The Functional Interaction of Mitochondrial Hsp70s with the Escort Protein Zim17 Is Critical for Fe/S Biogenesis and Substrate Interaction at the Inner Membrane Preprotein Translocase

The yeast protein Zim17 belongs to a unique class of co-chaperones that maintain the solubility of Hsp70 proteins in mitochondria and plastids of eukaryotic cells. However, little is known about the functional cooperation between Zim17 and mitochondrial Hsp70 proteins in vivo. To analyze the effects of a loss of Zim17 function in the authentic environment, we introduced novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae that allowed a recovery of temperature-sensitive but respiratory competent zim17 mutant cells. On fermentable growth medium, the mutant cells were prone to acquire respiratory deficits and showed a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. We show that the induction of the zim17 mutant phenotype leads to strong import defects for Ssc1-dependent matrix-targeted precursor proteins that correlate with a significantly reduced binding of newly imported substrate proteins to Ssc1. We conclude that Zim17 is not only required for the maintenance of mtHsp70 solubility but also directly assists the functional interaction of mtHsp70 with substrate proteins in a J-type co-chaperone-dependent manner.     

The Hsp40 J-domain Stimulates Hsp70 When Tethered by the Client to the ATPase Domain

The Escherichia coli Hsp40 DnaJ uses its J-domain (Jd) to couple ATP hydrolysis and client protein capture in Hsp70 DnaK. Fusion of the Jd to peptide p5 (as in Jdp5) dramatically increases the apparent affinity of the p5 moiety for DnaK in the presence of ATP, and Jdp5 stimulates ATP hydrolysis in DnaK by several orders of magnitude. NMR experiments with [¹⁵N]Jdp5 demonstrated that the peptide tethers the Jd to the ATPase domain. Thus, ATP hydrolysis and client protein binding in DnaK are coupled principally through the association of the client with DnaJ. Overexpression of a recombinant Jd was specifically toxic to cells that simultaneously expressed DnaK. No toxicity was observed when overexpressing Jdp5 or mutant Jd or when co-overexpressing the Jd and the nucleotide exchange factor GrpE. The results suggest that the Jd shifts DnaK to a client-bound form by stimulating the DnaK ATPase but only when the Jd is brought to DnaK by a client-Hsp40 complex.     

ATPase Activity and ATP-dependent Conformational Change in the Co-chaperone HSP70/HSP90-organizing Protein (HOP)

Co-chaperones help to maintain cellular homeostasis by modulating the activities of molecular chaperones involved in protein quality control. The HSP70/HSP90-organizing protein (HOP) is a co-chaperone that cooperates with HSP70 and HSP90 in catalysis of protein folding and maturation in the cytosol. We show here that HOP has ATP-binding activity comparable to that of HSP70/HSP90, and that HOP slowly hydrolyzes ATP. Analysis of deletion mutants revealed that the ATPase domain of HOP is in the N-terminal TPR1-DP1-TPR2A segment. In addition, HOP changes its conformation in the presence of ATP. These results indicate that HOP is a unique co-chaperone that undergoes an ATP-dependent conformational change.     

Hsp70 and Hsp40 Functionally Interact with Soluble Mutant Huntingtin Oligomers in a Classic ATP-dependent Reaction Cycle

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.     

Nucleolar Targeting of the Chaperone Hsc70 Is Regulated by Stress,Cell Signaling,and a Composite Targeting Signal Which Is Controlled by Autoinhibition

Hsc70s are constitutively synthesized members of the 70-kDa chaperone family; they are essential for viability and conserved among all organisms. When eukaryotic cells recover from stress, hsc70s accumulate in nucleoli by an unknown mechanism. Our studies were undertaken to characterize the signaling events and the targeting sequence required to concentrate hsc70 in the nucleoli of human cells. Here, we show that pharmacological inhibitors of phosphatidylinositol (PI) 3-kinase and MEK kinases as well as protein-tyrosine phosphatases abolished the stress-dependent nucleolar accumulation of hsc70. Furthermore, to identify the hsc70 nucleolar targeting sequence, green fluorescent protein-tagged fusion proteins with defined segments of hsc70 were generated and their subcellular distribution was analyzed in growing cells. These studies demonstrated that residues 225 to 297 serve as a heat-inducible nucleolar targeting signal. This segment directs green fluorescent protein to nucleoli in response to stress, but fails to do so under nonstress conditions. Fine mapping of the nucleolar targeting signal revealed that it has two separable functions. First, residues 225 to 262 direct reporter proteins constitutively to nucleoli, even without stress. Second, segment 263 to 287 functions as an autoinhibitory element that prevents hsc70 from concentrating in nucleoli when cells are not stressed. Taken together, PI 3-kinase and MEK kinase signaling as well as tyrosine dephosphorylation are essential for the accumulation of hsc70 in nucleoli of stressed cells. This process relies on a stress-dependent composite targeting signal that combines multiple functions.     

