Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation.H sp70 is a central component of the chaperone network in the cell with disparate cellular functions. Associated with the ribosome, Hsp70s foster proper de novo protein folding. In the cytoplasm, Hsp70s mediate the deoligomerization and recycling of native protein complexes (1, 2) and control key functions in evolution, cell morphogenesis (2), and apoptosis (3), often in association with Hsp90 (4). Hsp70 also serves as the central translocation motor in the posttranslational import of cytoplasmic proteins into mitochondria (5), chloroplasts (6, 7), and the endoplasmic reticulum (8). Moreover, Hsp70s can actively unfold, solubilize, and reactivate already formed, stable protein aggregates (9, 10) and may participate in targeting proteins to the degradation pathway (11, 12). Existing Models for Hsp70-Mediated Protein Translocation into MitochondriaThe translocation of proteins across the mitochondrial membrane, through the translocase of the outer membrane (TOM) and translocase of the inner membrane (TIM) translocation pores, is mediated by the presequence translocase-associated motor (PAM) complex consisting of matrix-localized Hsp70 (mtHsp70), membrane-associated J domain-containing proteins (three identified so far, PAM16͞Tim16, PAM18͞Tim14, and Mdj2) (13-19) and the nucleotide exchange factor Mge1. In the ATP-bound state, mtHsp70 is in the open (unlocked) state, which is as yet unbound to the translocating protein substrate, whereas mtHsp70 is found anchored to the mitochondrial import channel by way of its transient association with the mitochondrial peripheral inner-membrane protein Tim44. In the ADP-bound state, mtHsp70 is tightly bound (locked) onto the incoming polypeptide and is not associated to the m...

Molecular chaperones of the Hsp70 family are ubiquitous proteins that perform functions essential for cellular life, including protein folding, the assembly of protein complexes, protein degradation, the translocation of proteins across membranes and regulation of the heat shock response [1]. To carry out these different functions, Hsp70s rely on the ability to bind short hydrophobic peptide stretches in extended conformations that might become accessible within the sequence of a protein. Conservation among different members of the family is high and extends to both sequence and structure, as revealed by the available three-dimensional structures of isolated protein domains [2][3][4][5][6]. Thus, Hsp70s are composed of a highly homologous N-terminal ATPase domain of 45 kDa, connected by a short linker to a more variable peptide-binding domain (PBD) of 25 kDa, consisting of a conserved b-sandwich and a more variable a-helical subdomain [7]. The latter subdomain forms a lid that closes the binding site without contacting the peptide substrate [4,5]. The peptide binding site consists of a hydrophobic cavity formed by loops that protrude from the Among the eukaryotic members of the Hsp70 family, mitochondrial Hsp70 shows the highest degree of sequence identity with bacterial DnaK. Although they share a functional mechanism and homologous co-chaperones, they are highly specific and cannot be exchanged between Escherichia coli and yeast mitochondria. To provide a structural basis for this finding, we characterized both proteins, as well as two DnaK ⁄ mtHsp70 chimeras constructed by domain swapping, using biochemical and biophysical methods. Here, we show that DnaK and mtHsp70 display different conformational and biochemical properties. Replacing different regions of the DnaK peptide-binding domain with those of mtHsp70 results in chimeric proteins that: (a) are not able to support growth of an E. coli DnaK deletion strain at stress temperatures (e.g. 42°C); (b) show increased accessibility and decreased thermal stability of the peptide-binding pocket; and (c) have reduced activation by bacterial, but not mitochondrial co-chaperones, as compared with DnaK. Importantly, swapping the C-terminal a-helical subdomain promotes a conformational change in the chimeras to an mtHsp70-like conformation. Thus, interaction with bacterial co-chaperones correlates well with the conformation that natural and chimeric Hsp70s adopt in solution. Our results support the hypothesis that a specific protein structure might regulate the interaction of Hsp70s with particular components of the cellular machinery, such as Tim44, so that they perform specific functions.Abbreviations DSC, differential scanning spectroscopy; DTT, dithiothreitol; GdnHCl, guanidine hydrochloride; IR, infrared spectroscopy; mtHsp70, mitochondrial Hsp70; PBD, peptide binding domain.