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 andCMLS, Cell. Mol. Life Sci. 62 (2005) 670 -684 1420-682X/05/060670-15 DOI 10.1007/s00018-004-4464-6 © Birkhäuser Verlag, Basel, 2005 CMLS Cellular and Molecular Life Scienceshydrolysis 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.

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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.

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(Flynn et al., 1991; Landry et al., 1992; Hsp70 chaperones assist protein folding by ATP- Blond-Elguindi et al., 1993;Gragerov et al., 1994). With dependent association with linear peptide segments of respect to the binding motif and its amino acid composition, a large variety of folding intermediates. The molecular they yielded results in conflict with each other (Flynn et al., basis for this ability to differentiate between native 1991; Blond-Elguindi et al., 1993;Gragerov et al., 1994) and non-native conformers was investigated for the and the structure of the DnaK substrate binding pocket DnaK homolog of Escherichia coli. We identified bind- (Blond-Elguindi et al., 1993). Furthermore, they were not ing sites and the recognition motif in substrates by aimed at identifying Hsp70 binding sites in biologically screening 4360 cellulose-bound peptides scanning the relevant protein substrates. sequences of 37 biologically relevant proteins. DnaKThis study was performed to identify the binding sites binding sites in protein sequences occurred statistically within protein sequences and the substrate binding motif of every 36 residues. In the folded proteins these sites are the DnaK chaperone. For this purpose we screened cellumostly buried and in the majority found in β-sheet lose-bound peptide scans (Reineke et al., 1995) representing elements. The binding motif consists of a hydrophobic complete protein sequences for DnaK binding. This novel core of four to five residues enriched particularly in approach offers the advantages of (i) avoiding precipitation, Leu, but also in Ile, Val, Phe and Tyr, and two flanking in particular, of DnaK binding peptides anticipated to be regions enriched in basic residues. Acidic residues are hydrophobic, (ii) allowing identification of DnaK binding excluded from the core and disfavored in flanking sites in natural substrate sequences, (iii) allowing direct regions. The energetic contribution of all 20 amino identification of the recognition motif by sequence alignacids for DnaK binding was determined. On the basis ment of neighboring binding peptides and (iv) providing a of these data an algorithm was established that predicts large data set for binding as well as non-binding peptides. DnaK binding sites in protein sequences with highIt allowed the identification of the substrate binding motif accuracy.of DnaK and the establishment of an algorithm predicting Keywords: cellulose-bound peptide libraries/heat shock DnaK binding sites within protein sequences. proteins/Hsp70/protein folding/spot synthesis

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.

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.

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