Disruption of Ionic Interactions between the Nucleotide Binding Domain 1 (NBD1) and Middle (M) Domain in Hsp100 Disaggregase Unleashes Toxic Hyperactivity and Partial Independence from Hsp70

Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction.     

Allosteric communication between the nucleotide binding domains of caseinolytic peptidase B

ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K(d) = 3-7 μm) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the K(d) decreases from ≈160-300 to 50-60 μm) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.     

Importance of N- and C-terminal regions of IbpA, Escherichia coli small heat shock protein, for chaperone function and oligomerization

Small heat shock proteins are ubiquitous molecular chaperones that, during cellular stress, bind to misfolded proteins and maintain them in a refolding competent state. Two members of the small heat shock protein family, IbpA and IbpB, are present in Escherichia coli. Despite 48% sequence identity, the proteins have distinct activities in promoting protein disaggregation. Cooperation between IbpA and IbpB is crucial for prevention of the irreversible aggregation of proteins. In this study, we investigated the importance of the N- and C-terminal regions of IbpA for self-oligomerization and chaperone functions. Deletion of either the N- or C-terminal region of IbpA resulted in a defect in the IbpA fibril formation process. The deletions also impaired IbpA chaperone function, defined as the ability to stabilize, in cooperation with IbpB, protein aggregates in a disaggregation-competent state. Our results show that the defect in chaperone function, observed in truncated versions of IbpA, is due to the inability of these proteins to interact with substrate proteins and consequently to change the properties of aggregates. At the same time, these versions of IbpA interact with IbpB similarly to the wild type protein. Competition experiments performed with the pC peptide, which corresponds to the IbpA C terminus, suggested the importance of IbpA intermolecular interactions in the stabilization of aggregates in a state competent for disaggregation. Our results suggest that these interactions are not only dependent on the universally conserved IEI motif but also on arginine 133 neighboring the IEI motif. IbpA mutated at arginine 133 to alanine lacked chaperone activity.     

Role of DnaJ G/F-rich Domain in Conformational Recognition and Binding of Protein Substrates

DnaJ from Escherichia coli is a Type I Hsp40 that functions as a cochaperone of DnaK (Hsp70), stimulating its ATPase activity and delivering protein substrates. How DnaJ binds protein substrates is still poorly understood. Here we have studied the role of DnaJ G/F-rich domain in binding of several substrates with different conformational properties (folded, partially (un)folded and unfolded). Using partial proteolysis we find that RepE, a folded substrate, contacts a wide DnaJ area that involves part of the G/F-rich region and Zn-binding domain. Deletion of G/F-rich region hampers binding of native RepE and reduced the affinity for partially (un)folded substrates. However, binding of completely unfolded substrates is independent on the G/F-rich region. These data indicate that DnaJ distinguishes the substrate conformation and is able to adapt the use of the G/F-rich region to form stable substrate complexes.     

Understanding the Functional Interplay between Mammalian Mitochondrial Hsp70 Chaperone Machine Components

Mitochondria biogenesis requires the import of several precursor proteins that are synthesized in the cytosol. The mitochondrial heat shock protein 70 (mtHsp70) machinery components are highly conserved among eukaryotes, including humans. However, the functional properties of human mtHsp70 machinery components have not been characterized among all eukaryotic families. To study the functional interactions, we have reconstituted the components of the mtHsp70 chaperone machine (Hsp70/J-protein/GrpE/Hep) and systematically analyzed in vitro conditions for biochemical functions. We observed that the sequence-specific interaction of human mtHsp70 toward mitochondrial client proteins differs significantly from its yeast counterpart Ssc1. Interestingly, the helical lid of human mtHsp70 was found dispensable to the binding of P5 peptide as compared with the other Hsp70s. We observed that the two human mitochondrial matrix J-protein splice variants differentially regulate the mtHsp70 chaperone cycle. Strikingly, our results demonstrated that human Hsp70 escort protein (Hep) possesses a unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins. We observed that Hep binds with the C terminus of mtHsp70 in a full-length context and this interaction is distinctly different from unfolded client-specific or J-protein binding. In addition, we found that the interaction of Hep at the C terminus of mtHsp70 is regulated by the helical lid region. However, the interaction of Hep at the ATPase domain of the human mtHsp70 is mutually exclusive with J-proteins, thus promoting a similar conformational change that leads to ATPase stimulation. Additionally, we highlight the biochemical defects of the mtHsp70 mutant (G489E) associated with a myelodysplastic syndrome.     

