather than folding spontaneously, most newly synthesized polypeptide chains seem to acquire their native conformation in a reaction mediated by these versatile helper proteins. Understanding the structure and function of molecular chaperones is likely to yield useful applications for medicine and biotechnology in the future.     

Molecular chaperones in protein quality control

Proteins must fold into their correct three-dimensional conformation in order to attain their biological function. Conversely, protein aggregation and misfolding are primary contributors to many devastating human diseases, such as prion-mediated infections, Alzheimer's disease, type II diabetes and cystic fibrosis. While the native conformation of a polypeptide is encoded within its primary amino acid sequence and is sufficient for protein folding in vitro, the situation in vivo is more complex. Inside the cell, proteins are synthesized or folded continuously; a process that is greatly assisted by molecular chaperones. Molecular chaperones are a group of structurally diverse and mechanistically distinct proteins that either promote folding or prevent the aggregation of other proteins. With our increasing understanding of the proteome, it is becoming clear that the number of proteins that can be classified as molecular chaperones is increasing steadily. Many of these proteins have novel but essential cellular functions that differ from that of more "conventional" chaperones, such as Hsp70 and the GroE system. This review focuses on the emerging role of molecular chaperones in protein quality control, i.e. the mechanism that rids the cell of misfolded or incompletely synthesized polypeptides that otherwise would interfere with normal cellular function.     

An Essential Nonredundant Role for Mycobacterial DnaK in Native Protein Folding

Protein chaperones are essential in all domains of life to prevent and resolve protein misfolding during translation and proteotoxic stress. HSP70 family chaperones, including E. coli DnaK, function in stress induced protein refolding and degradation, but are dispensable for cellular viability due to redundant chaperone systems that prevent global nascent peptide insolubility. However, the function of HSP70 chaperones in mycobacteria, a genus that includes multiple human pathogens, has not been examined. We find that mycobacterial DnaK is essential for cell growth and required for native protein folding in Mycobacterium smegmatis. Loss of DnaK is accompanied by proteotoxic collapse characterized by the accumulation of insoluble newly synthesized proteins. DnaK is required for solubility of large multimodular lipid synthases, including the essential lipid synthase FASI, and DnaK loss is accompanied by disruption of membrane structure and increased cell permeability. Trigger Factor is nonessential and has a minor role in native protein folding that is only evident in the absence of DnaK. In unstressed cells, DnaK localizes to multiple, dynamic foci, but relocalizes to focal protein aggregates during stationary phase or upon expression of aggregating peptides. Mycobacterial cells restart cell growth after proteotoxic stress by isolating persistent DnaK containing protein aggregates away from daughter cells. These results reveal unanticipated essential nonredunant roles for mycobacterial DnaK in mycobacteria and indicate that DnaK defines a unique susceptibility point in the mycobacterial proteostasis network.     

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.     

Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.     

Roles of molecular chaperones in cytoplasmic protein folding

Newly synthesized polypeptide chains must fold and assemble into unique three-dimensional structures in order to become functionally active. In many cases productive folding depends on assistance from molecular chaperones, which act in preventing off-pathway reactions during folding that lead to aggregation. The inherent tendency of incompletely folded polypeptide chains to aggregate is thought to be strongly enhanced$L in vivo *I$Lby the high macromolecular concentration of the cellular solution, resulting in crowding effects, and by the close proximity of nascent polypeptide chains during synthesis on polyribosomes. The major classes of chaperones acting in cytoplasmic protein folding are the Hsp70s and the chaperonins. Hsp70 chaperones shield the hydrophobic regions of nascent and incompletely folded chains, whereas the chaperonins provide a sequestered environment in which folding can proceed unimpaired by intermolecular interactions between non-native polypeptides. These two principles of chaperone action can function in a coordinated manner to ensure the efficient folding of a subset of cytoplasmic proteins.     

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.