Bernd Bukau

The Hsp70 and Hsp60 Chaperone Machines

B. B. thanks members of his lab and J. Reinstein for critical reading of the manuscript and C. Gassler, T. Laufen, and S. Rudiger for figure preparation. A. H. thanks Wayne Fenton for critical reading and Zhaohui Xu for figure preparation. A. H. dedicates this work to Guenter Brueckner, always an inspiration.

Hsp70 chaperones: cellular functions and molecular mechanism.

Abstract.Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.

Molecular Chaperones and Protein Quality Control

In living cells, both newly made and preexisting polypeptide chains are at constant risk for misfolding and aggregation. In accordance with the wide diversity of misfolded forms, elaborate quality-control strategies have evolved to counter these inevitable mishaps. Recent reports describe the removal of aggregates from the cytosol; reveal mechanisms for protein quality control in the endoplasmic reticulum; and provide new insight into two classes of molecular chaperones, the Hsp70 system and the AAA+ (Hsp100) unfoldases.

Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.

Hsp70 chaperones assist protein folding by ATP‐dependent association with linear peptide segments of a large variety of folding intermediates. The molecular basis for this ability to differentiate between native and non‐native conformers was investigated for the DnaK homolog of Escherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose‐bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues. In the folded proteins these sites are mostly buried and in the majority found in β‐sheet elements. The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues. Acidic residues are excluded from the core and disfavored in flanking regions. The energetic contribution of all 20 amino acids for DnaK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.

Cellular strategies for controlling protein aggregation.

The aggregation of misfolded proteins is associated with the perturbation of cellular function, ageing and various human disorders. Mounting evidence suggests that protein aggregation is often part of the cellular response to an imbalanced protein homeostasis rather than an unspecific and uncontrolled dead-end pathway. It is a regulated process in cells from bacteria to humans, leading to the deposition of aggregates at specific sites. The sequestration of misfolded proteins in such a way is protective for cell function as it allows for their efficient solubilization and refolding or degradation by components of the protein quality-control network. The organized aggregation of misfolded proteins might also allow their asymmetric distribution to daughter cells during cell division.

Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB

We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150–200 species, corresponding to 15–25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (≥90 kDa) but only 18% of the small (≤30 kDa) cytosolic proteins and include essential proteins. The DnaK system in addition acts with ClpB to form a bi‐chaperone system that quantitatively solubilizes aggregates of most of these proteins. Efficient solubilization also occurred in an in vivo order‐of‐addition experiment in which aggregates were formed prior to induction of synthesis of the bi‐chaperone system. Our data indicate that large‐sized proteins are most vulnerable to thermal unfolding and aggregation, and that the DnaK system has central, dual protective roles for these proteins by preventing their aggregation and, cooperatively with ClpB, mediating their disaggregation.

DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.

Members of the conserved Hsp70 chaperone family are assumed to constitute a main cellular system for the prevention and the amelioration of stress‐induced protein damage, though little direct evidence exists for this function. We investigated the roles of the DnaK (Hsp70), DnaJ and GrpE chaperones of Escherichia coli in prevention and repair of thermally induced protein damage using firefly luciferase as a test substrate. In vivo, luciferase was rapidly inactivated at 42 degrees C, but was efficiently reactivated to 50% of its initial activity during subsequent incubation at 30 degrees C. DnaK, DnaJ and GrpE did not prevent luciferase inactivation, but were essential for its reactivation. In vitro, reactivation of heat‐inactivated luciferase to 80% of its initial activity required the combined activity of DnaK, DnaJ and GrpE as well as ATP, but not GroEL and GroES. DnaJ associated with denatured luciferase, targeted DnaK to the substrate and co‐operated with DnaK to prevent luciferase aggregation at 42 degrees C, an activity that was required for subsequent reactivation. The protein repair function of DnaK, GrpE and, in particular, DnaJ is likely to be part of the role of these proteins in regulation of the heat shock response.

Trigger factor and DnaK cooperate in folding of newly synthesized proteins

The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood. In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures. The major protein associated with nascent polypeptides is ribosome-bound trigger factor,, which displays chaperone and prolyl isomerase activities in vitro,,. Here we show that Δtig::kan mutants lacking trigger factor have no defects in growth or protein folding. However, combined Δtig::kan and ΔdnaK mutations cause synthetic lethality. Depletion of DnaK in the Δtig::kan mutant results in massive aggregation of cytosolic proteins. In Δtig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK. These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70. Trigger factor and DnaK cooperate to promote proper folding of a variety of E. coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.

Thermotolerance Requires Refolding of Aggregated Proteins by Substrate Translocation through the Central Pore of ClpB

Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.

Getting Newly Synthesized Proteins into Shape

Recent progress made in the analysis of in vivo folding of cytosolic proteins suggests that folding of cytosolic proteins occurs via multiple chaperone-assisted, as well as unassisted pathways. In the case of individual proteins, certain pathways may be highly favored. But, the cellular folding machinery also shows significant redundancy and flexibility, resulting in a variable network of folding pathways having alternative routes and backup systems.‡To whom correspondence should be addressed (e-mail: ecraig@facstaff.wisc.edu [E. A. C.], bukau@sun2.ruf.uni-freiburg.de [B. B.]).