Ordered assembly of heat shock proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on expanded polyglutamine fragments revealed by chemical probes

In Saccharomyces cerevisae, expanded polyglutamine (polyQ) fragments are assembled into discrete cytosolic aggregates in a process regulated by the molecular chaperones Hsp26, Hsp70, Hsp90, and Hsp104. To better understand how the different chaperones might cooperate during polyQ aggregation, we used sequential immunoprecipitations and mass spectrometry to identify proteins associated with either soluble (Q25) or aggregation-prone (Q103) fragments at both early and later times after induction of their expression. We found that Hsp26, Hsp70, Hsp90, and other chaperones interact with Q103, but not Q25, within the first 2 h. Further, Hsp70 and Hsp90 appear to be partially released from Q103 prior to the maturation of the aggregates and before the recruitment of Hsp104. To test the importance of this seemingly ordered process, we used a chemical probe to artificially enhance Hsp70 binding to Q103. This treatment retained both Hsp70 and Hsp90 on the polyQ fragment and, interestingly, limited subsequent exchange for Hsp26 and Hsp104, resulting in incomplete aggregation. Together, these results suggest that partial release of Hsp70 may be an essential step in the continued processing of expanded polyQ fragments in yeast.     

Recognition of mitochondrial targeting sequences by the import receptors Tom20 and Tom22

The Tom20 and Tom22 receptor subunits of the TOM (translocase of the outer mitochondrial membrane) complex recognize N-terminal presequences of proteins that are to be imported into the mitochondrion. In plants, Tom20 is C-terminally anchored in the mitochondrial membrane, whereas Tom20 is N-terminally anchored in animals and fungi. Furthermore, the cytosolic domain of Tom22 in plants is smaller than its animal/fungal counterpart and contains fewer acidic residues. Here, NMR spectroscopy was used to explore presequence interactions with the cytosolic regions of receptors from the plant Arabidopsis thaliana and the fungus Saccharomyces cerevisiae (i.e., AtTom20, AtTom22, and ScTom22). It was found that AtTom20 possesses a discontinuous bidentate hydrophobic binding site for presequences. The presequences on plant mitochondrial proteins comprise two or more hydrophobic binding regions to match this bidentate site. NMR data suggested that while these presequences bind to ScTom22, they do not bind to AtTom22. AtTom22, however, binds to AtTom20 at the same binding site as presequences, suggesting that this domain competes with the presequences of imported proteins, thereby enabling their progression along the import pathway.     

Distribution of binding sequences for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequence-carrying preprotein and a non-cleavable preprotein.

Preproteins destined for mitochondria either are synthesized with amino-terminal signal sequences, termed presequences, or possess internal targeting information within the protein. The preprotein translocase of the outer mitochondrial membrane (designated Tom) contains specific import receptors. The cytosolic domains of three import receptors, Tom20, Tom22, and Tom70, have been shown to interact with preproteins. Little is known about the internal targeting information in preproteins and the distribution of binding sequences for the three import receptors. We have studied the binding of the purified cytosolic domains of Tom20, Tom22, and Tom70 to cellulose-bound peptide scans derived from a presequence-carrying cleavable preprotein, cytochrome c oxidase subunit IV, and a non-cleavable preprotein with internal targeting information, the phosphate carrier. All three receptor domains are able to bind efficiently to linear 13-mer peptides, yet with different specificity. Tom20 preferentially binds to presequence segments of subunit IV. Tom22 binds to segments corresponding to the carboxyl-terminal part of the presequence and the amino-terminal part of the mature protein. Tom70 does not bind efficiently to any region of subunit IV. In contrast, Tom70 and Tom20 bind to multiple segments within the phosphate carrier, yet the amino-terminal region is excluded. Both charged and uncharged peptides derived from the phosphate carrier show specific binding properties for Tom70 and Tom20, indicating that charge is not a critical determinant of internal targeting sequences. This feature contrasts with the crucial role of positively charged amino acids in presequences. Our results demonstrate that linear peptide segments of preproteins can serve as binding sites for all three receptors with differential specificity and imply different mechanisms for translocation of cleavable and non-cleavable preproteins.     

