Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling

Abstract

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...

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

“…Previous studies have shown that DnaK and Ssc1 can reactivate stable protein aggregates when assisted by bacterial DnaJ and GrpE co-chaperones (De Los Rios et al 2006;Mogk et al 1999). However, the ability of mortalin to reactivate preformed protein aggregates has not been demonstrated so far.…”

“…This particular function of the chaperone occurs through association of the ATP-bound state to the TIM23 import channel, by way of the specific protein anchors, Tim44 and PAM16/18. Dissociation of ADP-bound Ssc1 from the anchors takes place as soon as it has locked onto an incoming polypeptide (De Los Rios et al 2006;D'Silva et al 2004;Neupert and Herrmann 2007). Ssc1 plays an additional important role in mediating the refolding of matrix-localized proteins, a function which is carried out with the help of two matrix-localized co-chaperones Mdj1 and Mge1, which are homologous to the Escherichia coli DnaJ and GrpE, respectively (Deloche et al 1997;Horst et al 1997;Liu et al 2001;Rowley et al 1994) (Supplemental Fig.…”

“…2). Using purified proteins, it was shown that Ssc1 can mediate ATP-dependent disaggregation and proper refolding of stably misfolded reporter proteins, when supplemented with bacterial DnaJ and GrpE (De Los Rios et al 2006). Ssq1 is known to play a role in the biogenesis of iron sulfur clusters and the function of Ecm10 is still unclear (Baumann et al 2000;Dutkiewicz et al 2003).…”

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

“…Whereas Hsp70/40 is essential for Hsp104 protein disaggregation, the mechanistic role of Hsp70 remained unclear. It has been reported that Hsp70/40 possesses some protein disaggregating activity (45), which can be stimulated by Hsp110 (an Hsp70-related nucleotide exchange factor of Hsp70), but which is less efficient than the Hsp104 bichaperone system (46).…”

Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis

“…While there is some information available on the role that Hsp72 plays in chaperoning proteins through intracellular membranes (De Los Rios et al 2006) and remodeling complex protein structures such as clathrin-coated pits (Prasad et al 1994), the effect of Hsp72 on intracellular signaling remains largely undefined. Hsp72 has been shown to bind to a variety of different kinases and signaling molecules, and so may have an impact on a number of different aspects of cell regulation including the cell cycle (Diehl et al 2003) and p53 signaling (Fourie et al 1997).…”