Effect of Constitutive 70-kDa Heat Shock Protein Polymerization on Its Interaction with Protein Substrate

Abstract

Constitutive 70-kDa heat shock protein (hsc70) is a mixture of monomers and oligomers in ADP, while in ATP it is monomeric unless certain DnaJ homologs are present which induce hsc70 to form large polymers in an ATP-dependent reaction. A key question regarding polymerized hsc70 is whether it is able to bind protein substrates. Polymerized BiP, the hsc70 present in the endoplasmic reticulum, has been found to bind substrates in vitro although substrates appear to bind only to monomeric BiP in vivo. In this study, we investigated whether substrate binds to polymerized cytoplasmic hsc70 in vitro. Although both stoichiometric ATP and high concentrations of cytochrome c peptide monomerized hsc70, direct binding studies provided no evidence that cytochrome c peptide binds to polymerized hsc70. Furthermore, the time course of cytochrome c peptide and clathrin binding to hsc70 suggested that rather than binding to polymerized hsc70, they monomerized it by reducing free monomer, thereby shifting the monomerpolymer equilibrium toward monomer. We conclude that peptide and protein substrates bind at least an order of magnitude more weakly to polymerized hsc70 than to monomer, suggesting that polymerization of hsc70 in vivo, perhaps by DnaJ homologs, may store it in an inactive form. hsp70s1 belong to one of the most prominent classes of proteins produced by the cell under stress. In addition, numerous cognate forms of the hsp70s (hsc70s) are expressed constitutively in the cell. Interactions of the hsc70s with their protein substrates are involved in many important cellular functions including protein translocation across membranes of cell organelles, nascent protein folding and multiunit protein assembly, antigen presentation, protein degradation in the lysosome, and uncoating of clathrin-coated vesicles (for a review, see Hendrick and Hartl, 1993). In each of these processes, the hsc70s act as molecular chaperones which interact with protein substrates in a nucleotide-dependent manner. In general, hsc70-ATP appears to bind and release protein substrates rapidly and hsc70-ADP appears to bind and release protein substrates very slowly, suggesting that hydrolysis of ATP to ADP traps protein substrates on the hsc70 (Prasad et al., 1994;Greene et al., 1995;McCarty et al., 1995).ATP and ADP also affect the polymerization of hsc70. Most studies suggest that purified hsc70 occurs as a mixture of monomers, dimers, and higher order polymers in ADP, while in ATP it occurs mostly as monomers (Schlossman et al., 1984;Schmid et al., 1985;Carlino et al., 1992;Toledo et al., 1993;Palleros et al., 1993). There is evidence that polymerization of hsc70 may be important in vivo, in particular, in storing hsc70 in an inactive form. Studying BiP, the hsc70 protein found in the endoplasmic reticulum, Freiden et al. (1992) found that BiP was predominantly monomeric in cells accumulating nontransportable proteins, and only monomeric BiP was complexed with protein substrates, while in control cells BiP appeared to be mostly polymerized and free of...

Hop Modulates hsp70/hsp90 Interactions in Protein Folding

“…However, the hsp70 state could change during the course of an experiment. Our results agree with observations that Ydj is able to cause hsp70 polymerization which lowers its affinity for substrates (42), but Hop does not shift the hsp70 population toward the monomer form either in the presence or absence of Ydj (not shown). On the other hand, Hop does suppress the inhibitory effect resulting at high hsp70 concentrations and broadens the operating range of hsp70.…”

“…Also, we have never observed more hsp70 than Hop in immune precipitations of these proteins. Hop's effect could also be explained as changing the oligomeric state of hsp70 which has recently been shown to alter its ability to bind substrate (42). Shifting the hsp70 population toward monomer form would be expected to increase its affinity for substrate and increase refolding efficiency.…”

Heteromeric complexes of heat shock protein 70 (HSP70) family members, including Hsp70B′, in differentiated human neuronal cells

“…HSP70 family members including Hsp70, Hsc70, and the endoplasmic reticulum member BiP have been shown to self-associate (Freiden et al 1992;Benaroudj et al 1995;Wei et al 1995;Gao et al 1996;Angelidis et al 1999;Nemoto et al 2006). In the present report, we demonstrate that Hsp70B′, Hsp70, and Hsc70 form heteromeric complexes in differentiated human neuronal cells.…”

Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate

“…Attempts to add higher levels of Hsc70 to RRL from these concentrated Hsc70 stock solutions did not protect translation from inhibition in heme-deficient RRL, and inhibited translation when added to heme-supplemented RRL. In some experiments, partially aggregated Hsc70 was activated by preincubation in hemin-supplemented RRL in the presence of a creatine phosphate/creatine phosphokinase ATP-regenerating system [33] for 10 min prior to the addition of agents that activate HRI. Controls were incubated for 10 min without the addition of Hsc70.…”