Characterization of a human import component of the mitochondrial outer membrane,TOMM70A

Functional mitochondria require up to 1000 proteins to function properly, with 99% synthesized as precursors in the cytoplasm and transported into the mitochondria with the aid of cytosolic chaperones and mitochondrial translocators (import components). Proteins to be imported are chaperoned to the mitochondria by the cytosolic heat shock protein (cHSP70) and are immediately pursued by Translocators of the Outer Membrane (TOMs), followed by transient interactions of the unfolded proteins with Translocators of the Inner Membrane (TIMs). In the present study, we describe a human gene, TOMM70A, orthologous to the yeast Tom70 import component. TOMM70A is ubiquitously expressed in human tissues, maps on chromosome 3q13.1-q13.2 and consists of 12 coding exons spanning over 37 kb. TOMM70A localizes in the mitochondria of COS-7 cells, and in organello import assays confirmed its presence in the Outer Mitochondrial membrane (OM) of rat liver mitochondria. TOMM70A could play a significant role in the import of nuclear-encoded mitochondrial proteins with internal targeting sites such as ADP/ATP carriers and the uncoupling proteins.     

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein.     

Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates

Structurally and sequence-wise, the Hsp110s belong to a subfamily of the Hsp70 chaperones. Like the classical Hsp70s, members of the Hsp110 subfamily can bind misfolding polypeptides and hydrolyze ATP. However, they apparently act as a mere subordinate nucleotide exchange factors, regulating the ability of Hsp70 to hydrolyze ATP and convert stable protein aggregates into native proteins. Using stably misfolded and aggregated polypeptides as substrates in optimized in vitro chaperone assays, we show that the human cytosolic Hsp110s (HSPH1 and HSPH2) are bona fide chaperones on their own that collaborate with Hsp40 (DNAJA1 and DNAJB1) to hydrolyze ATP and unfold and thus convert stable misfolded polypeptides into natively refolded proteins. Moreover, equimolar Hsp70 (HSPA1A) and Hsp110 (HSPH1) formed a powerful molecular machinery that optimally reactivated stable luciferase aggregates in an ATP- and DNAJA1-dependent manner, in a disaggregation mechanism whereby the two paralogous chaperones alternatively activate the release of bound unfolded polypeptide substrates from one another, leading to native protein refolding.     

Multiple Molecules of Hsc70 and a Dimer of DjA1 Independently Bind to an Unfolded Protein

Protein folding is a prominent chaperone function of the Hsp70 system. Refolding of an unfolded protein is efficiently mediated by the Hsc70 system with either type 1 DnaJ protein, DjA1 or DjA2, and a nucleotide exchange factor. A surface plasmon resonance technique was applied to investigate substrate recognition by the Hsc70 system and demonstrated that multiple Hsc70 proteins and a dimer of DjA1 initially bind independently to an unfolded protein. The association rate of the Hsc70 was faster than that of DjA1 under folding-compatible conditions. The Hsc70 binding involved a conformational change, whereas the DjA1 binding was bivalent and substoichiometric. Consistently, we found that the bound ¹⁴C-labeled Hsc70 to the unfolded protein became more resistant to tryptic digestion. The gel filtration and cross-linking experiments revealed the predominant presence of the DjA1 dimer. Furthermore, the Hsc70 and DjA1 bound to distinct sets of peptide array sequences. All of these findings argue against the generality of the widely proposed hypothesis that the DnaJ-bound substrate is targeted and transferred to Hsp70. Instead, these results suggest the importance of the bivalent binding of DjA1 dimer that limits unfavorable transitions of substrate conformations in protein folding.     

Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.     

Mitochondrial Heat Shock Protein (Hsp) 70 and Hsp10 Cooperate in the Formation of Hsp60 Complexes

Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings.     

Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL

Many proteins contain a thioredoxin (Trx)-like domain fused with one or more partner domains that diversify protein function by the modular construction of new molecules. The Escherichia coli protein YbbN is a Trx-like protein that contains a C-terminal domain with low homology to tetratricopeptide repeat motifs. YbbN has been proposed to act as a chaperone or co-chaperone that aids in heat stress response and DNA synthesis. We report the crystal structure of YbbN, which is an elongated molecule with a mobile Trx domain and four atypical tetratricopeptide repeat motifs. The Trx domain lacks a canonical CXXC active site architecture and is not a functional oxidoreductase. A variety of proteins in E. coli interact with YbbN, including multiple ribosomal protein subunits and a strong interaction with GroEL. YbbN acts as a mild inhibitor of GroESL chaperonin function and ATPase activity, suggesting that it is a negative regulator of the GroESL system. Combined with previous observations that YbbN enhances the DnaK-DnaJ-GrpE chaperone system, we propose that YbbN coordinately regulates the activities of these two prokaryotic chaperones, thereby helping to direct client protein traffic initially to DnaK. Therefore, YbbN may play a role in integrating the activities of different chaperone pathways in E. coli and related bacteria.