Yeast Hsp104 and its bacterial homolog, ClpB, are Clp/Hsp100 molecular chaperones and AAA+ ATPases. Hsp104 and ClpB collaborate with the Hsp70 and DnaK chaperone systems, respectively, to retrieve and reactivate stress-denatured proteins from aggregates. The action of Hsp104 and ClpB in promoting cell survival following heat stress is species-specific: Hsp104 cannot function in bacteria and ClpB cannot act in yeast. To determine the regions of Hsp104 and ClpB necessary for this specificity, we tested chimeras of Hsp104 and ClpB in vivo and in vitro. We show that the Hsp104 and ClpB middle domains dictate the species-specificity of Hsp104 and ClpB for cell survival at high temperature. In protein reactivation assays in vitro, chimeras containing the Hsp104 middle domain collaborate with Hsp70 and those with the ClpB middle domain function with DnaK. The region responsible for the specificity is within helix 2 and helix 3 of the middle domain. Additionally, several mutants containing amino acid substitutions in helix 2 of the ClpB middle domain are defective in protein disaggregation in collaboration with DnaK. In a bacterial two-hybrid assay, DnaK interacts with ClpB and with chimeras that have the ClpB middle domain, implying that species-specificity is due to an interaction between DnaK and the middle domain of ClpB. Our results suggest that the interaction between Hsp70/DnaK and helix 2 of the middle domain of Hsp104/ClpB determines the specificity required for protein disaggregation both in vivo and in vitro, as well as for cellular thermotolerance.Hsp40 | DnaJ | M-domain | GrpE | nucleotide exchange factor

Saccharomyces cerevisiae Hsp104 and Escherichia coli ClpB are Hsp100 family AAA+ chaperones that provide stress tolerance by cooperating with Hsp70 and Hsp40 to solubilize aggregated protein. Hsp104 also remodels amyloid in vitro and promotes propagation of amyloid prions in yeast, but ClpB does neither, leading to a view that Hsp104 evolved these activities. Although biochemical analyses identified disaggregation machinery components required for resolubilizing proteins, interactions among these components required for in vivo functions are not clearly defined. We express prokaryotic chaperones in yeast to address these issues and find ClpB supports both prion propagation and thermotolerance in yeast if it is modified to interact with yeast Hsp70 or if E. coli Hsp70 and its cognate nucleotide exchange factor (NEF) are present. Our findings show prion propagation and thermotolerance in yeast minimally require cooperation of species-specific Hsp100, Hsp70, and NEF with yeast Hsp40. The functions of this machinery in prion propagation were directed primarily by Hsp40 Sis1p, while thermotolerance relied mainly on Hsp40 Ydj1p. Our results define cooperative interactions among these components that are specific or interchangeable across life kingdoms and imply Hsp100 family disaggregases possess intrinsic amyloid remodeling activity.

SummaryTo examine the relationship between folding and aggregation in the periplasm of Escherichia coli, we have analysed the cellular fates of exported proteins fused to either the wild-type maltose-binding protein (MalE) or the aggregation-prone variant MalE31. The propensity of fusion proteins to aggregate in the periplasm was determined by the intrinsic folding characteristics of the upstream protein. When b-lactamase or alkaline phosphatase was linked to the C-terminus of MalE31, the resultant fusion proteins accumulated in an insoluble form, but retained their catalytic activity. In addition, these protein aggregates induced an extracytoplasmic stress response, similar to unfused MalE31. However, using a fluorescent substrate, we found that alkaline phosphatase activity was present inside periplasmic aggregates. These results suggest that periplasmic inclusion body formation may result in intermolecular interactions between participating proteins without loss of function of the fused enzymes.

The DnaK/Hsp70 chaperone system and ClpB/Hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. The teamwork is specific: E. coli DnaK interacts with E. coli ClpB and yeast Hsp70, Ssa1, interacts with yeast Hsp104. This interaction is between the M-domains of hexameric ClpB/Hsp104 and the DnaK/Hsp70 nucleotide-binding domain (NBD). To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomain IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Computational modeling of the DnaK-ClpB hexamer complex indicated that one DnaK monomer contacts two adjacent ClpB protomers simultaneously. The model and the experiments support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation.

BackgroundCaseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium.ResultsWe report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in Escherichia coli. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides.We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers.ConclusionDespite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual mechanism of self-assembly. Therefore, the mechanism underlying their peptidase and proteolytic activities might differ from that of other ClpP proteolytic complexes.

The proper functioning of extracytoplasmic proteins requires their export to, and productive folding in, the correct cellular compartment. All proteins in Escherichia coli are initially synthesized in the cytoplasm, then follow a pathway that depends upon their ultimate cellular destination. Many proteins destined for the periplasm are synthesized as precursors carrying an N-terminal signal sequence that directs them to the general secretion machinery at the inner membrane. After translocation and signal sequence cleavage, the newly exported mature proteins are folded and assembled in the periplasm. Maintaining quality control over these processes depends on chaperones, folding catalysts, and proteases. This article summarizes the general principles which control protein folding in the bacterial periplasm by focusing on the periplasmic maltose-binding protein. ReviewAlthough all the information necessary for a protein to reach a native structure is contained in its amino acid sequence, in vivo protein folding requires the participation of protein factors; molecular chaperones, folding catalysts and proteases, that monitor or regulate this process through many cellular functions. Under physiological conditions, these factors maintain quality control of protein biosynthesis, and errors or failures of the protein folding process are rare. However, incorrectly folded or misfolded proteins can appear as a result of (i) spontaneous or inducible mutations that affect protein folding pathways, (ii) exposure of the cell to environmental stress, such as elevated temperatures or hyperosmolarity, and (iii) overexpression of recombinant genes. In these cases, the polypeptide chain, instead of folding into the native biologically active state, misfolds, and eventually induces a stress response [1]. The resulting misfolded proteins may either be degraded by proteases, repaired by chaperones, or aggregated and sequestered as inclusion bodies (IBs), when escaping protein folding quality control [2].In the cytoplasm of Escherichia coli, this control is performed mainly by a set of stress-inducible chaperones and proteases collectively known as heat shock proteins (Hsps). The presence of cellular membrane-bounded compartments in E. coli further complicates these issues of protein biosynthesis, and implies the existence of distinct folding and degradation machineries, quality control systems, for extracytoplasmic proteins [3]. The bacterial periplasm is separated from the extracellular milieu only by the porous outer membrane, and hence is more susceptible to changes in the environment than the cytoplasm. Thus, protein quality control in the periplasm is crucial for the survival of bacterial cells. Here we summarize the main features of protein folding in the periplasm of E. coli, and focus on studies using a model system, the maltosebinding protein. Biosynthesis of periplasmic proteinsIn E. coli, exported proteins with an ultimate destination of the periplasm and outer membrane are synthesized as precursors with a cleavabl...

To eliminate unavoidable contamination of purified recombinant proteins by DnaK, we present a unique approach employing a BL21(DE3) ⌬dnaK strain of Escherichia coli. Selected representative purified proteins remained soluble, correctly assembled, and active. This finding establishes DnaK dispensability for protein production in BL21(DE3), which is void of Lon protease, key to eliminating unfolded proteins.Obtaining substantial amounts of pure protein is essential in innumerable biological studies and indispensable to the biochemical characterization of proteins. The ease of growth, well-characterized genetics, and the large number of tools for gene expression have long made Escherichia coli the organism of choice for protein overproduction. The BL21(DE3) strain is widely used for recombinant protein production because of its engineered capacity to produce T7 polymerase and its deficiency in Lon and OmpT proteases.DnaK is an abundant protein (about 1% of the total protein of E. coli) (17) that interacts with a wide range of newly synthesized polypeptides (28) and assists their proper folding and assembly into oligomers by preventing protein aggregation.

Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation.Furthermore Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone decreases S109 phosphorylation, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.